( A , B ) ( A ) Coronal sections of P14 brains stained for calbindin and VgluT1 . Infrapyramidal bundle ( IPB ) progresses into hippocampal CA3 region in both WT and crmp2 - / - mice . Nuclei are counterstained with Hoechst 33342 . ( B ) Details from depicted regions in ( A ) , arrowheads show the course of IPB . Scale bars : 100 μm .

O O O O O O O O B-TISSUE I-TISSUE O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-GENEPROD O O O B-ORGANISM O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O

(A, B) (A) Coronal sections of P14 brains stained for calbindin and VgluT1. Infrapyramidal bundle (IPB) progresses into hippocampal CA3 region in both WT and crmp2-/- mice. Nuclei are counterstained with Hoechst 33342. (B) Details from depicted regions in (A), arrowheads show the course of IPB. Scale bars: 100 μm.


( C , D ) ( C ) Staining for calbindin shows unpruned infrapyramidal bundle in adult crmp2 - / - mice . No VGluT2 signal in hippocampal CA3 region is present in WT and crmp2 - / - suggesting mature synapses . Scale bars : 500 μm . ( D ) Details from depicted regions in ( C ) , arrowheads show the course of IPB . Scale bars : 100 μm .

O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O

(C, D) (C) Staining for calbindin shows unpruned infrapyramidal bundle in adult crmp2-/- mice. No VGluT2 signal in hippocampal CA3 region is present in WT and crmp2-/- suggesting mature synapses. Scale bars: 500 μm. (D) Details from depicted regions in (C), arrowheads show the course of IPB. Scale bars: 100 μm.


( E , F ) Staining for calbindin and VgluT1 shows mature synapses in the main bundle in both genotypes and in the IPB in crmp2 - / - mice ( arrowheads ) . Scale bars : 100 μm .

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(E, F) Staining for calbindin and VgluT1 shows mature synapses in the main bundle in both genotypes and in the IPB in crmp2-/- mice (arrowheads). Scale bars: 100 μm.


( G ) Quantification of IPB index ( length of the IPB vs . main bundle , WT 0 . 43 ± 0 . 05 , crmp2 - / - 0 . 78 ± 0 . 05 , p < 0 . 001 ) counted from adult coronal sections ( WT n = 4 mice , crmp2 - / - n = 5 mice ) . Mean ± SD , * * * p < 0 . 001 , t - test .

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(G) Quantification of IPB index (length of the IPB vs. main bundle, WT 0.43±0.05, crmp2-/- 0.78±0.05, p<0.001) counted from adult coronal sections (WT n=4 mice, crmp2-/- n=5 mice). Mean ± SD, ***p<0.001, t-test.


( A ) Upper row : DiI tracing of the visual cortex axons at P9 ( before pruning ) , sagittal sections . Branching point of the tract is shown . Lower row : visual cortex axons in adult mice after pruning period . Note significantly reduced number of axons continuing into pyramidal tract in WT ( dashed arrow ) . ( arrows - 2 branches of corticospinal visual axons ) . Maximum projections are shown . Scale bars : 200 μm .

O O O O O O B-SMALL_MOLECULE O O O B-TISSUE I-TISSUE B-SUBCELLULAR O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-TISSUE I-TISSUE B-SUBCELLULAR O O B-ORGANISM O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O B-TISSUE I-TISSUE O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O

(A) Upper row: DiI tracing of the visual cortex axons at P9 (before pruning), sagittal sections. Branching point of the tract is shown. Lower row: visual cortex axons in adult mice after pruning period. Note significantly reduced number of axons continuing into pyramidal tract in WT (dashed arrow). (arrows - 2 branches of corticospinal visual axons). Maximum projections are shown. Scale bars: 200 μm.


( C ) Quantification of VP ( visual pruning ) index ( fluorescence intensity of pyramidal axons after vs . before the branch point ) . In WT animals after the pruning period , only a minor part of axons descends towards the pyramidal tract ( WT : P9 0 . 65 ± 0 . 09 , adults 0 . 17 ± 0 . 04 , p = 0 . 006 ) . However , in crmp2 - / - mice , corticospinal axons are still largely present , and their VP indexes are not significantly different between adult and P9 stages ( crmp2 - / - : P9 0 . 54 ± 0 . 18 , adults 0 . 62 ± 0 . 27 , p = 0 . 24 ) . Mean ± SD , * * p < 0 . 01 , 2 - way ANOVA with Bonferroni ' s multiple comparisons test . P9 : n = 3 pups / genotype , adults : n = 5 mice / genotype )

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O

(C) Quantification of VP (visual pruning) index (fluorescence intensity of pyramidal axons after vs. before the branch point). In WT animals after the pruning period, only a minor part of axons descends towards the pyramidal tract (WT: P9 0.65±0.09, adults 0.17±0.04, p=0.006). However, in crmp2-/- mice, corticospinal axons are still largely present, and their VP indexes are not significantly different between adult and P9 stages (crmp2-/-: P9 0.54±0.18, adults 0.62±0.27, p=0.24). Mean ± SD, **p<0.01, 2-way ANOVA with Bonferroni's multiple comparisons test. P9: n=3 pups/genotype, adults: n=5 mice/genotype)


( A ) Time - lapse imaging of DIV7 cultured hippocampal neurons before and after Semaphorin stimulation . Upper panel : Axon growth without Semaphorin stimulation . Middle panel : stimulation with Sema3A ( 0 hours ) ( 1nM , n = 669 axons for WT , 761 axons for knockout ) causes retraction of both WT and crmp2 - / - neurons . Lower panel : stimulation with Sema3F ( 5 nM , n = 602 axons for WT , 955 axons for knockout ) causes axon retraction in WT , but not in crmp2 - / - neurons . Red triangles depict growing axons , yellow retracting axons and blue asterisks indicate steady non - growing axons . Scale bars : 100 μm ( whole image field ) and 50 μm ( magnified ) .

O O O O O O B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-EXP_ASSAY B-SUBCELLULAR O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O O O O O O O O

(A) Time-lapse imaging of DIV7 cultured hippocampal neurons before and after Semaphorin stimulation. Upper panel: Axon growth without Semaphorin stimulation. Middle panel: stimulation with Sema3A (0 hours) (1nM, n=669 axons for WT, 761 axons for knockout) causes retraction of both WT and crmp2-/- neurons. Lower panel: stimulation with Sema3F (5 nM, n=602 axons for WT, 955 axons for knockout) causes axon retraction in WT, but not in crmp2-/- neurons. Red triangles depict growing axons, yellow retracting axons and blue asterisks indicate steady non-growing axons. Scale bars: 100 μm (whole image field) and 50 μm (magnified).


( C ) Quantification of retracting axons ( number of retracting vs . steady axons , 3 experiments ) . WT : control 13 . 4 ± 5 % , Sema3A 31 . 2 ± 6 % ( p < 0 . 001 ) , Sema3F 36 . 9 ± 8 . 7 % ( p < 0 . 001 ) ; crmp2 - / - : control 19 . 8 ± 1 . 8 % , Sema3A 28 . 5 ± 4 . 4 % ( p < 0 . 05 ) , Sema3F 22 . 4 ± 4 . 1 % ( p > 0 . 99 ) , mean ± SD are shown . * * * p < 0 . 001 , * p < 0 . 05 , 2 - way ANOVA with Bonferroni ' s multiple comparisons test .

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(C) Quantification of retracting axons (number of retracting vs. steady axons, 3 experiments). WT: control 13.4±5%, Sema3A 31.2±6% (p<0.001), Sema3F 36.9±8.7% (p<0.001); crmp2-/-: control 19.8±1.8%, Sema3A 28.5±4.4% (p<0.05), Sema3F 22.4±4.1% (p>0.99), mean ± SD are shown. ***p<0.001, *p<0.05, 2-way ANOVA with Bonferroni's multiple comparisons test.


( A , B ) Dendritic spine density in DiOlistically labeled DG granule cells is similar in WT and crmp2 - / - mice at P30 . In adults , however , spine density in crmp2 - / - granule cells is increased comparing to WT . Scale bars : 5 μm . ( C ) Quantification of dendritic spine density in DG granule cells in the inner molecular layer ( 50 - 100 μm away from the soma ) . WT , P30 : 12 . 5 ± 2 . 2 spines / 10 μm , adults : 9 . 95 ± 2 . 3 ; crmp2 - / - , P30 : 12 . 86 ± 2 . 1 ( p > 0 . 99 ) , adults : 12 . 88 ± 2 . 9 ( p < 0 . 001 ) , mean ± SD , * * * p < 0 . 001 , 2 - way ANOVA with Bonferroni correction . P30 : 3 animals / genotype , WT = 61 dendrites , knockout = 64 dendrites ; adults : 3 animals / genotype , WT = 41 dendrites , knockout = 37 dendrites .

O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-ORGANISM O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O

(A, B) Dendritic spine density in DiOlistically labeled DG granule cells is similar in WT and crmp2-/- mice at P30. In adults, however, spine density in crmp2-/- granule cells is increased comparing to WT. Scale bars: 5 μm. (C) Quantification of dendritic spine density in DG granule cells in the inner molecular layer (50 - 100 μm away from the soma). WT, P30: 12.5±2.2 spines/10 μm, adults: 9.95±2.3; crmp2-/-, P30: 12.86±2.1 (p>0.99), adults: 12.88±2.9 (p<0.001), mean ± SD, ***p<0.001, 2-way ANOVA with Bonferroni correction. P30: 3 animals/genotype, WT=61 dendrites, knockout=64 dendrites; adults: 3 animals/genotype, WT=41 dendrites, knockout=37 dendrites.


( D , E ) Analysis of branching of DiOlistically labeled DG granule cell dendrites in adult WT and crmp2 - / - mice ( n = 3 animals , ≥ 25 dendrites ) . Quantification of granule cell branching by Sholl analysis showed no significant differences ( p > 0 . 99 ) . Scale bars : 50 μm , mean ± SEM , 2 - way ANOVA with Bonferroni correction .

O O O O O O O B-EXP_ASSAY O O O B-TISSUE I-TISSUE I-TISSUE B-SUBCELLULAR O O O O B-GENEPROD O O O B-ORGANISM O O O O B-ORGANISM O O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Analysis of branching of DiOlistically labeled DG granule cell dendrites in adult WT and crmp2-/- mice (n=3 animals, ≥25 dendrites). Quantification of granule cell branching by Sholl analysis showed no significant differences (p>0.99). Scale bars: 50 μm, mean ± SEM, 2-way ANOVA with Bonferroni correction.


( F ) Defects in synapse elimination in the inner molecular layer revealed by double immunostaining with PSD95 and VGluT2 antibodies ( n = 5 mice / genotype ) . GL indicates granule cell layer , IML / OML indicate inner / outer molecular layer , respectively . Density of colocalized PSD95 / VGLuT2 puncta was counted . IML : WT 109 ± 21 , crmp2 - / - 156 ± 15 ( p = 0 . 004 ) . OML : WT 281 ± 38 , crmp2 - / - 297 ± 80 ( p = 0 . 7 ) . Scale bars : 10 μm , mean ± SD , * p < 0 . 05 , t - test .

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(F) Defects in synapse elimination in the inner molecular layer revealed by double immunostaining with PSD95 and VGluT2 antibodies (n=5 mice/genotype). GL indicates granule cell layer, IML/OML indicate inner/outer molecular layer, respectively. Density of colocalized PSD95/VGLuT2 puncta was counted. IML: WT 109±21, crmp2-/- 156±15 (p=0.004). OML: WT 281±38, crmp2-/- 297±80 (p=0.7). Scale bars: 10 μm, mean ± SD, *p<0.05, t-test.


( G ) Spine density in DiOlistically labeled prefrontal cortex ( PFC ) is similar in WT and crmp2 - / - mice in both juvenile and adult mice . Scale bars : 5 μm . ( H ) Quantification of dendritic spine density of PFC pyramidal neurons , basal dendrites ( WT , juvenile : 8 . 63 ± 1 . 44 spines / 10 μm , adults : 7 . 3 ± 1 . 4 ; crmp2 - / - , juvenile : 9 . 05 ± 1 . 73 , p = 0 . 97 , adults : 6 . 98 ± 1 . 53 , p > 0 . 99 ) , mean ± SD , 2 - way ANOVA with Bonferroni ' s multiple comparisons test . P25 : n = 3 , ≥ 50 dendrites ; adults : n = 3 , ≥ 50 dendrites .

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(G) Spine density in DiOlistically labeled prefrontal cortex (PFC) is similar in WT and crmp2-/- mice in both juvenile and adult mice. Scale bars: 5 μm. (H) Quantification of dendritic spine density of PFC pyramidal neurons, basal dendrites (WT, juvenile: 8.63±1.44 spines/10 μm, adults: 7.3±1.4; crmp2-/-, juvenile: 9.05±1.73, p=0.97, adults: 6.98±1.53, p>0.99), mean ± SD, 2-way ANOVA with Bonferroni's multiple comparisons test. P25: n=3, ≥50 dendrites; adults: n=3, ≥50 dendrites.


( A - B ) Ultrasonic vocalization was measured at P6 , P8 and P12 ( WT n = 14 pups , crmp2 - / - n = 13 pups ) . In crmp2 - / - mice , there is a significant decrease in the rate and duration of calls at P8 ( call rate , WT 352 ± 58 / 5 min , crmp2 - / - 164 ± 41 / 5 min , p = 0 . 015 ; call duration , WT 9 . 52 ± 2 ms , crmp2 - / - 3 . 36 ± 1 ms , p = 0 . 011 ) and P12 ( call rate , WT 46 ± 16 / 5 min crmp2 - / - 1 . 54 ± 1 . 24 / 5 min , p = 0 . 002 ; call duration , WT 0 . 68 ± 0 . 35 ms , crmp2 - / - 0 . 007 ± 0 . 006 ms , p = 0 . 002 ) . Mean ± SEM , * p < 0 . 05 , t - test ( P6 , P8 ) , Mann - Whitney test ( P12 ) . ( C ) Representative sonograms of the P8 mice .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O

(A - B) Ultrasonic vocalization was measured at P6, P8 and P12 (WT n=14 pups, crmp2-/- n=13 pups). In crmp2-/- mice, there is a significant decrease in the rate and duration of calls at P8 (call rate, WT 352±58/5 min, crmp2-/- 164±41/5 min, p=0.015; call duration, WT 9.52±2 ms, crmp2-/- 3.36±1 ms, p=0.011) and P12 (call rate, WT 46±16/5 min crmp2-/- 1.54±1.24/5 min, p=0.002; call duration, WT 0.68±0.35 ms, crmp2-/- 0.007±0.006 ms, p=0.002). Mean ± SEM, *p<0.05, t-test (P6, P8), Mann-Whitney test (P12). (C) Representative sonograms of the P8 mice.


( D ) 3 - chamber test ( WT n = 11 mice , crmp2 - / - n = 13 mice ) . In sociability phase , WT mice spent significantly more time with a social partner ( stranger 1 ) , unlike knockouts . ( WT - stranger 1 : 0 . 14 ± 0 . 01 , object : 0 . 07 ± 0 . 007 ( p = 0 . 0001 ) ; crmp2 - / - - stranger 1 : 0 . 07 ± 0 . 01 , object : 0 . 04 ± 0 . 006 ( p = 0 . 07 ) ) . In social novelty phase , when an object was substituted with a second social partner ( stranger 2 ) , both WTs and knockouts preferred novel mice to known mice . ( WT - stranger 1 : 0 . 03 ± 0 . 006 , stranger 2 : 0 . 09 ± 0 . 008 ( p < 0 . 0001 ) ; crmp2 - / - - stranger 1 : 0 . 02 ± 0 . 004 , stranger 2 : 0 . 05 ± 0 . 01 ( p = 0 . 02 ) ) . Mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , t - test .

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(D) 3-chamber test (WT n=11 mice, crmp2-/- n=13 mice). In sociability phase, WT mice spent significantly more time with a social partner (stranger 1), unlike knockouts. (WT - stranger 1: 0.14±0.01, object: 0.07±0.007 (p=0.0001); crmp2-/- - stranger 1: 0.07±0.01, object: 0.04±0.006 (p=0.07)). In social novelty phase, when an object was substituted with a second social partner (stranger 2), both WTs and knockouts preferred novel mice to known mice. (WT - stranger 1: 0.03±0.006, stranger 2: 0.09±0.008 (p<0.0001); crmp2-/- - stranger 1: 0.02±0.004, stranger 2: 0.05±0.01 (p=0.02)). Mean ± SEM, *p<0.05, ***p<0.001, t-test.


Elevated plus maze test ( n = 10 mice / genotype ) . ( E ) Total distance walked is similar in WT and crmp2 - / - ( WT 7 . 9 ± 0 . 45 m , crmp2 - / - 8 . 6 ± 0 . 5 m , p = 0 . 3 ) . ( F ) Frequency of open arm ( OA ) visits is increased in crmp2 - / - mice suggesting decreased anxiety . CA denotes closed arms , MID denotes the transition zone between arms , Total arm visits represents a sum of visits in all four arms . ( CA frequency : WT 13 . 7 ± 0 . 9 / 5 min , crmp2 - / - 10 . 5 ± 1 . 1 / 5 min , p = 0 . 04 ; OA frequency : WT 7 . 7 ± 0 . 8 / 5 min , crmp2 - / - 14 . 6 ± 1 . 7 / 5 min , p = 0 . 002 ; MID frequency : WT 20 . 6 ± 1 / 5 min , crmp2 - / - 24 . 6 ± 1 . 6 / 5 min , p = 0 . 06 mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , t - test .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-ORGANISM O O B-DISEASE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Elevated plus maze test (n=10 mice/genotype). (E) Total distance walked is similar in WT and crmp2-/- (WT 7.9±0.45 m, crmp2-/- 8.6±0.5 m, p=0.3). (F) Frequency of open arm (OA) visits is increased in crmp2-/- mice suggesting decreased anxiety. CA denotes closed arms, MID denotes the transition zone between arms, Total arm visits represents a sum of visits in all four arms. (CA frequency: WT 13.7±0.9/5 min, crmp2-/- 10.5±1.1/5 min, p=0.04; OA frequency: WT 7.7±0.8/5 min, crmp2-/- 14.6±1.7/5 min, p=0.002; MID frequency: WT 20.6±1/5 min, crmp2-/- 24.6±1.6/5 min, p=0.06 mean ± SEM, *p<0.05, ***p<0.001, t-test.


Elevated plus maze test ( n = 10 mice / genotype ) . ( G ) duration of open arm ( OA ) visits is increased in crmp2 - / - mice suggesting decreased anxiety . CA denotes closed arms , MID denotes the transition zone between arms , Total arm visits represents a sum of visits in all four arms . Total arm visits : WT 21 . 4 ± 1 / 5 min , crmp2 - / - 25 . 1 ± 1 . 5 / 5 min , p = 0 . 07 ; CA duration : WT 177 ± 17 s . , crmp2 - / - 75 . 3 ± 12 . 6 s , p < 0 . 001 ; OA duration : WT 42 ± 12 . 6 s , crmp2 - / - 144 . 4 ± 21 s , p < 0 . 001 ; MID duration : WT 80 . 26 ± 11 . 5 s . , crmp2 - / - 80 . 17 ± 12 . 6 s . , p = 0 . 99 ) , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 001 , t - test .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-ORGANISM O O B-DISEASE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Elevated plus maze test (n=10 mice/genotype). (G) duration of open arm (OA) visits is increased in crmp2-/- mice suggesting decreased anxiety. CA denotes closed arms, MID denotes the transition zone between arms, Total arm visits represents a sum of visits in all four arms. Total arm visits: WT 21.4±1/5 min, crmp2-/- 25.1±1.5/5 min, p=0.07; CA duration: WT 177±17 s., crmp2-/- 75.3±12.6 s, p<0.001; OA duration: WT 42±12.6 s, crmp2-/- 144.4±21 s, p<0.001; MID duration: WT 80.26±11.5 s., crmp2-/- 80.17±12.6 s., p=0.99), mean ± SEM, *p<0.05, ***p<0.001, t-test.


( H , I ) Y maze test ( WT n = 9 mice , crmp2 - / - n = 8 mice ) . Decreased alternations between arms of the maze indicates impaired working memory ( number of visits , WT : 23 . 1 ± 1 . 9 , crmp2 - / - 30 . 4 ± 3 . 8 , p = 0 . 12 ; alternations , WT 68 . 8 ± 2 . 5 % , crmp2 - / - 57 . 3 ± 2 . 5 % , p = 0 . 006 ) , mean ± SEM , * p < 0 . 05 , t - test .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H, I) Y maze test (WT n=9 mice, crmp2-/- n=8 mice). Decreased alternations between arms of the maze indicates impaired working memory (number of visits, WT: 23.1±1.9, crmp2-/- 30.4±3.8, p=0.12; alternations, WT 68.8±2.5%, crmp2-/- 57.3±2.5%, p=0.006), mean ± SEM, *p<0.05, t-test.


( a ) MN - 1 cells were transfected with mCherry - EGFP - LC3 vector , fixed and imaged after 24 h . Numbers of yellow puncta ( autophagosomes ) and red puncta ( autolysosomes ) per cell were counted . Autophagosomes : n = 3 independent experiments , F = 3 . 56 ; autolysosomes : n = 3 independent experiments , F = 20 . 13 . One - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * * P 0 . 001 . n = 34 cells per genotype .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) MN-1 cells were transfected with mCherry-EGFP-LC3 vector, fixed and imaged after 24 h. Numbers of yellow puncta (autophagosomes) and red puncta (autolysosomes) per cell were counted. Autophagosomes: n = 3 independent experiments, F = 3.56; autolysosomes: n = 3 independent experiments, F = 20.13. One-way ANOVA with post hoc Tukey test. *P 0.05, ***P 0.001. n = 34 cells per genotype.


( b ) We transfected MEFs stably expressing mCherry - EGFP - LC3 with BFP - tagged AR25Q or AR125Q . Cells were imaged after 24 h , and yellow puncta ( autophagosomes ) and red puncta ( autolysosomes ) in AR - expressing ( blue ) cells were counted . Autophagosomes : n = 3 independent experiments , F = 20 . 65 ; autolysosomes : n = 3 independent experiments , F = 4 . 49 . One - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 . n = 33 cells per genotype .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) We transfected MEFs stably expressing mCherry-EGFP-LC3 with BFP-tagged AR25Q or AR125Q. Cells were imaged after 24 h, and yellow puncta (autophagosomes) and red puncta (autolysosomes) in AR-expressing (blue) cells were counted. Autophagosomes: n = 3 independent experiments, F = 20.65; autolysosomes: n = 3 independent experiments, F = 4.49. One-way ANOVA with post hoc Tukey test. *P 0.05, **P 0.01, ***P 0.001. n = 33 cells per genotype.


( c ) MN - 1 cells were immunoblotted for LC3 in the presence or absence of ammonium chloride to evaluate autophagic flux . A representative LC3 western blot is shown . All ratios were normalized to MN - 1 WT cells at baseline , which was set to 1 . ( d ) The ratio of LC3 - II : actin for c determined by densitometry analysis using ImageJ . n = 3 independent experiments , F = 0 . 4501 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) MN-1 cells were immunoblotted for LC3 in the presence or absence of ammonium chloride to evaluate autophagic flux. A representative LC3 western blot is shown. All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (d) The ratio of LC3-II:actin for c determined by densitometry analysis using ImageJ. n = 3 independent experiments, F = 0.4501, one-way ANOVA with post hoc Tukey test. *P 0.05.


( e ) MN - 1 cells were treated with ammonium chloride to block lysosomal activity , and then ammonium chloride was removed and cells were allowed to degrade accumulated autophagy cargo during a 6 - h recovery period . We performed p62 western blot analysis on MN - 1 cells at baseline ( - ) , after ammonium chloride treatment ( + ) and after the recovery period ( Rec ) . All ratios were normalized to MN - 1 WT cells at baseline , which was set to 1 . ( f ) On the basis of the p62 values obtained in e , we calculated the efficiency of degraded accumulated autophagic cargo by dividing p62 at baseline by p62 at recovery and normalized the baseline / recovery values to baseline / recovery for MN - 1 WT cells . n = 3 independent experiments , F = 5 . 45 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 , * * P 0 . 01 . Data are presented as mean ± s . e . m . Scale bar , 20 μm . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) MN-1 cells were treated with ammonium chloride to block lysosomal activity, and then ammonium chloride was removed and cells were allowed to degrade accumulated autophagy cargo during a 6-h recovery period. We performed p62 western blot analysis on MN-1 cells at baseline (-), after ammonium chloride treatment (+) and after the recovery period (Rec). All ratios were normalized to MN-1 WT cells at baseline, which was set to 1. (f) On the basis of the p62 values obtained in e, we calculated the efficiency of degraded accumulated autophagic cargo by dividing p62 at baseline by p62 at recovery and normalized the baseline/recovery values to baseline/recovery for MN-1 WT cells. n = 3 independent experiments, F = 5.45, one-way ANOVA with post hoc Tukey test. *P 0.05, ** P 0.01. Data are presented as mean ± s.e.m. Scale bar, 20 μm. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a , b ) Electron micrographs of motor neuron perinuclear regions from age - matched non - transgenic ( Nt ) , YAC AR20 and YAC AR100 transgenic mice before disease onset ( a , 6 months of age ) and after prominent neuromuscular and molecular pathology is apparent ( b , 14 months of age ) . ( a ) At 6 months of age , occasional autophagosomes ( yellow arrowheads ) are noted in Nt , YAC AR20 and YAC AR100 at roughly equivalent frequency . Autolysosomes ( red arrowheads ) are much more common and are present in higher numbers in YAC AR20 and YAC AR100 motor neurons . ( b ) At 14 months of age , when YAC AR100 mice display signs of motor neuronopathy and molecular pathology , we observed many YAC AR100 motor neuron micrographs with frequent autophagosomes ( yellow arrowheads ) . Despite this increase in autophagosomes , autolysosomes were fewer in number at this age in YAC AR100 motor neurons , such that autophagosome numbers approached autolysosome numbers in YAC AR100 motor neurons . This was never the case for Nt motor neurons or YAC AR20 motor neurons . Main panels are at original magnification 2 , 200 × and insets are at original magnification 3 , 700 × .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O

(a,b) Electron micrographs of motor neuron perinuclear regions from age-matched non-transgenic (Nt), YAC AR20 and YAC AR100 transgenic mice before disease onset (a, 6 months of age) and after prominent neuromuscular and molecular pathology is apparent (b, 14 months of age). (a) At 6 months of age, occasional autophagosomes (yellow arrowheads) are noted in Nt, YAC AR20 and YAC AR100 at roughly equivalent frequency. Autolysosomes (red arrowheads) are much more common and are present in higher numbers in YAC AR20 and YAC AR100 motor neurons. (b) At 14 months of age, when YAC AR100 mice display signs of motor neuronopathy and molecular pathology, we observed many YAC AR100 motor neuron micrographs with frequent autophagosomes (yellow arrowheads). Despite this increase in autophagosomes, autolysosomes were fewer in number at this age in YAC AR100 motor neurons, such that autophagosome numbers approached autolysosome numbers in YAC AR100 motor neurons. This was never the case for Nt motor neurons or YAC AR20 motor neurons. Main panels are at original magnification 2,200× and insets are at original magnification 3,700×.


( a ) Mean number of autophagosomes per motor neuron field in electron micrographs from non - transgenic ( Nt ) , YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age , ± s . e . m . 6 months : n = 3 independent experiments , F = 0 . 63 , one - way ANOVA with post hoc Tukey test . P = 0 . 534 . 14 months : n = 3 independent experiments , F = 4 . 32 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . ( b ) Mean number of autolysosomes per motor neuron field in electron micrographs from Nt , YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age , ± s . e . m . 6 months : n = 3 independent experiments , F = 4 . 18 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . 14 months : n = 3 independent experiments , F = 4 . 56 , one - way ANOVA with post hoc Tukey test . * P 0 . 05 . ( c ) Mean autophagy index , dividing number of autolysosome by number of autophagosomes for each motor neuron field , ± s . e . m . At 6 months , YAC AR100 motor neurons displayed a markedly increased autophagy index ; n = 3 independent experiments , F = 129 . 81 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . However , once YAC AR100 mice develop disease pathology at 14 months , the autophagy index for YAC AR100 motor neurons is significantly decreased ; n = 3 independent experiments , F = 82 . 82 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O O O O O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-CELL_TYPE I-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Mean number of autophagosomes per motor neuron field in electron micrographs from non-transgenic (Nt), YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age, ± s.e.m. 6 months: n = 3 independent experiments, F = 0.63, one-way ANOVA with post hoc Tukey test. P = 0.534. 14 months: n = 3 independent experiments, F = 4.32, one-way ANOVA with post hoc Tukey test. *P 0.05. (b) Mean number of autolysosomes per motor neuron field in electron micrographs from Nt, YAC AR20 and YAC AR100 transgenic mice at 6 and 14 months of age, ± s.e.m. 6 months: n = 3 independent experiments, F = 4.18, one-way ANOVA with post hoc Tukey test. *P 0.05. 14 months: n = 3 independent experiments, F = 4.56, one-way ANOVA with post hoc Tukey test. *P 0.05. (c) Mean autophagy index, dividing number of autolysosome by number of autophagosomes for each motor neuron field, ± s.e.m. At 6 months, YAC AR100 motor neurons displayed a markedly increased autophagy index; n = 3 independent experiments, F = 129.81, one-way ANOVA with post hoc Tukey test. **P 0.01. However, once YAC AR100 mice develop disease pathology at 14 months, the autophagy index for YAC AR100 motor neurons is significantly decreased; n = 3 independent experiments, F = 82.82, one-way ANOVA with post hoc Tukey test. **P 0.01. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) HEK293 cells were transfected with TFEB and AR25Q or AR125Q , as indicated . Immunoprecipitation ( IP ) was performed with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O

(a) HEK293 cells were transfected with TFEB and AR25Q or AR125Q, as indicated. Immunoprecipitation (IP) was performed with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).


( b ) We transfected MN - 1 cells with the 4X - CLEAR luciferase reporter and treated cells with sucrose to induce TFEB activation . Untreated : n = 3 independent experiments , F = 3 . 48 , one - way ANOVA with post hoc Tukey test . * * P 0 . 01 . Sucrose treatment : n = 3 independent experiments , * * P 0 . 01 , t - test , WT t ( 10 ) = 8 . 133 ; * * * P 0 . 001 , t - test , AR 24Q t ( 10 ) = 6 . 11 ; P not significant ( n . s . ) , AR 65Q t ( 10 ) = 0 . 644 . Sucrose - treated MN - 1 AR24Q cells displayed significantly higher 4X - CLEAR luciferase activity : n = 3 independent experiments , F = 2 . 49 , one - way ANOVA with post hoc Tukey test . * * * P 0 . 001 . All ratios were normalized to untreated MN - 1 WT cells , whose value was set to 1 .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O

(b) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and treated cells with sucrose to induce TFEB activation. Untreated: n = 3 independent experiments, F = 3.48, one-way ANOVA with post hoc Tukey test. **P 0.01. Sucrose treatment: n = 3 independent experiments, **P 0.01, t-test, WT t(10) = 8.133; ***P 0.001, t-test, AR 24Q t(10) = 6.11; P not significant (n.s.), AR 65Q t(10) = 0.644. Sucrose-treated MN-1 AR24Q cells displayed significantly higher 4X-CLEAR luciferase activity: n = 3 independent experiments, F = 2.49, one-way ANOVA with post hoc Tukey test. ***P 0.001. All ratios were normalized to untreated MN-1 WT cells, whose value was set to 1.


( c ) We exposed MN - 1 cells to ammonium chloride , an inhibitor of lysosomal activity , to determine the capacity for TFEB induction . RT - PCR analysis revealed lack of TFEB target gene induction in MN - 1 AR65Q cells for four gene targets : Lamp1 ( lysosomal - associated membrane protein 1 ) , F = 37 . 51 ; Atp6v1h ( vesicular ATPase V1 subunit H ) , F = 57 . 26 ; Mcoln1 ( mucolipin 1 ) , F = 251 . 0 ; and Gla ( galactosidase - α ) , F = 75 . 51 ; n = 3 independent experiments , * P 0 . 05 , * * P 0 . 01 , ANOVA with post hoc Tukey test . For Lamp1 and Mcoln1 , MN - 1 AR24Q cells yielded significantly higher expression . Differences in the expression of TFEB target genes at baseline were negligible .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O

(c) We exposed MN-1 cells to ammonium chloride, an inhibitor of lysosomal activity, to determine the capacity for TFEB induction. RT-PCR analysis revealed lack of TFEB target gene induction in MN-1 AR65Q cells for four gene targets: Lamp1 (lysosomal-associated membrane protein 1), F = 37.51; Atp6v1h (vesicular ATPase V1 subunit H), F = 57.26; Mcoln1 (mucolipin 1), F = 251.0; and Gla (galactosidase-α), F = 75.51; n = 3 independent experiments, *P 0.05, **P 0.01, ANOVA with post hoc Tukey test. For Lamp1 and Mcoln1, MN-1 AR24Q cells yielded significantly higher expression. Differences in the expression of TFEB target genes at baseline were negligible.


( d ) We measured expression levels of TFEB target genes in E13 motor neurons derived from non - transgenic ( Nt ) , YAC AR20 and YAC AR100 mice by RT - PCR analysis : Ctsf ( cathepsin F ) , F = 4 . 63 ; Mcoln1 , F = 6 . 64 ; Atp6v1h , F = 5 . 6 ; and Gla , F = 5 . 65 ; n = 3 independent experiments , * P 0 . 05 , ANOVA with post hoc Tukey test . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O O O O O O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) We measured expression levels of TFEB target genes in E13 motor neurons derived from non-transgenic (Nt), YAC AR20 and YAC AR100 mice by RT-PCR analysis: Ctsf (cathepsin F), F = 4.63; Mcoln1, F = 6.64; Atp6v1h, F = 5.6; and Gla, F = 5.65; n = 3 independent experiments, *P 0.05, ANOVA with post hoc Tukey test. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) We transfected MN - 1 cells with the 4X - CLEAR luciferase reporter and with BFP - tagged TFEB or BFP empty vector , and then measured luciferase activity . Marked induction of luciferase activity occurred with BFP - TFEB ; n = 3 independent experiments , * * * P 0 . 001 , t - test , WT t ( 4 ) = 48 . 79 ; * * * P 0 . 001 , t - test , AR 24Q t ( 4 ) = 22 . 91 ; * * * P 0 . 001 , t - test , AR 65Q t ( 4 ) = 14 . 02 . Results for each line were normalized to the BFP - empty luciferase activity .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O

(a) We transfected MN-1 cells with the 4X-CLEAR luciferase reporter and with BFP-tagged TFEB or BFP empty vector, and then measured luciferase activity. Marked induction of luciferase activity occurred with BFP-TFEB; n = 3 independent experiments, ***P 0.001, t-test, WT t(4) = 48.79; ***P 0.001, t-test, AR 24Q t(4) = 22.91; ***P 0.001, t-test, AR 65Q t(4) = 14.02. Results for each line were normalized to the BFP-empty luciferase activity.


( b ) We transfected MN - 1 AR65Q cells with the 4X - CLEAR luciferase reporter and with BFP - TFEB or BFP empty vector and subjected the MN - 1 AR65Q cells to conditions that promote TFEB activation , as shown . MN - 1 WT cells were also transfected with the 4X - CLEAR luciferase reporter and BFP empty vector and subjected to the identical treatments for control purposes . Results are shown normalized to untreated MN - 1 WT cells expressing BFP - empty . Untreated , F = 182 . 7 ; starvation , F = 63 . 64 ; NH4Cl , F = 291 . 9 ; Rapamycin , F = 3128 ; ANOVA with post hoc Tukey test . * * P 0 . 01 , * * * P 0 . 001 .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) We transfected MN-1 AR65Q cells with the 4X-CLEAR luciferase reporter and with BFP-TFEB or BFP empty vector and subjected the MN-1 AR65Q cells to conditions that promote TFEB activation, as shown. MN-1 WT cells were also transfected with the 4X-CLEAR luciferase reporter and BFP empty vector and subjected to the identical treatments for control purposes. Results are shown normalized to untreated MN-1 WT cells expressing BFP-empty. Untreated, F = 182.7; starvation, F = 63.64; NH4Cl, F = 291.9; Rapamycin, F = 3128; ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001.


( c ) We transfected MN - 1 AR65Q cells with the mCherry - EGFP - LC3 construct and with either BFP - empty or BFP - TFEB . Autophagic vesicles forming in BFP - expressing cells were then detected as red , green or yellow puncta , as shown . Scale bar , 20 μm . ( d ) Left , quantification of autophagic vesicle type . Note marked reduction in autophagosomes in MN - 1 AR65Q cells expressing BFP - TFEB ; n = 3 independent experiments , F = 16 . 596 , ANOVA with post hoc Tukey test . * * P 0 . 01 , * * * P 0 . 001 . Right , calculation of the autophagy index ; n = 3 independent experiments , F = 24 . 43 , ANOVA with post hoc Tukey test . * * P 0 . 01 . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) We transfected MN-1 AR65Q cells with the mCherry-EGFP-LC3 construct and with either BFP-empty or BFP-TFEB. Autophagic vesicles forming in BFP-expressing cells were then detected as red, green or yellow puncta, as shown. Scale bar, 20 μm. (d) Left, quantification of autophagic vesicle type. Note marked reduction in autophagosomes in MN-1 AR65Q cells expressing BFP-TFEB; n = 3 independent experiments, F = 16.596, ANOVA with post hoc Tukey test. **P 0.01, ***P 0.001. Right, calculation of the autophagy index; n = 3 independent experiments, F = 24.43, ANOVA with post hoc Tukey test. **P 0.01. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( b ) HEK293 cells were transfected with TFEB and influenza hemagglutinin ( HA ) - tagged full - length AR25 ( FL ) or a C - terminal AR mutant ( m1 , m2 , m3 , m4 ) . Immunoprecipitation ( IP ) was performed with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O

(b) HEK293 cells were transfected with TFEB and influenza hemagglutinin (HA)-tagged full-length AR25 (FL) or a C-terminal AR mutant (m1, m2, m3, m4). Immunoprecipitation (IP) was performed with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).


( c ) HEK293 cells were transfected with TFEB and HA - tagged N - terminal AR mutant containing a 19Q or 112Q tract ( NT19Q , NT112Q ) . Immunoprecipitation was performed as in b . with anti - TFEB antibody , followed by immunoblotting ( IB ) for TFEB and AR . Untransfected HEK293 cells served as a negative control , and no AR was observed with IgG antibody only ( not shown ) .

O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O

(c) HEK293 cells were transfected with TFEB and HA-tagged N-terminal AR mutant containing a 19Q or 112Q tract (NT19Q, NT112Q). Immunoprecipitation was performed as in b. with anti-TFEB antibody, followed by immunoblotting (IB) for TFEB and AR. Untransfected HEK293 cells served as a negative control, and no AR was observed with IgG antibody only (not shown).


( d ) MN - 1 WT , MN - 1 AR24Q and MN - 1 AR65Q cells were cultured in standard medium ( Ctl ) or in medium supplemented with R1881 and then immunostained with antibodies to AR and to TFEB . Cells were scored for nuclear or cytosolic localization of TFEB . Scale bars , 20 μm . ( e ) Quantification of d . Untreated , F = 0 . 56 ; R1881 , F = 5 . 51 ; ANOVA with post hoc Tukey test . * * P 0 . 01 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

(d) MN-1 WT, MN-1 AR24Q and MN-1 AR65Q cells were cultured in standard medium (Ctl) or in medium supplemented with R1881 and then immunostained with antibodies to AR and to TFEB. Cells were scored for nuclear or cytosolic localization of TFEB. Scale bars, 20 μm. (e) Quantification of d. Untreated, F = 0.56; R1881, F = 5.51; ANOVA with post hoc Tukey test. **P 0.01.


( f ) We transfected MN - 1 WT cells with the 4X - CLEAR luciferase reporter and either BFP - empty or AR25Q - BFP , under standard culture conditions ( control ) , ammonium chloride treatment or rapamycin treatment . Control , t ( 3 ) = 15 . 73 ; NH4Cl , t ( 4 ) = 4 . 108 ; rapamycin . t ( 4 ) = 4 . 58 ; t - test . * * P 0 . 01 .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

(f) We transfected MN-1 WT cells with the 4X-CLEAR luciferase reporter and either BFP-empty or AR25Q-BFP, under standard culture conditions (control), ammonium chloride treatment or rapamycin treatment. Control, t(3) = 15.73; NH4Cl, t(4) = 4.108; rapamycin. t(4) = 4.58; t-test. **P 0.01.


( g ) We transfected MN - 1 AR24Q cells with either scrambled shRNA control vector or AR shRNA vector and measured 4X - CLEAR luciferase reporter activity under baseline conditions ( control ) or after rapamycin . Control , t ( 4 ) = 0 . 51 ; rapamycin , t ( 4 ) = 7 . 15 ; t - test . * * P 0 . 01 .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

(g) We transfected MN-1 AR24Q cells with either scrambled shRNA control vector or AR shRNA vector and measured 4X-CLEAR luciferase reporter activity under baseline conditions (control) or after rapamycin. Control, t(4) = 0.51; rapamycin, t(4) = 7.15; t-test. **P 0.01.


( h ) RT - PCR analysis of MN - 1 AR24Q cells transfected with scrambled shRNA control or AR shRNA vector : Gla , t ( 4 ) = 4 . 77 ; Atp6v1h , t ( 4 ) = 3 . 75 ; Mcoln1 , t ( 4 ) = 13 . 74 ; Ctsd ( cathepsin D ) , t ( 4 ) = 7 . 91 ; and Lamp1 , t ( 4 ) = 6 . 46 ; n = 3 independent experiments , * P 0 . 05 , * * P 0 . 01 , t - test . Data are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) RT-PCR analysis of MN-1 AR24Q cells transfected with scrambled shRNA control or AR shRNA vector: Gla, t(4) = 4.77; Atp6v1h, t(4) = 3.75; Mcoln1, t(4) = 13.74; Ctsd (cathepsin D), t(4) = 7.91; and Lamp1, t(4) = 6.46; n = 3 independent experiments, *P 0.05, **P 0.01, t-test. Data are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) We performed filter - trap assays to determine the extent of accumulation of insoluble AR protein in NPC lines from three different controls and three different SBMA patients . Significantly higher levels of insoluble AR protein were present in the SBMA NPC lines , as based on densitometry ; n = 3 independent experiments , P 0 . 05 , t - test , t ( 4 ) = 4 . 026 . AU , arbitrary units .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) We performed filter-trap assays to determine the extent of accumulation of insoluble AR protein in NPC lines from three different controls and three different SBMA patients. Significantly higher levels of insoluble AR protein were present in the SBMA NPC lines, as based on densitometry; n = 3 independent experiments, P 0.05, t-test, t(4) = 4.026. AU, arbitrary units.


( b ) NPCs from three different controls and three different SBMA patients were cultured in the presence of JC - 1 dye . Results are presented as a Tukey box plots with box corresponding to first to third quartiles from the median and whiskers corresponding to 1 . 5 quartiles from the median ; n = 3 independent experiments , * P 0 . 05 , t - test , t ( 4 ) = 3 . 983 .

O O O B-CELL_TYPE O O O O O O O O B-ORGANISM O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) NPCs from three different controls and three different SBMA patients were cultured in the presence of JC-1 dye. Results are presented as a Tukey box plots with box corresponding to first to third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(4) = 3.983.


( c ) We transfected NPC lines derived from SBMA patients and controls ( three different NPC lines per patient ) with the mCherry - EGFP - LC3 vector . Autophagic vesicles were detected as colored puncta , as shown . Scale bar , 10 μm . ( d ) Quantification of autophagic vesicle type from c . SBMA NPCs exhibit a significant increase in autophagosomes ; n = 18 different samples : 3 clones per line , 3 lines per group , * P 0 . 05 , t - test ; t ( 18 ) = 2 . 3799 ( autophagosomes ) .

O O O O O B-CELL_TYPE O O O O B-ORGANISM O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O

(c) We transfected NPC lines derived from SBMA patients and controls (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector. Autophagic vesicles were detected as colored puncta, as shown. Scale bar, 10 μm. (d) Quantification of autophagic vesicle type from c. SBMA NPCs exhibit a significant increase in autophagosomes; n = 18 different samples: 3 clones per line, 3 lines per group, *P 0.05, t-test; t(18) = 2.3799 (autophagosomes).


( e ) We isolated RNAs from SBMA and control NPC lines ( three different NPC lines per human subject ) and then measured the expression levels of TFEB target genes including CYCS ( cytochrome c ) , t ( 4 ) = 2 . 844 ; MCOLN1 , t ( 4 ) = 3 . 246 ; GLA , t ( 4 ) = 3 . 174 ; ACADM ( acyl - CoA dehydrogenase ) , t ( 4 ) = 4 . 405 ; COX6A1 ( cytochrome oxidase 6A1 ) , t ( 4 ) = 5 . 584 ; and the TFEB gene itself , t ( 4 ) = 0 . 593 . Significant expression reductions were noted in SBMA NPC lines for all tested TFEB target genes . * P 0 . 05 , * * P 0 . 01 . Data in d , e are presented as mean ± s . e . m . Individual P values and degrees of freedom are available in the Supplementary Methods Checklist .

O O O O O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e) We isolated RNAs from SBMA and control NPC lines (three different NPC lines per human subject) and then measured the expression levels of TFEB target genes including CYCS (cytochrome c), t(4) = 2.844; MCOLN1, t(4) = 3.246; GLA, t(4) = 3.174; ACADM (acyl-CoA dehydrogenase), t(4) = 4.405; COX6A1 (cytochrome oxidase 6A1), t(4) = 5.584; and the TFEB gene itself, t(4) = 0.593. Significant expression reductions were noted in SBMA NPC lines for all tested TFEB target genes. *P 0.05, **P 0.01. Data in d,e are presented as mean ± s.e.m. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.


( a ) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone ( DHT ) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation ( IP ) . Immunoblotting ( IB ) for AR confirmed expression of AR in control ( Ctrl ) and SBMA NPCs , and immunoblotting for TFEB confirmed the interaction of AR with TFEB in both control and SBMA NPCs .

O O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O

(a) Control and SBMA NPC lines were maintained in the presence or absence of the AR ligand dihydrotestosterone (DHT) and then harvested for immunoblotting analysis after TFEB antibody immunoprecipitation (IP). Immunoblotting (IB) for AR confirmed expression of AR in control (Ctrl) and SBMA NPCs, and immunoblotting for TFEB confirmed the interaction of AR with TFEB in both control and SBMA NPCs.


( b ) We transfected SBMA NPC lines ( three different NPC lines per patient ) with either the BFP - empty vector or BFP - TFEB expression construct , treated the NPCs with JC - 1 dye and counted the percentage of cells with depolarized mitochondria . Results are presented as Tukey box plots with boxes corresponding to first and third quartiles from the median and whiskers corresponding to 1 . 5 quartiles from the median ; n = 3 independent experiments , * P 0 . 05 , t - test , t ( 6 ) = 3 . 217 .

O O O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O B-ORGANISM O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(b) We transfected SBMA NPC lines (three different NPC lines per patient) with either the BFP-empty vector or BFP-TFEB expression construct, treated the NPCs with JC-1 dye and counted the percentage of cells with depolarized mitochondria. Results are presented as Tukey box plots with boxes corresponding to first and third quartiles from the median and whiskers corresponding to 1.5 quartiles from the median; n = 3 independent experiments, *P 0.05, t-test, t(6) = 3.217.


( c ) Representative images from transfection of SBMANPCs with the mCherry - EGFP - LC3 vector and either BFP - empty vector or BFP - TFEB expression construct . BFP - empty vector - expressing SBMANPCs display a preponderance of yellow puncta ( autophagosomes ) , while BFP - TFEB - expressing SBMANPCs exhibit a shift toward almost entirely red puncta ( autolysosomes ) , consistent with rescue of the autophagic flux defect . Scale bars , 20 μm . ( d ) We transfected SBMANPC lines ( three different NPC lines per patient ) with the mCherry - EGFP - LC3 vector and either BFP - empty vector or BFP - TFEB expression construct , counted the number of autophagosomes and autolysosomes in BFP - expressing NPCs and determined the number of autophagic vesicles per cell . TFEB overexpression markedly reduced autophagosome formation in SBMANPCs . * * * P 0 . 001 ; t - test , t ( 30 ) = 4 . 373 . Data in d are presented as mean ± s . e . m . Individual P values are available in the Supplementary Methods Checklist .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c) Representative images from transfection of SBMANPCs with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct. BFP-empty vector-expressing SBMANPCs display a preponderance of yellow puncta (autophagosomes), while BFP-TFEB-expressing SBMANPCs exhibit a shift toward almost entirely red puncta (autolysosomes), consistent with rescue of the autophagic flux defect. Scale bars, 20 μm. (d) We transfected SBMANPC lines (three different NPC lines per patient) with the mCherry-EGFP-LC3 vector and either BFP-empty vector or BFP-TFEB expression construct, counted the number of autophagosomes and autolysosomes in BFP-expressing NPCs and determined the number of autophagic vesicles per cell. TFEB overexpression markedly reduced autophagosome formation in SBMANPCs. ***P 0.001; t-test, t(30) = 4.373. Data in d are presented as mean ± s.e.m. Individual P values are available in the Supplementary Methods Checklist.


Detection of NRPB2WT - FLAG , NRPB2P979S - FLAG and NRPB2Y732F - FLAG protein by western blotting in NRPB2WT - FLAG Col - 0 , NRPB2P979S - FLAG Col - 0 and NRPB2Y732F - FLAG Col - 0 plants . Untagged NRPB2 ( Col - 0 ) was used as a negative control . Histone H3 was used as an internal control and total protein level detected by stain - free blot was used as a loading control . Quantification was done by normalizing to the loading control and anti - H3 blot based on 3 independent replicates .

O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O

Detection of NRPB2WT-FLAG, NRPB2P979S-FLAG and NRPB2Y732F-FLAG protein by western blotting in NRPB2WT-FLAG Col-0, NRPB2P979S-FLAG Col-0 and NRPB2Y732F-FLAG Col-0 plants. Untagged NRPB2 (Col-0) was used as a negative control. Histone H3 was used as an internal control and total protein level detected by stain-free blot was used as a loading control. Quantification was done by normalizing to the loading control and anti-H3 blot based on 3 independent replicates.


Transmission rate of nrpb2 - 2 allele in nrpb2 - 2 + / - line ( n = 197 ) and nrpb2 - + / - lines combined with homozygous NRPB2P979S - FLAG + / + ( n = 280 ) , NRPB2Y732F - FLAG + / + ( n = 240 ) and NRPB2WT - FLAG + / + ( n = 210 ) , respectively . Fisher ´ s exact test was used as a statistic test , three asterisks denote p < 0 . 001 between samples and n . s . stands for not significant .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Transmission rate of nrpb2-2 allele in nrpb2-2 +/- line (n=197) and nrpb2- +/- lines combined with homozygous NRPB2P979S-FLAG +/+ (n=280), NRPB2Y732F-FLAG +/+ (n=240) and NRPB2WT-FLAG +/+ (n=210), respectively. Fisher´s exact test was used as a statistic test, three asterisks denote p<0.001 between samples and n.s. stands for not significant.


Image of homozygous mutant nrpb2 - 2 fully complemented by NRPB2WT - FLAG ( top , NRPB2WT + / + nrpb2 - 2 - / - ) and partially complemented by NRPB2Y732F - FLAG ( bottom , NRPB2WT + / + nrpb2 - 2 - / - ) . Plants were grown for 4 weeks in soil . Scale bars represent 1 cm .

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Image of homozygous mutant nrpb2-2 fully complemented by NRPB2WT-FLAG (top, NRPB2WT +/+ nrpb2-2 -/-) and partially complemented by NRPB2Y732F-FLAG (bottom, NRPB2WT +/+ nrpb2-2 -/-). Plants were grown for 4 weeks in soil. Scale bars represent 1 cm.


Schematic drawing of the experimental design to investigate RNAPII transcription speed in vivo . In brief , Arabidopsis seedlings of NRPB2WT - FLAG Col - 0 and NRPB2Y732F - FLAG Col - 0 were grown on MS media for 12 days and then were transferred to MS liquid media for 2 days . Flagellin peptides ( flagellin 22 ) were added into media and treated samples were collected in a 0 minute ( no treatment ) , 2 minutes and 4 minutes time course . The nascent RNA was isolated and used for reverse transcription and qPCR analyses to reveal RNAPII accumulation at different region in genes . See Methods for technical details .

O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O O

Schematic drawing of the experimental design to investigate RNAPII transcription speed in vivo. In brief, Arabidopsis seedlings of NRPB2WT-FLAG Col-0 and NRPB2Y732F-FLAG Col-0 were grown on MS media for 12 days and then were transferred to MS liquid media for 2 days. Flagellin peptides (flagellin 22) were added into media and treated samples were collected in a 0 minute (no treatment), 2 minutes and 4 minutes time course. The nascent RNA was isolated and used for reverse transcription and qPCR analyses to reveal RNAPII accumulation at different region in genes. See Methods for technical details.


Nascent RNA profile of AT5G41750 . Nascent RNA RT - qPCR assay measuring RNAPII signal at 3 positions ( dark red bars : probe 1 , 2 and 3 ) on the gene upon flagellin 22 treatment in a 0 minute , 2 minutes , 3 minutes and 4 minutes time course . Nascent RNA signal values were normalized to reference gene ACT2 . Error bars represent SEM from 3 independent replicates . The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by the two - sided Student ' s t - test . n . s . denotes not significant ; * denotes p < 0 . 05 and * * denotes p < 0 . 01 . Scale bar ( black ) represent 0 . 5 kb .

O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Nascent RNA profile of AT5G41750. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by the two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represent 0.5 kb.


Nascent RNA profile of AT4G19520 . Nascent RNA RT - qPCR assay measuring RNAPII signal at 3 positions ( dark red bars : probe 1 , 2 and 3 ) on the gene upon flagellin 22 treatment in a 0 minute , 2 minutes , 3 minutes and 4 minutes time course . Nascent RNA signal values were normalized to reference gene ACT2 . Error bars represent SEM from 3 independent replicates . The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by a two - sided Student ' s t - test . n . s . denotes not significant ; * denotes p < 0 . 05 and * * denotes p < 0 . 01 . Scale bar ( black ) represents 0 . 5 kb .

Nascent RNA profile of AT4G19520. Nascent RNA RT-qPCR assay measuring RNAPII signal at 3 positions (dark red bars: probe 1, 2 and 3) on the gene upon flagellin 22 treatment in a 0 minute, 2 minutes, 3 minutes and 4 minutes time course. Nascent RNA signal values were normalized to reference gene ACT2. Error bars represent SEM from 3 independent replicates. The statistical significance of differences between NRPB2Y732F and NRPB2WT at the same time point were assessed by a two-sided Student's t-test. n.s. denotes not significant; * denotes p<0.05 and ** denotes p<0.01. Scale bar (black) represents 0.5 kb.


plaNET - seq signal of RNAPII in the promoter proximal region of AT1G70600 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Arrows indicate the RNAPII signal at the region of promoter - proximal stalling .

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plaNET-seq signal of RNAPII in the promoter proximal region of AT1G70600 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Arrows indicate the RNAPII signal at the region of promoter-proximal stalling.


Metagene profile of plaNET - seq mean signal of RNAPII in a 1 Kb window centered at the + 1 nucleosome in Arabidopsis genes ( n = 25474 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . The significance of differences of plaNET - seq signal in the region from - 25 bins to + 25 bins around + 1 nucleosome between NRPB2WT and NRPB2Y732F were assessed by a two - sided Mann - Whitney U - test , p = 5 . 20e - 10 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O B-ORGANISM O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

Metagene profile of plaNET-seq mean signal of RNAPII in a 1 Kb window centered at the +1 nucleosome in Arabidopsis genes (n=25474) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). The significance of differences of plaNET-seq signal in the region from -25 bins to +25 bins around +1 nucleosome between NRPB2WT and NRPB2Y732F were assessed by a two-sided Mann-Whitney U-test, p=5.20e-10.


RNAPII stalling index calculated for all the genes with plaNET - Seq FPKM ≥ 10 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) ( n = 6596 ) . Medians of the stalling index are 1 . 891 and 1 . 222 for NRPB2WT and NRPB2Y732F , respectively . * * * denotes p - value < 0 . 001 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RNAPII stalling index calculated for all the genes with plaNET-Seq FPKM ≥ 10 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red) (n=6596). Medians of the stalling index are 1.891 and 1.222 for NRPB2WT and NRPB2Y732F, respectively. *** denotes p-value <0.001 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


Metagene profile of plaNET - seq mean signal over whole genes ( length from 0 . 5 Kb to 5 Kb , scaled to 500 bins , n = 27042 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O

Metagene profile of plaNET-seq mean signal over whole genes (length from 0.5 Kb to 5 Kb, scaled to 500 bins, n=27042) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).


Metagene profile of plaNET - seq mean signal of RNAPII in exons ( length from 50 bp to 300 bp , scaled to 100 bins , n = 73925 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Pink dashed line rectangle illustrates the amplitude of differences between the minimum and the maximum of RNAPII signal across the exons . A two - sided Mann - Whitney U - test was used to assess the plaNET - seq signal of NRPB2WT ( blue ) and NRPB2Y732F ( red ) in exons , p < 1e - 16 . Metagene profile of plaNET - seq mean signal of RNAPII in introns ( 50 bp to 300 bp , scaled to 100 bins , n = 102260 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . A two - sided Mann - Whitney U - test was used to assess the plaNET - seq signal of NRPB2WT ( blue ) and NRPB2Y732F ( red ) in introns , p < 1e - 16 .

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Metagene profile of plaNET-seq mean signal of RNAPII in exons (length from 50 bp to 300 bp, scaled to 100 bins, n=73925) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Pink dashed line rectangle illustrates the amplitude of differences between the minimum and the maximum of RNAPII signal across the exons. A two-sided Mann-Whitney U-test was used to assess the plaNET-seq signal of NRPB2WT (blue) and NRPB2Y732F (red) in exons, p<1e-16. Metagene profile of plaNET-seq mean signal of RNAPII in introns (50 bp to 300 bp, scaled to 100 bins, n=102260) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). A two-sided Mann-Whitney U-test was used to assess the plaNET-seq signal of NRPB2WT (blue) and NRPB2Y732F (red) in introns, p<1e-16.


Bar charts showing the fractions of 3 ' and 5 ' splicing intermediate reads from plaNET - seq in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) .

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Bar charts showing the fractions of 3' and 5' splicing intermediate reads from plaNET-seq in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).


Genome browser snapshots illustrating enhanced intron splicing in NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) compared to NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) . Scale bars denote 0 . 5 Kb .

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Genome browser snapshots illustrating enhanced intron splicing in NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F, red) compared to NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT ,blue). Scale bars denote 0.5 Kb.


The fraction of RNA - seq intronic reads calculated for all genes ( n = 24912 ) in NRPB2WT + / + nrpb2 - 2 - / - and NRPB2Y732F + / + nrpb2 - 2 - / - . Two - sided Mann - Whitney U test : * * * * denotes p - value < 2 . 2e - 16 . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The fraction of RNA-seq intronic reads calculated for all genes (n=24912) in NRPB2WT +/+ nrpb2-2 -/- and NRPB2Y732F +/+ nrpb2-2 -/-. Two-sided Mann-Whitney U test: **** denotes p-value<2.2e-16. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


Differentially expressed ( DE ) exons and introns in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - based on RNA - seq results . Numbers of DE exons and introns were shown in plot . Quantification ( log fold change of FPKM from RNA - seq ) of differentially expressed ( DE ) exons and non - DE exons in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - . * * denotes p - value < 0 . 01 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Differentially expressed (DE) exons and introns in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/- based on RNA-seq results. Numbers of DE exons and introns were shown in plot. Quantification (log fold change of FPKM from RNA-seq) of differentially expressed (DE) exons and non-DE exons in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/-. ** denotes p-value <0.01 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


Genome browser snapshots illustrating enhanced exon skipping in NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) compared to NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) . Scale bars denote 0 . 5 Kb . Quantification ( log fold change of FPKM from RNA - seq ) of differentially expressed ( DE ) introns and non - DE exons in NRPB2Y732F + / + nrpb2 - 2 - / - compared to NRPB2WT + / + nrpb2 - 2 - / - . * * * * denotes p - value < 0 . 0001 by Wilcoxon signed - rank test . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Genome browser snapshots illustrating enhanced exon skipping in NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F, red) compared to NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT ,blue). Scale bars denote 0.5 Kb. Quantification (log fold change of FPKM from RNA-seq) of differentially expressed (DE) introns and non-DE exons in NRPB2Y732F +/+ nrpb2-2 -/- compared to NRPB2WT +/+ nrpb2-2 -/-. **** denotes p-value <0.0001 by Wilcoxon signed-rank test. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


plaNET - seq signal of RNAPII at 3 ' end of AT2G21410 in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Arrows indicate the RNAPII signal peaks at PAS stalling region .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O

plaNET-seq signal of RNAPII at 3' end of AT2G21410 in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Arrows indicate the RNAPII signal peaks at PAS stalling region.


Metagene profile of plaNET - seq mean signal of RNAPII in a 1 kb window centered at PAS ( n = 24448 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . The significance of differences of plaNET - seq signal in the region from PAS to + 100 bins between NRPB2WT and NRPB2Y732F were assessed by Two - sided Mann - Whitney U - test , p = 1 . 53e - 06 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

Metagene profile of plaNET-seq mean signal of RNAPII in a 1 kb window centered at PAS (n=24448) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). The significance of differences of plaNET-seq signal in the region from PAS to +100 bins between NRPB2WT and NRPB2Y732F were assessed by Two-sided Mann-Whitney U-test, p = 1.53e-06.


Histogram of transcriptional read - through length ( nt ) from PAS of protein - coding gene ( plaNET - seq FPKM > 5 , n = 9316 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) .

O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O

Histogram of transcriptional read-through length (nt) from PAS of protein-coding gene (plaNET-seq FPKM>5, n=9316) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red).


Box plot shows the RNAPII transcriptional read - through length from PAS of protein - coding genes ( plaNET - seq FPKM > 5 n = 9316 ) called based on statistic model ( see Methods ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Median of read - through length in NRPB2WT and NRPB2Y732F mutant are 534 nt and 649 nt . Two - sided Mann - Whitney U - test : * * * denotes p = 9 . 9e - 62 . The solid horizontal lines and box limits represent medians , lower and upper quartiles of data values in each group . The upper and lower whiskers extend to the largest or smallest value , respectively , no further than 1 . 5 * IQR from the relevant quartile .

O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Box plot shows the RNAPII transcriptional read-through length from PAS of protein-coding genes (plaNET-seq FPKM>5 n=9316) called based on statistic model (see Methods) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Median of read-through length in NRPB2WT and NRPB2Y732F mutant are 534 nt and 649 nt. Two-sided Mann-Whitney U-test: *** denotes p = 9.9e-62. The solid horizontal lines and box limits represent medians, lower and upper quartiles of data values in each group. The upper and lower whiskers extend to the largest or smallest value, respectively, no further than 1.5 * IQR from the relevant quartile.


Metagene plot of RNAPII signal by plaNET - seq anchored at both PAS of upstream genes and TSS of downstream genes for tandemly oriented genes ( n = 5753 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Red arrow denotes the direction of transcriptional read - through . Pink dashed line rectangle indicates the region corresponding to the second half of PAS - TSS gaps along 5 ' to 3 ' direction . Metagene plot of RNAPII signal by plaNET - seq anchored at PASs of both upstream genes and downstream genes for gene pairs located in " tail to tail " orientation ( n = 1384 ) in NRPB2WT + / + nrpb2 - 2 - / - ( NRPB2WT , blue ) and NRPB2Y732F + / + nrpb2 - 2 - / - ( NRPB2Y732F , red ) . Red arrows denote the directions of transcriptional read - through from both PASs . Pink dashed line rectangles indicate the region corresponding to the second half of PAS - TSS gaps along 5 ' to 3 ' direction .

O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Metagene plot of RNAPII signal by plaNET-seq anchored at both PAS of upstream genes and TSS of downstream genes for tandemly oriented genes (n=5753) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Red arrow denotes the direction of transcriptional read-through. Pink dashed line rectangle indicates the region corresponding to the second half of PAS-TSS gaps along 5' to 3' direction. Metagene plot of RNAPII signal by plaNET-seq anchored at PASs of both upstream genes and downstream genes for gene pairs located in "tail to tail" orientation (n=1384) in NRPB2WT +/+ nrpb2-2 -/- (NRPB2WT, blue) and NRPB2Y732F +/+ nrpb2-2 -/- (NRPB2Y732F ,red). Red arrows denote the directions of transcriptional read-through from both PASs. Pink dashed line rectangles indicate the region corresponding to the second half of PAS-TSS gaps along 5' to 3' direction.


A . Binding of ubiquitin chains to immobilized GST - fusion proteins . Indicated proteins were purified from E . coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains , which were either K48 - or K63 - linked . The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS ‑ PAGE and fluorescence scan . Total fluorescence was quantified per lane and normalized to the first lane ( rel . fluo . ) . Asterisk ( * ) indicates lane from the same gel that was cropped and moved .

O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Binding of ubiquitin chains to immobilized GST-fusion proteins. Indicated proteins were purified from E. coli and incubated with equal amounts of preformed fluorescently labeled ubiquitin chains, which were either K48- or K63-linked. The fraction of ubiquitin chains interacting with the bait protein was analyzed by SDS‑PAGE and fluorescence scan. Total fluorescence was quantified per lane and normalized to the first lane (rel. fluo.). Asterisk (*) indicates lane from the same gel that was cropped and moved.


B . At indicated time points , samples were taken from the reaction mix and analyzed by SDS ‑ PAGE and fluorescence scan . Product fluorescence over total fluorescence per lane was quantified ( rel . fluo . ) . D . Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full ‑ length Ubc1 or Ubc1 - ΔUBA ( aa1 ‑ 150 ) . The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin , while acceptor sites were blocked by introducing the K48R substitution . Reactions were performed in triplicate .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

B. At indicated time points, samples were taken from the reaction mix and analyzed by SDS‑PAGE and fluorescence scan. Product fluorescence over total fluorescence per lane was quantified (rel. fluo.). D. Initial reaction rates were determined for a panel of acceptor ubiquitin molecules in reactions with either full‑length Ubc1 or Ubc1-ΔUBA (aa1‑150). The binding of the UBA domain to individual ubiquitin molecules was disrupted by introducing an R42A amino acid exchange in ubiquitin, while acceptor sites were blocked by introducing the K48R substitution. Reactions were performed in triplicate.


b . L143E and / or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC / UBA domain interface . Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63 ‑ Ub2 with Ub ( K48R ) at the distal position or monoubiquitin as acceptor . Experiment and initial rate

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O

b. L143E and/or L147E amino acid substitutions were introduced into Ubc1 to disrupt the hydrophobic UBC/UBA domain interface. Single turnover ubiquitination experiments were performed with these Ubc1 variants in presence of K63‑Ub2 with Ub(K48R) at the distal position or monoubiquitin as acceptor. Experiment and initial rate determination


c . Ubc1 variants were generated with deletions in the linker region ( aa152 ‑ 167 ) , which connects the UBA domain and the UBC domain . Single turnover ubiquitination reaction mixes were incubated for 5 min with K63 - Ub2 or for 20 min with monoubiquitin as acceptor . Final product formation was analyzed by SDS - PAGE and fluorescence scan . Product fluorescence over total fluorescence ( rel . fluo . ) was quantified .

O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O

c. Ubc1 variants were generated with deletions in the linker region (aa152‑167), which connects the UBA domain and the UBC domain. Single turnover ubiquitination reaction mixes were incubated for 5 min with K63-Ub2 or for 20 min with monoubiquitin as acceptor. Final product formation was analyzed by SDS-PAGE and fluorescence scan. Product fluorescence over total fluorescence (rel. fluo.) was quantified.


d . C - terminally blocked M1 - linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full - length Ubc1 or Ubc1 - ΔUBA . Reactions with acceptor K63 - Ub2 were carried out controls . Initial rate

O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O

d. C-terminally blocked M1-linked diubiquitin with or without a K48R amino acid substitution at the distal or proximal position were employed in single turnover experiments with full-length Ubc1 or Ubc1-ΔUBA. Reactions with acceptor K63-Ub2 were carried out controls. Initial rate determination


e . CSPs of I44 were plotted over UBA domain concentration . f . KD values for binding of the UBA domain were determined from results in ( E ) assuming two independent but different binding sites .

O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O

e. CSPs of I44 were plotted over UBA domain concentration. f. KD values for binding of the UBA domain were determined from results in (E) assuming two independent but different binding sites.


a . The activity of Ubc1 was compared with the K48 - linked chain building enzyme Ubc7 / Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin , K48 ‑ Ub2 or K63 - Ub2 . Initial rates were

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O B-GENEPROD O O O O

a. The activity of Ubc1 was compared with the K48-linked chain building enzyme Ubc7/Cue1 in single turnover ubiquitination experiments in presence of either monoubiquitin, K48‑Ub2 or K63-Ub2. Initial rates were determined