( C ) Complexome analysis of MEFmitochondria ( n = 3 ) . Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARLimmunoprecipitates at a FDR level of 0 . 05 ( A ) and identified by mass spectrometry after BN - PAGE analysis are shown after hierarchical clustering .

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(C) Complexome analysis of MEFmitochondria (n=3). Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARLimmunoprecipitates at a FDR level of 0.05 (A) and identified by mass spectrometry after BN-PAGE analysis are shown after hierarchical clustering.


( D ) BN - PAGE analysis of mitochondria lacking SLP2 , YME1L or PARL . Mitochondria isolated from corresponding MEFs were solubilized using 1 . 5 % ( w / v ) digitonin at a protein concentration of 2 . 5 mg / ml . Solubilized proteins were separated by a 3 - 13 % blue native gradient gel and analyzed using SLP2 - , YME1L - and PARL - specific antibodies .

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(D) BN-PAGE analysis of mitochondria lacking SLP2, YME1L or PARL. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-13% blue native gradient gel and analyzed using SLP2-, YME1L- and PARL-specific antibodies.


( E ) High resolution complexome analysis of SPY complex members SLP2 , YME1L and PARL using MEFmitochondria ( n = 3 ) . Heat maps and migration profiles are shown after separation using a 3 - 9 % BN - PAGE . SLP2 , YME1L and PARL migrate in a high molecular weight complex ( 2 MDa ) , whereas SLP2 additionally is present in complexes of 1 . 6 MDa ) .

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(E) High resolution complexome analysis of SPY complex members SLP2, YME1L and PARL using MEFmitochondria (n=3). Heat maps and migration profiles are shown after separation using a 3-9% BN-PAGE. SLP2, YME1L and PARL migrate in a high molecular weight complex (2 MDa), whereas SLP2 additionally is present in complexes of 1.6 MDa).


( A ) Co - immunoprecipitation of endogenous SLP2 , PARL and YME1L in mitochondria isolated from human FITR293T cells using either SLP2 - or PARL - specific antibodies . IgG was used as a negative control . In , input ( 10 % ) .

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(A) Co-immunoprecipitation of endogenous SLP2, PARL and YME1L in mitochondria isolated from human FITR293T cells using either SLP2- or PARL-specific antibodies. IgG was used as a negative control. In, input (10%).


( B , C ) Co - immunoprecipitation of endogenous SLP2 and YME1L with PARL - specific antibodies in mitochondria isolated from wild type MEFs ( WT ) and MEFs lacking YME1L , SLP2 or PARL . In , input ( 10 % ) .

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(B, C) Co-immunoprecipitation of endogenous SLP2 and YME1L with PARL-specific antibodies in mitochondria isolated from wild type MEFs (WT) and MEFs lacking YME1L, SLP2 or PARL. In, input (10%).


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( D ) Assembly of SLP2 , PARL and YME1L into a high molecular weight complex . Mitochondria isolated from FITR293T cells ( Con ) and cells inducibly expressing SLP2 - FLAG ( FLAG ) were solubilized in digitonin and subjected to immunoprecipitation using FLAG - specific antibodies . Native eluates of the precipitate were analyzed by BN - PAGE and immunoblotting using SLP2 - , PARL - , YME1L - and PHB2 - specific antibodies . In , input ( 8 % ) ; E , eluate ( 100 % ) .

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(D) Assembly of SLP2, PARL and YME1L into a high molecular weight complex. Mitochondria isolated from FITR293T cells (Con) and cells inducibly expressing SLP2-FLAG (FLAG) were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, PARL-, YME1L- and PHB2-specific antibodies. In, input (8%); E, eluate (100%).


( E ) High resolution BN - PAGE analysis of mitochondria lacking PARL , YME1L or SLP2 . Mitochondria isolated from corresponding MEFs were solubilized using 1 . 5 % ( w / v ) digitonin at a protein concentration of 2 . 5 mg / ml . Solubilized proteins were separated by a 3 - 9 % gradient gel containing 0 - 10 % glycerol and analyzed using SLP2 - specific antibodies .

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(E) High resolution BN-PAGE analysis of mitochondria lacking PARL, YME1L or SLP2. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-9% gradient gel containing 0-10% glycerol and analyzed using SLP2-specific antibodies.


( F , G ) Submitochondrial localization of SLP2 . ( F ) Mitochondria were isolated from MEFs and fractionated in the presence or absence of proteinase K as indicated . Fractions were analyzed by SDS - PAGE and immunoblotting . MFN2 served as OM marker , TIMM23 , SMAC / DIABLO , and YME1L as IMS markers , and AFG3L1 and AFG3L2 as matrix marker proteins .

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(F, G) Submitochondrial localization of SLP2. (F) Mitochondria were isolated from MEFs and fractionated in the presence or absence of proteinase K as indicated. Fractions were analyzed by SDS-PAGE and immunoblotting. MFN2 served as OM marker, TIMM23, SMAC/DIABLO, and YME1L as IMS markers, and AFG3L1 and AFG3L2 as matrix marker proteins.


( F , G ) Submitochondrial localization of SLP2 . ( G ) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet ( P ) and supernatant ( S ) fraction by centrifugation . Fractions were analyzed by SDS - PAGE and immunoblotting .

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(F, G) Submitochondrial localization of SLP2. (G) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet (P) and supernatant (S) fraction by centrifugation. Fractions were analyzed by SDS-PAGE and immunoblotting.


( A ) Steady state levels of proteolytic substrates of YME1L . Whole cell extracts of Yme1l - / - , Parl - / - , and Slp2 - / - MEFs were analyzed by SDS - PAGE and immunoblotting using the indicated antibodies .

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(A) Steady state levels of proteolytic substrates of YME1L. Whole cell extracts of Yme1l-/-, Parl-/-, and Slp2-/- MEFs were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies.


( B ) PGAM5 processing depends on proteolytically active PARL . PARL and catalytic inactive PARLS277A harboring C - terminal FLAG - epitopes were expressed under the control of a tetracycline ( tet ) - inducible promoter in wild type ( WT ) and PARL - / - FITR293T cells as indicated . Processing of L - PGAM5 to S - PGAM5 was monitored by immunoblotting .

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(B) PGAM5 processing depends on proteolytically active PARL. PARL and catalytic inactive PARLS277A harboring C-terminal FLAG-epitopes were expressed under the control of a tetracycline (tet)-inducible promoter in wild type (WT) and PARL-/- FITR293T cells as indicated. Processing of L-PGAM5 to S-PGAM5 was monitored by immunoblotting.


( C ) Accelerated processing of L - PGAM5 in Slp2 - / - cells . Processing of L - PGAM5 was analyzed by immunoblotting in wild type ( WT ) , Yme1l - / - , Parl - / - and Slp2 - / - MEFs after inhibition of cytosolic protein synthesis by cycloheximide ( CHX ) . A quantification of L - PGAM5 levels at different time points is shown in the lower panel . Two - way ANOVA analysis ( n = 3 ; * * * * , p < 0 . 0001 ) .

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(C) Accelerated processing of L-PGAM5 in Slp2-/- cells. Processing of L-PGAM5 was analyzed by immunoblotting in wild type (WT), Yme1l-/-, Parl-/- and Slp2-/-MEFs after inhibition of cytosolic protein synthesis by cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown in the lower panel. Two-way ANOVA analysis (n=3; ****, p<0.0001).


( D ) The accelerated processing of L - PGAM5 in Slp2 - / - cells is mediated by PARL . PARL was depleted from Slp2 - / - cells by RNAi prior to CHX treatment . A quantification of L - PGAM5 levels at different time points is shown . Two - way ANOVA analysis ( n = 3 ; * * , p < 0 . 01 . * * * * , p < 0 . 0001 ) .

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(D) The accelerated processing of L-PGAM5 in Slp2-/- cells is mediated by PARL. PARL was depleted from Slp2-/- cells by RNAi prior to CHX treatment. A quantification of L-PGAM5 levels at different time points is shown. Two-way ANOVA analysis (n=3; **, p<0.01. ****, p<0.0001).


( E ) PGAM5 associates with the SPY complex harboring proteolytically inactive PARL . Mitochondria isolated from FITR293T cells ( WT ) or PARL - / - FITR293T cells expressing PARL - FLAG ( PARL - WT ) or PARLS277A - FLAG ( PARL - S277A ) were solubilized in digitonin were analyzed by BN - PAGE and immunoblotting using FLAG - and PGAM5 - specific antibodies .

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(E) PGAM5 associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin were analyzed by BN-PAGE and immunoblotting using FLAG- and PGAM5-specific antibodies.


( A ) PARL dependent cleavage of PINK1 - HA is reduced in Slp2 - / - and Yme1l - / - MEFs . Whole cell extracts of Slp2 - / - , Parl - / - and Yme1l - / - MEFs expressing PINK1 - HA were analysed by SDS - PAGE and immunoblotting using the indicated antibodies . Cells were treated with 20 µM MG132 or 20 µM CCCP for 4 h . ( B ) Quantification of the protein ratio ( Log2 ) PINK1 - HA 52 kDa / 63 kDa ( n = 3 ; * , p < 0 . 05 ; One - way ANOVA ) . n . s . , not significant . Error bars indicate SEM .

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(A) PARL dependent cleavage of PINK1-HA is reduced in Slp2-/- and Yme1l-/- MEFs. Whole cell extracts of Slp2-/-, Parl-/ -and Yme1l-/- MEFs expressing PINK1-HA were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Cells were treated with 20 µM MG132 or 20 µM CCCP for 4 h.(B) Quantification of the protein ratio (Log2) PINK1-HA 52 kDa/63 kDa (n=3; *, p<0.05; One-way ANOVA). n.s., not significant. Error bars indicate SEM.


( C ) PINK1 - HA associates with the SPY complex harboring proteolytically inactive PARL . Mitochondria isolated from FITR293T cells ( WT ) or PARL - / - FITR293T cells expressing PARL - FLAG ( PARL - WT ) or PARLS277A - FLAG ( PARL - S277A ) were solubilized in digitonin and analysed by BN - PAGE and immunoblotting using FLAG - , HA - and SLP2 - specific antibodies .

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(C) PINK1-HA associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using FLAG-, HA- and SLP2-specific antibodies.


( D ) PINK1 associates with SLP2 . Mitochondria isolated from FITR293T cells ( WT ) or PARL - / - FITR293T cells , depleted of SLP2 by siRNA as indicated , were solubilized in digitonin and analysed by BN - PAGE and immunoblotting using SLP2 - and PINK1 - specific antibodies .

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(D) PINK1 associates with SLP2. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells, depleted of SLP2 by siRNA as indicated, were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using SLP2- and PINK1-specific antibodies.


( A ) Processing of L - PGAM5 to S - PGAM5 in depolarized mitochondria of wild type ( WT ) , Slp2 - / - , Yme11 - / - , and Parl - / - MEFs . PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide ( CHX ) . A quantification of L - PGAM5 levels at different time points is shown . L - PGAM5 levels at t = 0 was set to 100 % . Two - way ANOVA analysis ( n = 3 ; * , p < 0 . 05 , * * , p < 0 . 01 . * * * * , p < 0 . 0001 ) . Arrowheads denote intermediate PGAM5 cleavage products .

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(A) Processing of L-PGAM5 to S-PGAM5 in depolarized mitochondria of wild type (WT), Slp2-/-, Yme11-/-, and Parl-/- MEFs. PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown. L-PGAM5 levels at t=0 was set to 100%. Two-way ANOVA analysis (n=3; *, p<0.05, **, p<0.01. ****, p<0.0001). Arrowheads denote intermediate PGAM5 cleavage products.


( B ) OMA1 - mediated processing of PGAM5 in depolarized Parl - / - mitochondria . Processing of L - PGAM5 to S - PGAM5 in Parl - / mitochondria was assessed as in ( A ) in the presence or absence of CCCP ( 20 µM ) , which were depleted of OMA1 by siRNA as indicated . The arrowhead denotes an intermediate PGAM5 cleavage product . SCR , scrambled .

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(B) OMA1-mediated processing of PGAM5 in depolarized Parl-/-mitochondria. Processing of L-PGAM5 to S-PGAM5 in Parl-/ mitochondria was assessed as in (A) in the presence or absence of CCCP (20 µM), which were depleted of OMA1 by siRNA as indicated. The arrowhead denotes an intermediate PGAM5 cleavage product. SCR, scrambled.


( C ) Impaired PGAM5 processing in depolarized Oma1 - / - mitochondria lacking PARL . Processing of L - PGAM5 to S - PGAM5 was examined as in ( A ) after 2 h in wild type ( WT ) and Oma1 - / - mitochondria in the presence or absence of CCCP ( 20 µM , 2 h ) , which were depleted of PARL by siRNA as indicated . * unspecific cross - reaction .

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(C) Impaired PGAM5 processing in depolarized Oma1-/- mitochondria lacking PARL. Processing of L-PGAM5 to S-PGAM5 was examined as in (A) after 2 h in wild type (WT) and Oma1-/- mitochondria in the presence or absence of CCCP (20 µM, 2 h), which were depleted of PARL by siRNA as indicated. * unspecific cross-reaction.


( D ) OMA1 mediates accelerated processing of L - OPA1 in Slp2 - / - MEFs . Processing of OPA1 and PGAM5 was monitored in Slp2 - / - , Oma1 - / - , and Slp2 - / - Oma1 - / - cells by immunoblotting after inhibition of cytosolic protein synthesis with cycloheximide ( CHX ) .

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(D) OMA1 mediates accelerated processing of L-OPA1 in Slp2-/- MEFs. Processing of OPA1 and PGAM5 was monitored in Slp2-/-, Oma1-/-, and Slp2-/-Oma1-/- cells by immunoblotting after inhibition of cytosolic protein synthesis with cycloheximide (CHX).


( E ) OMA1 interacts with SLP2 . OMA1 - / - FITR293T cells ectopically expressing OMA1 - myc were transfected with pcDNA5 ( Control ) or pcDNA5 - SLP2 - FLAG ( FLAG ) . Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG - specific antibodies . Native eluates of the precipitate were analyzed by BN - PAGE and immunoblotting using SLP2 - , YME1L - , PARL - , and myc - specific antibodies . In , input ( 8 % ) ; E , eluate ( 100 % ) . Samples were analyzed by SDS - PAGE in parallel ( lower panel ) . ( F ) Quantification of immunoprecipitation efficiencies in ( E ) .

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(E) OMA1 interacts with SLP2. OMA1-/-FITR293T cells ectopically expressing OMA1-myc were transfected with pcDNA5 (Control) or pcDNA5-SLP2-FLAG (FLAG). Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, YME1L-, PARL-, and myc-specific antibodies. In, input (8%); E, eluate (100%). Samples were analyzed by SDS-PAGE in parallel (lower panel).(F) Quantification of immunoprecipitation efficiencies in (E).


B , C , D . WT , ∆ cpt1 ∆ ept1 ( B ) , Δcho2 ( C ) or Δopi3 ( D ) cells expressing GFP - Atg8 were grown to logarithmic phase in SD - URA , and shifted to SD - N for 4 h of starvation . Cells were harvested at indicated time points and subjected to western blotting ( left panel ) . Pgk1 was used as a loading control . Middle panel - Autophagic activity during starvation was quantified by calculating the ratio of free GFP to total GFP ( GFP - Atg8 + free GFP ) . Statistical analysis was done by student ' s t - test ( paired , two tailed ) ( ns - not significant , * * * * , p ≤ 0 . 0001 ) , error bars represent SEM of at least 3 independent experiments . Right panel - Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount , during SD and SD - N .

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B, C, D. WT, ∆cpt1∆ept1 (B), Δcho2 (C) or Δopi3 (D) cells expressing GFP-Atg8 were grown to logarithmic phase in SD-URA, and shifted to SD-N for 4 h of starvation. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was used as a loading control. Middle panel- Autophagic activity during starvation was quantified by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by student's t-test (paired, two tailed) (ns- not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount, during SD and SD-N.


E . WT , Δopi3 , and Δcho2 as well as Δatg1 ( negative autophagy control ) cells expressing chromosomally - tagged Fba1 - GFP were grown to log phase in SD , and starved in SD - N for 4 hours . Cells were harvested at indicated starvation time points and subjected to western blotting ( left panel ) . Pgk1 was monitored as a loading control . Right panel - Autophagic activity during starvation was quantified , by calculating the ratio of free GFP to total GFP ( Fba1 - GFP + free GFP ) .

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E. WT, Δopi3, and Δcho2 as well as Δatg1 (negative autophagy control) cells expressing chromosomally-tagged Fba1-GFP were grown to log phase in SD, and starved in SD-N for 4 hours. Cells were harvested at indicated starvation time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Right panel- Autophagic activity during starvation was quantified, by calculating the ratio of free GFP to total GFP (Fba1-GFP + free GFP).


F . WT , Δopi3 , Δcho2 and Δatg1 cells on the background of pho8Δ60 pho13Δ strain , were grown to log phase in SD , and starved in SD - N for 4 hours . Pho8 activity was measured by the alkaline phosphatase assay .

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F. WT, Δopi3, Δcho2 and Δatg1 cells on the background of pho8Δ60 pho13Δ strain, were grown to log phase in SD, and starved in SD-N for 4 hours. Pho8 activity was measured by the alkaline phosphatase assay.


A . Representative images of GFP - Atg8 ( green ) and vacuole ( red , stained with FM4 - 64 ) . WT , Δopi3 and Δcho2 cells expressing GFP - Atg8 were grown to log phase in SD - URA medium and shifted to SD - N for 3 h . Cells were observed by confocal microscopy before ( SD ) and during nitrogen starvation ( SD - N ) ( left panel ) . Scale bar 1 µm . Right panel - Quantification of of cells with or without GFP inside vacuoles . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s , compared to WT ( * * * * , p ≤ 0 . 0001 ) ( ns - not significant ) , error bars represent SEM of at least 3 independent experiments . Number of cells analyzed for each strain and condition : SD ( WT ( n = 446 ) , Δcho2 ( n = 272 ) , Δopi3 ( n = 272 ) ) , SD - N ( WT ( n = 398 ) , Δcho2 ( n = 224 ) , Δopi3 ( n = 213 ) ) .

O O O O O O O B-MEASURED_VAR 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT, Δopi3 and Δcho2 cells expressing GFP-Atg8 were grown to log phase in SD-URA medium and shifted to SD-N for 3 h. Cells were observed by confocal microscopy before (SD) and during nitrogen starvation (SD-N) (left panel). Scale bar 1 µm. Right panel- Quantification of of cells with or without GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001) (ns- not significant), error bars represent SEM of at least 3 independent experiments. Number of cells analyzed for each strain and condition: SD (WT (n=446), Δcho2 (n=272), Δopi3 (n=272)), SD-N (WT (n=398), Δcho2 (n=224), Δopi3 (n=213)).


A , B . WT and Δopi3 cells expressing GFP - Atg8 were grown to log phase in SD - URA , and shifted to SD - N for 4 h , choline ( 1 mM ) was not supplemented ( - choline ) or supplemented to SD and SD - N ( + choline SD , SD - N ) or only to SD - N ( + choline , SD - N ) as indicated . Cells were harvested at indicated time points and subjected to western blotting ( left panel ) . Pgk1 was monitored as a loading control . Middle panel - Autophagic activity was quantified during starvation by calculating the ratio of free GFP to total GFP ( GFP - Atg8 + free GFP ) . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s , compared to WT ( ns - not significant , * * * * , p ≤ 0 . 0001 ) , error bars represent SEM of at least 3 independent experiments . Right panel - Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount in Δopi3 cells at growth ( SD ) or during starvation ( SD - N ) , with choline supplemented as indicated . Statistical analysis was done by Sidak ' s multiple comparisons test compared to WT ( ns - not significant , * , p ≤ 0 . 05 , * * , p ≤ 0 . 005 , * * * * , p ≤ 0 . 0001 ) , error bars represent SEM of at least 3 independent experiments .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B. WT and Δopi3 cells expressing GFP-Atg8 were grown to log phase in SD-URA, and shifted to SD-N for 4 h, choline (1 mM) was not supplemented (-choline) or supplemented to SD and SD-N (+choline SD, SD-N) or only to SD-N (+choline, SD-N) as indicated. Cells were harvested at indicated time points and subjected to western blotting (left panel). Pgk1 was monitored as a loading control. Middle panel- Autophagic activity was quantified during starvation by calculating the ratio of free GFP to total GFP (GFP-Atg8 + free GFP). Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (ns-not significant, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments. Right panel- Ape1 maturation was quantified by measuring the mApe1 level out of the total Ape1 amount in Δopi3 cells at growth (SD) or during starvation (SD-N), with choline supplemented as indicated. Statistical analysis was done by Sidak's multiple comparisons test compared to WT (ns-not significant, *, p≤0.05, **, p≤0.005, ****, p≤0.0001), error bars represent SEM of at least 3 independent experiments.


C . Representative images of GFP - Atg8 ( green ) and vacuole ( red , stained with FM4 - 64 ) . WT and Δopi3 cells expressing GFP - Atg8 were grown in SD - URA and shifted to SD - N . Choline ( 1mM ) was either excluded ( - choline ) , added to growth and starvation medium ( + choline SD , SD - N ) or added only to starvation medium ( + choline , SD - N ) . Cells were observed using confocal microscopy during starvation ( left panel ) . Scale bar 1 µm . Right panel - Quantification of cells with GFP inside vacuoles . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s , compared to WT ( * * * * , p ≤ 0 . 0001 , ns - not significant ) , error bars represent SEM of 3 independent experiments . Number of cells analyzed for each strain condition ; - Choline : WT ( n = 487 ) , Δopi3 ( n = 316 ) , Choline SD , SD - N : WT ( n = 251 ) , Δopi3 ( n = 361 ) , Choline SD - N : WT ( n = 320 ) , Δopi3 ( n = 267 ) . D . PC levels determined by shotgun lipidomics for WT and Δopi3 cells expressing GFP - Atg8 , with or without choline ( 1 mM ) supplementation to SD - N . Samples were taken after 4 h in SD - N medium .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O 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 of GFP-Atg8 (green) and vacuole (red, stained with FM4-64). WT and Δopi3 cells expressing GFP-Atg8 were grown in SD-URA and shifted to SD-N. Choline (1mM) was either excluded (-choline), added to growth and starvation medium (+choline SD, SD-N) or added only to starvation medium (+choline, SD-N). Cells were observed using confocal microscopy during starvation (left panel). Scale bar 1 µm. Right panel- Quantification of cells with GFP inside vacuoles. Statistical analysis was done by Anova multiple comparisons test- Sidak's, compared to WT (****, p≤0.0001, ns- not significant), error bars represent SEM of 3 independent experiments. Number of cells analyzed for each strain condition; -Choline: WT (n=487), Δopi3 (n=316), Choline SD, SD-N: WT (n=251), Δopi3 (n=361), Choline SD-N: WT (n=320), Δopi3 (n=267). D. PC levels determined by shotgun lipidomics for WT and Δopi3 cells expressing GFP-Atg8, with or without choline (1 mM) supplementation to SD-N. Samples were taken after 4 h in SD-N medium.


A . Representative Images of Atg5 - mNG with sfTq2 - Atg8 during SD - N . Δatg3 and Δatg3Δopi3 cells were grown to log phase in SD medium , shifted to SD - N for 4 h , and observed by Widefield microscopy ( left panel ) , scale bar 1 µm . Right panel - quantification of percentage of cells with Atg5 puncta during different time points . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s ns - not significant ) , error bars represent SEM of 2 independent experiments . Number of cells analyzed for each strain and time point ( 0 hour : Δatg3 ( n = 296 ) , Δatg3Δopi3 ( n = 111 ) , 1 hour : Δatg3 ( n = 325 ) , Δatg3Δopi3 ( n = 110 ) , 2 hours : Δatg3 ( n = 338 ) , Δatg3Δopi3 ( n = 135 ) , 4 hours : Δatg3 ( n = 309 ) , Δatg3Δopi3 ( n = 167 ) .

O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative Images of Atg5-mNG with sfTq2-Atg8 during SD-N. Δatg3 and Δatg3Δopi3 cells were grown to log phase in SD medium, shifted to SD-N for 4 h, and observed by Widefield microscopy (left panel), scale bar 1 µm. Right panel- quantification of percentage of cells with Atg5 puncta during different time points. Statistical analysis was done by Anova multiple comparisons test- Sidak's ns- not significant), error bars represent SEM of 2 independent experiments. Number of cells analyzed for each strain and time point (0 hour: Δatg3 (n=296), Δatg3Δopi3 (n=111), 1 hour: Δatg3 (n=325), Δatg3Δopi3 (n=110), 2 hours: Δatg3 (n=338), Δatg3Δopi3 (n=135), 4 hours: Δatg3 (n=309), Δatg3Δopi3 (n=167).


C . Representative images of WT and Δopi3 cells expressing GFP - Atg8 . Cells were grown to log phase in SD - URA medium , shifted to SD - N for 3 h and observed by Airyscan microscopy ( left panel ) . White dashes indicate cell boundaries , yellow dashes indicate phagophores . For each cell - Magnification of yellow dashed area ( top right ) , schematic representation ( bottom right ) . Scale bar 1 µm . Right panel - quantification of the percentage of cells with cup shaped GFP - Atg8 structures imaged by Airyscan microscopy . Statistical analysis was done by student ' s t - test ( unpaired ) ( * * p ≤ 0 . 05 ) , error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain , WT ( n = 45 ) , Δopi3 ( n = 110 ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of WT and Δopi3 cells expressing GFP-Atg8. Cells were grown to log phase in SD-URA medium, shifted to SD-N for 3 h and observed by Airyscan microscopy (left panel). White dashes indicate cell boundaries, yellow dashes indicate phagophores. For each cell - Magnification of yellow dashed area (top right), schematic representation (bottom right). Scale bar 1 µm. Right panel- quantification of the percentage of cells with cup shaped GFP-Atg8 structures imaged by Airyscan microscopy. Statistical analysis was done by student's t-test (unpaired) (** p≤0.05), error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain, WT (n=45), Δopi3 (n=110).


D . Representative CLEM images of WT and Δopi3 cells expressing GFP - Atg8 , grown to log phase in SD - URA medium and shifted to SD - N for 3h . Cells were harvested , deep frozen and processed for CLEM ( left panel ) , V - vacuole . For Δopi3 cell - Magnification of yellow dashed area of GFP - Atg8 positive phagophore ( top right ) , TEM image only ( middle right ) , schematic representation of a phagophore ( bottom right ) . Scale bar 1 µm . Right panel - quantification of the percentage of cells with cup shaped GFP - Atg8 positive cup shaped membrane structures imaged by CLEM . Statistical analysis was done by student ' s t - test ( unpaired ) ( * * * p ≤ 0 . 005 ) , error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain , WT ( n = 20 ) , Δopi3 ( n = 21 ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative CLEM images of WT and Δopi3 cells expressing GFP-Atg8, grown to log phase in SD-URA medium and shifted to SD-N for 3h. Cells were harvested, deep frozen and processed for CLEM (left panel), V-vacuole. For Δopi3 cell - Magnification of yellow dashed area of GFP-Atg8 positive phagophore (top right), TEM image only (middle right), schematic representation of a phagophore (bottom right). Scale bar 1 µm. Right panel- quantification of the percentage of cells with cup shaped GFP-Atg8 positive cup shaped membrane structures imaged by CLEM. Statistical analysis was done by student's t-test (unpaired) (*** p≤0.005), error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain, WT (n=20), Δopi3 (n=21).


A . Representative images of Δypt7 , Δopi3Δypt7 expressing GFP - Atg8 ( z - stack projection ) . Cells were grown to log phase in SD - URA medium and starved in SD - N , images were taken during starvation in indicated time points by widefield microscopy ( left panel ) . Scale bar 1 µm . Right panel - quantification of GFP - Atg8 puncta per cell in Δypt7 , Δopi3Δypt7 at indicated time points . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s ( * * * * , p ≤ 0 . 0001 , ns - not significant ) , compared to WT . Error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain and time point , 0 hours : Δypt7 ( n = 322 ) , Δopi3Δypt7 ( n = 194 ) , 1 . 5 hours : Δypt7 ( n = 220 ) , Δopi3Δypt7 ( n = 155 ) , 3 . 5 hours Δypt7 ( n = 216 ) , Δopi3Δypt7 ( n = 307 ) . B . Quantification of cup - shaped phagophores in Δypt7 and Δopi3Δypt7 cells expressing GFP - Atg8 during starvation ( 1 - 3h ) . Statistical analysis was done by students t - test ( * * * , p ≤ 0 . 001 ) , error bars represent SEM from at least 3 independent experiments . Number of cells analyzed for each strain , Δypt7 ( n = 347 ) , Δopi3Δypt7 ( n = 474 ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of Δypt7, Δopi3Δypt7 expressing GFP-Atg8 (z-stack projection). Cells were grown to log phase in SD-URA medium and starved in SD-N, images were taken during starvation in indicated time points by widefield microscopy (left panel). Scale bar 1 µm. Right panel- quantification of GFP-Atg8 puncta per cell in Δypt7, Δopi3Δypt7 at indicated time points. Statistical analysis was done by Anova multiple comparisons test- Sidak's (****, p≤0.0001, ns- not significant), compared to WT. Error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain and time point, 0 hours: Δypt7 (n=322), Δopi3Δypt7 (n=194), 1.5 hours: Δypt7 (n=220), Δopi3Δypt7 (n=155), 3.5 hours Δypt7 (n=216), Δopi3Δypt7 (n=307). B. Quantification of cup-shaped phagophores in Δypt7 and Δopi3Δypt7 cells expressing GFP-Atg8 during starvation (1-3h). Statistical analysis was done by students t-test (***, p≤0.001), error bars represent SEM from at least 3 independent experiments. Number of cells analyzed for each strain, Δypt7 (n= 347), Δopi3Δypt7 (n= 474).


C . Representative images of mNG - tagged Atg8 colocalized with mScarletI - PXVam7 . WT , Δopi3 , Δatg1 and Δopi3Δatg1 cells on the background of Δymr1 , expressing mScarletI - PXVam7 under the CUP1 promoter from the knocked - out VPS38 locus , were grown to log phase in SD medium in the presence of 10 µM copper sulfate , and shifted to SD - N in the presence of 10 µM copper sulfate . Images were taken during SD - N ( 1 - 3 h ) by widefield microscopy . D . Quantification of autophagic structure diameters in WT and Δopi3 cells on the background of Δymr1Δvps38 ( C ) by ImageJ . Statistical analysis was done by student ' s t - test , unpaired , two sided ( ns - not significant ) , error bars represent SEM of at least three independent experiments . Number of cells analyzed for each strain , WT ( n = 51 ) , Δopi3 ( n = 28 ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of mNG-tagged Atg8 colocalized with mScarletI-PXVam7. WT, Δopi3, Δatg1 and Δopi3Δatg1 cells on the background of Δymr1, expressing mScarletI-PXVam7 under the CUP1 promoter from the knocked-out VPS38 locus, were grown to log phase in SD medium in the presence of 10 µM copper sulfate, and shifted to SD-N in the presence of 10 µM copper sulfate. Images were taken during SD-N (1-3 h) by widefield microscopy. D. Quantification of autophagic structure diameters in WT and Δopi3 cells on the background of Δymr1Δvps38 (C) by ImageJ. Statistical analysis was done by student's t-test, unpaired, two sided (ns- not significant), error bars represent SEM of at least three independent experiments. Number of cells analyzed for each strain, WT (n=51), Δopi3 (n=28).


E . Representative images of Δypt7 , Δopi3Δypt7 cells expressing GFP - Atg8 ( projection ) . Cells were grown to log phase in SD - URA medium and starved in SD - N , with supplementation of choline ( 1 mM ) to SD and SD - N as indicated . Images were taken during growth and starvation by confocal microscopy ( left panel ) . Scale bar 1 µm . Right panel - quantification of cell with more than two GFP - Atg8 puncta , in Δypt7 and Δopi3Δypt7 at growth ( SD ) or starvation ( SD - N ) in the presence ( + choline : SD , SD - N ) or absence ( - choline ) of choline ( 1 mM ) . Incidence were normalized to Δypt7 ( SD - N ) control . Statistical analysis was done by Anova multiple comparisons test - Sidak ' s ( * * * , p ≤ 0 . 001 ) . Error bars represent SEM of least 3 independent experiments . Number of cells analyzed for each strain and condition , SD : Δypt7 ( n = 209 ) , Δopi3Δypt7 ( n = 111 ) , SD - N : Δypt7 ( n = 118 ) , Δopi3Δypt7 ( n = 103 ) , SD + Choline : Δypt7 ( n = 107 ) , Δopi3Δypt7 ( n = 120 ) , Δypt7 ( n = 84 ) , Δopi3Δypt7 ( n = 74 ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of Δypt7, Δopi3Δypt7 cells expressing GFP-Atg8 (projection). Cells were grown to log phase in SD-URA medium and starved in SD-N, with supplementation of choline (1 mM) to SD and SD-N as indicated. Images were taken during growth and starvation by confocal microscopy (left panel). Scale bar 1 µm. Right panel- quantification of cell with more than two GFP-Atg8 puncta, in Δypt7 and Δopi3Δypt7 at growth (SD) or starvation (SD-N) in the presence (+choline: SD, SD-N) or absence (-choline) of choline (1 mM). Incidence were normalized to Δypt7 (SD-N) control. Statistical analysis was done by Anova multiple comparisons test- Sidak's (***, p≤0.001). Error bars represent SEM of least 3 independent experiments. Number of cells analyzed for each strain and condition, SD: Δypt7 (n=209), Δopi3Δypt7 (n=111), SD-N: Δypt7 (n=118), Δopi3Δypt7 (n=103), SD+Choline: Δypt7 (n=107), Δopi3Δypt7 (n=120), Δypt7 (n=84), Δopi3Δypt7 (n=74).


( B ) Phase - contrast images of wild - type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA - mediated knockdown . Scale bar , 100 μm . ( C ) Total numbers of viable cells in ( B ) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild - type HeLa cells . Error bars represent the SEM of three biological replicates .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phase-contrast images of wild-type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA-mediated knockdown. Scale bar, 100 μm. (C) Total numbers of viable cells in (B) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild-type HeLa cells. Error bars represent the SEM of three biological replicates.


( C ) Phase - contrast images of PABPDHFR cells , or PABPDHFR cells transfected with a plasmid coding for wild - type PABPC1 , 48 hours post - TMP removal . Scale bar , 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

(C) Phase-contrast images of PABPDHFR cells, or PABPDHFR cells transfected with a plasmid coding for wild-type PABPC1, 48 hours post-TMP removal. Scale bar, 100 μm.


( D ) Western blot analysis of lysates derived from cells imaged in ( C ) .

(D) Western blot analysis of lysates derived from cells imaged in (C).


( B ) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP . Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide ( control ) , lysed and equal protein amounts were resolved by SDS - PAGE . Western blot analysis was performed using a monoclonal antibody against puromycin or actin ( loading control ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP. Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide (control), lysed and equal protein amounts were resolved by SDS-PAGE. Western blot analysis was performed using a monoclonal antibody against puromycin or actin (loading control).


( D ) Scatter plot comparing log2 fold - changes [ ( - ) PABP vs ( + ) PABP ] in polysome - associated mRNA ( y - axis ) to corresponding changes in total cytoplasmic mRNA . Transcripts identified as regulated via altered translation efficiency ( orange and red ) or abundance ( light and dark green ) according to anota2seq are visualized together with non - regulated transcripts ( grey ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Scatter plot comparing log2 fold-changes [(-) PABP vs (+) PABP] in polysome-associated mRNA (y-axis) to corresponding changes in total cytoplasmic mRNA. Transcripts identified as regulated via altered translation efficiency (orange and red) or abundance (light and dark green) according to anota2seq are visualized together with non-regulated transcripts (grey).


Boxplots of 5 ´ UTR ( A ) , 3 ´ UTR ( B ) lengths for mRNAs whose abundance decreased ( ' DOWN ' , dark green ) or increased ( ' UP ' , light green ) in PABP - depleted cells as compared to cells expressing PABP . Solid horizonal middle lines in box plots denote the median ; lower and upper box limits correspond to first and third quartiles ; whiskers extend from box limits to the most extreme values . Wilcoxon - Mann - Whitney tests ( two - sided ) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background ( mRNAs whose abundance did not change in the absence of PABP relative to PABP - expressing cells ) .

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Boxplots of 5´UTR (A), 3´UTR (B) lengths for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP. Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values. Wilcoxon-Mann-Whitney tests (two-sided) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background (mRNAs whose abundance did not change in the absence of PABP relative to PABP-expressing cells).


( A ) Boxplots of mRNA half - lives ( hrs ) assessed by metabolic labeling of normal human umbilical vein endothelial cells for mRNAs whose abundance decreased ( ' DOWN ' , dark green ) or increased ( ' UP ' , light green ) in PABP - depleted cells as compared to cells expressing PABP comparing ( Tiana et al . , 2020 ) . Solid horizonal middle lines in box plots denote the median ; lower and upper box limits correspond to first and third quartiles ; whiskers extend from box limits to the most extreme values but not further than 1 . 5 × the inter - quartile range ( IQR ) . ( B - H ) PABPDHFR cells were cultured in the presence or absence of TMP for 12 hours . The stabilities of select mRNAs was assessed by using actinomycin D ( 5 μg / ml ) for the indicated amounts of time . Total RNA was isolated , reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR . mRNA decay rates were normalized to an in vitro synthesized spike - in RNA with the zero time point set at 1 . Error bars represent the SEM of three independent experiments . First order exponential decay trend lines were generated by non - linear regression analysis .

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(A) Boxplots of mRNA half-lives (hrs) assessed by metabolic labeling of normal human umbilical vein endothelial cells for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP comparing (Tiana et al., 2020). Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values but not further than 1.5× the inter-quartile range (IQR). (B- H) PABPDHFR cells were cultured in the presence or absence of TMP for 12 hours. The stabilities of select mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.


( B ) PABPDHFR cells were transfected with plasmids encoding RL - 6xB or RL - 6xBMUT . 24 hours after transfections , the cells were split and cultured for an additional 12 hours in the presence or absence of TMP . The stabilities of reporter mRNAs was assessed by using actinomycin D ( 5 μg / ml ) for the indicated amounts of time . Total RNA was isolated , reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR . mRNA decay rates were normalized to an in vitro synthesized spike - in RNA with the zero time point set at 1 . Error bars represent the SEM of three independent experiments . First order exponential decay trend lines were generated by non - linear regression analysis .

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(B) PABPDHFR cells were transfected with plasmids encoding RL-6xB or RL-6xBMUT. 24 hours after transfections, the cells were split and cultured for an additional 12 hours in the presence or absence of TMP. The stabilities of reporter mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.


( A ) Western blot analysis PABPDHFR cells depleted of LSM1 , DCP2 or XRN1 by siRNA - mediated knockdown . siGFP represents a negative control .

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(A) Western blot analysis PABPDHFR cells depleted of LSM1, DCP2 or XRN1 by siRNA-mediated knockdown. siGFP represents a negative control.


( B ) Phase - contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP ( control ) , LSM1 , DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours . Total numbers of viable cells , denoted in the upper right hand corner of images , were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP , along with the SEM . Scale bar , 100 μm .

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(B) Phase-contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP (control), LSM1, DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours. Total numbers of viable cells, denoted in the upper right hand corner of images, were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP, along with the SEM. Scale bar, 100 μm.


( A ) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA - mediated knockdown . Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP .

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(A) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA-mediated knockdown. Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP.


( C and D ) Poly ( A ) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in ( B ) was determined by extension poly ( A ) tail ( ePAT ) . PCR products were run on high - resolution agarose gels . ' A12 ' represents the size of ePAT RT - PCR amplicons derived from a mRNA with a fixed ( A12 ) - tail .

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(C and D) Poly(A) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in (B) was determined by extension poly(A) tail (ePAT). PCR products were run on high-resolution agarose gels. 'A12' represents the size of ePAT RT-PCR amplicons derived from a mRNA with a fixed (A12)-tail.


C Similar to CSR - 1 , the majority of small RNAs interacting with CSR - 2 in vivo have 5 ′ PPP ends . The RNAs in the immunopurified Argonaute complexes were treated with or without Vaccinia virus capping enzyme followed by an alkaline phosphatase , and finally labeled with [ γ - 32P ] ATP and a polynucleotide kinase . RNA products were resolved by 15 % sequencing gels .

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C Similar to CSR-1, the majority of small RNAs interacting with CSR-2 in vivo have 5′PPP ends. The RNAs in the immunopurified Argonaute complexes were treated with or without Vaccinia virus capping enzyme followed by an alkaline phosphatase, and finally labeled with [γ-32P]ATP and a polynucleotide kinase. RNA products were resolved by 15% sequencing gels.


G Three csr - 1 hypomorphic mutants showed a diminished RNAi response to exo - dsRNA . The WT , the csr - 1 ( fj150 ) mutant , and several strains were injected with dsRNA ( 1 . 25 µg / µL ) targeting the maternal gene pos - 1 , essential for embryogenesis . The incidence of lethal embryos in the F1 progeny was examined . The number of F1 progeny counted is indicated by n . Error bars represent 95 % confidence intervals ( CIs ) .

O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Three csr-1 hypomorphic mutants showed a diminished RNAi response to exo-dsRNA. The WT, the csr-1(fj150) mutant, and several strains were injected with dsRNA (1.25 µg/µL) targeting the maternal gene pos-1, essential for embryogenesis. The incidence of lethal embryos in the F1 progeny was examined. The number of F1 progeny counted is indicated by n. Error bars represent 95% confidence intervals (CIs).


A Nuclei of the WT strain were stained by the immuno - FISH method using an anti - histone H3K9me2 antibody and a probe against the right region of the X chromosome . B Shapes of H3K9me2 - positive chromosomes in male testes of the WT and the csr - 2 mutant . The relative frequency of extended chromosomes in H3K9me2 - positive chromosomes was examined . H3K9me2 - positive chromosomes ≥ 2 . 5 µm were interpreted to be in an extended state . The number of nuclei counted is indicated by n . Error bars represent 95 % CIs .

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A Nuclei of the WT strain were stained by the immuno-FISH method using an anti-histone H3K9me2 antibody and a probe against the right region of the X chromosome. B Shapes of H3K9me2-positive chromosomes in male testes of the WT and the csr-2 mutant. The relative frequency of extended chromosomes in H3K9me2-positive chromosomes was examined. H3K9me2-positive chromosomes ≥ 2.5 µm were interpreted to be in an extended state. The number of nuclei counted is indicated by n. Error bars represent 95% CIs.


E Immunofluorescence images of H3K9me2 and SYP - 1 in male nuclei of the WT and the Argonaute mutants . High - resolution fluorescence images , 3D reconstruction images ( 3D CG ) , and maximum intensity projection ( MIP ) images of segmented H3K9me2 signals are presented . In the male nuclei of the csr - 1 mutant and the csr - 2 ; csr - 1 double mutant , some H3K9me2 segments showed abnormally large volumes ( narrow arrow ) and / or overlapped partially with SYP - 1 signals ( arrowhead ) . In the double mutant , DAPI signals were often detected in an aggregated state ( dashed line ) .

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E Immunofluorescence images of H3K9me2 and SYP-1 in male nuclei of the WT and the Argonaute mutants. High-resolution fluorescence images, 3D reconstruction images (3D CG), and maximum intensity projection (MIP) images of segmented H3K9me2 signals are presented. In the male nuclei of the csr-1 mutant and the csr-2; csr-1 double mutant, some H3K9me2 segments showed abnormally large volumes (narrow arrow) and/or overlapped partially with SYP-1 signals (arrowhead). In the double mutant, DAPI signals were often detected in an aggregated state (dashed line).


Immunofluorescence experiments were performed on the pachytene nuclei of X0 males . A The affinities of the recombinant chromodomain peptides of CEC - 5 and CEC - 8 for the N - terminus of histone H3 were assayed by in vitro pull - down experiments . CEC - 4 served as a control .

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Immunofluorescence experiments were performed on the pachytene nuclei of X0 males. A The affinities of the recombinant chromodomain peptides of CEC-5 and CEC-8 for the N-terminus of histone H3 were assayed by in vitro pull-down experiments. CEC-4 served as a control.


Immunofluorescence experiments were performed on the pachytene nuclei of X0 males . C H3K9me2 signals at the pachytene region in the males of cec mutants and cohesin mutants . Worms were cultured at 20 ° C , except for the smc - 1 ( e870 ) mutant that was cultured at 23 ° C . The intensity of the H3K9me2 signal in the him - 8 mutant male was similar to that in the WT male .

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Immunofluorescence experiments were performed on the pachytene nuclei of X0 males. C H3K9me2 signals at the pachytene region in the males of cec mutants and cohesin mutants. Worms were cultured at 20°C, except for the smc-1(e870) mutant that was cultured at 23°C. The intensity of the H3K9me2 signal in the him-8 mutant male was similar to that in the WT male.


Immunofluorescence experiments were performed on the pachytene nuclei of X0 males . D The nuclear intensities of H3K9me2 signals were measured from wide - field ( WF ) images . The image quantification was performed E The nuclear intensities of H3K9me2 and H3K9me3 signals were measured from deconvolved ( DV ) images . The H3K9me2 signals in the major strains were double - checked using deconvolution microscopy .

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Immunofluorescence experiments were performed on the pachytene nuclei of X0 males. D The nuclear intensities of H3K9me2 signals were measured from wide-field (WF) images. The image quantification was performed E The nuclear intensities of H3K9me2 and H3K9me3 signals were measured from deconvolved (DV) images. The H3K9me2 signals in the major strains were double-checked using deconvolution microscopy.


A The meiosis - specific kleisin ( s ) COH - 3 / 4 was detected on paired chromosomes and the heterochromatinized unpaired X chromosome . The testes of WT males were immunostained with anti - COH - 3 / 4 antibodies and an anti - H3K9me2 antibody .

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A The meiosis-specific kleisin(s) COH-3/4 was detected on paired chromosomes and the heterochromatinized unpaired X chromosome. The testes of WT males were immunostained with anti-COH-3/4 antibodies and an anti-H3K9me2 antibody.


B Immunofluorescence images of COH - 3 / 4 and SYP - 1 on paired chromosomes in the gonads of WT hermaphrodites . COH - 3 / 4 kleisin ( s ) was detected laterally to the string - like signal of SYP - 1 , which corresponds to the central region of the SC .

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B Immunofluorescence images of COH-3/4 and SYP-1 on paired chromosomes in the gonads of WT hermaphrodites. COH-3/4 kleisin(s) was detected laterally to the string-like signal of SYP-1, which corresponds to the central region of the SC.


C SC formation in the WT and multiple mutants were analyzed by immunostaining against SYP - 1 and COH - 3 / 4 . The SC formed string - like structures in the csr - 2 ; csr - 1 double mutant and the ego - 1 mutant , but the superstructure of the SC was often abnormally branched .

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C SC formation in the WT and multiple mutants were analyzed by immunostaining against SYP-1 and COH-3/4. The SC formed string-like structures in the csr-2; csr-1 double mutant and the ego-1 mutant, but the superstructure of the SC was often abnormally branched.


A Multicolor FISH for the 5S rRNA region ( rDNA , green ) of autosome V and the right region of the X chromosome ( LGX - R , magenta ) in nuclei of the WT strain , coh - 3 / 4 double mutant , csr - 1 mutants , csr - 2 ; csr - 1 double mutant , and ego - 1 mutant . Arrows indicate occasional colocalizations of the 5S rDNA and X - R loci detected in the mutant strains .

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A Multicolor FISH for the 5S rRNA region (rDNA, green) of autosome V and the right region of the X chromosome (LGX-R, magenta) in nuclei of the WT strain, coh-3/4 double mutant, csr-1 mutants, csr-2; csr-1 double mutant, and ego-1 mutant. Arrows indicate occasional colocalizations of the 5S rDNA and X-R loci detected in the mutant strains.


D Simultaneous staining of the 5S rRNA region ( rDNA , green ) by FISH , and SYP - 1 ( magenta ) by immunofluorescence .

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D Simultaneous staining of the 5S rRNA region (rDNA, green) by FISH, and SYP-1 (magenta) by immunofluorescence.


E csr - 1 ( fj67 ) and csr - 1 ( fj126 ) in - frame mutations reduced the abundance of CSR - 1 at the chromosomal regions in pachytene nuclei . The WT strain and the csr - 1 mutants were stained by anti - CSR - 1 antibodies .

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E csr-1(fj67) and csr-1(fj126) in-frame mutations reduced the abundance of CSR-1 at the chromosomal regions in pachytene nuclei. The WT strain and the csr-1 mutants were stained by anti-CSR-1 antibodies.


A , B ChIP - seq data for CSR - 1 , SMC - 1 , COH - 3 / 4 , and histone H3K9me2 were compared with sub - populations in CSR - 1 - interacting small RNAs . Adult worms were used for experiments . ChIP signals on autosome III ( A ) and the X chromosome ( B ) from the WT hermaphrodites were visualized by aggregation plots with a bin size of 20 kbp . H3K9me2 ChIP signals derived from the him - 8 mutant population rich in X0 males are shown in the lower part . The common ChIP regions computed from the technical duplicates were used for panels A , B , In the adult body , about half of the nuclei correspond to germ cells . Most H3K9me2 ChIP signals from the WT hermaphrodites were likely to originate in somatic cells because staining signals of H3K9me2 in adult hermaphrodites were detected abundantly in somatic cells and very weakly in the germline . Most meiosis - specific COH - 3 / 4 ChIP signals were thought to originate in the germline . For comparison , aggregation plots were also constructed from the following sub - populations of CSR - 1 - interacting small RNAs : small RNAs mapped in sense orientation to coding genes ( exons and introns ) , and repeat - derived small RNAs . The small RNA data merged from the technical duplicates were used for panels A and B . Densities of protein - coding exons , all non - simple repeats , and five major repeats on the reference genome are attached . The CeRep55 sequences were partially degenerate , and no clear ChIP signal for CSR - 1 was detected in CeRep55 cluster # 4 on the X chromosome in the ce10 reference genome .

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A, B ChIP-seq data for CSR-1, SMC-1, COH-3/4, and histone H3K9me2 were compared with sub-populations in CSR-1-interacting small RNAs. Adult worms were used for experiments. ChIP signals on autosome III (A) and the X chromosome (B) from the WT hermaphrodites were visualized by aggregation plots with a bin size of 20 kbp. H3K9me2 ChIP signals derived from the him-8 mutant population rich in X0 males are shown in the lower part. The common ChIP regions computed from the technical duplicates were used for panels A, B, In the adult body, about half of the nuclei correspond to germ cells. Most H3K9me2 ChIP signals from the WT hermaphrodites were likely to originate in somatic cells because staining signals of H3K9me2 in adult hermaphrodites were detected abundantly in somatic cells and very weakly in the germline. Most meiosis-specific COH-3/4 ChIP signals were thought to originate in the germline. For comparison, aggregation plots were also constructed from the following sub-populations of CSR-1-interacting small RNAs: small RNAs mapped in sense orientation to coding genes (exons and introns), and repeat-derived small RNAs. The small RNA data merged from the technical duplicates were used for panels A and B. Densities of protein-coding exons, all non-simple repeats, and five major repeats on the reference genome are attached. The CeRep55 sequences were partially degenerate, and no clear ChIP signal for CSR-1 was detected in CeRep55 cluster #4 on the X chromosome in the ce10 reference genome.


G Frequencies of small RNAs expressed from the major non - simple repeats were counted in the CSR - 1 and CSR - 2 complexes and the input cell lysates . The small RNA reads obtained by the experiments in were analyzed . The data from technical duplicates ( # 1 and # 2 ) are individually presented . The y - axis indicates reads per million ( rpm ) . Error bars represent 95 % CIs . The red border indicates the expected value of non - specific recovery , which was predicted with the recovery rate of miRNAs .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Frequencies of small RNAs expressed from the major non-simple repeats were counted in the CSR-1 and CSR-2 complexes and the input cell lysates. The small RNA reads obtained by the experiments in were analyzed. The data from technical duplicates (#1 and #2) are individually presented. The y-axis indicates reads per million (rpm). Error bars represent 95% CIs. The red border indicates the expected value of non-specific recovery, which was predicted with the recovery rate of miRNAs.


F The CeRep55 lncRNA signals partially colocalized with those of COH - 3 / 4 in hermaphrodites . Pachytene nuclei in the WT strain and the csr - 2 ; csr - 1 double mutant were stained .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O

F The CeRep55 lncRNA signals partially colocalized with those of COH-3/4 in hermaphrodites. Pachytene nuclei in the WT strain and the csr-2; csr-1 double mutant were stained.


H The CeRep55 lncRNA signal was also detected at a region of the unpaired X chromosome where the CEC - 5 and COH - 3 / 4 signals overlapped . The pachytene chromosome showing the strongest CEC - 5 enrichment was considered to be the unpaired X chromosome in males . The RNA - directed in situ hybridizations were performed with stringent conditions that detected only long RNAs .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O 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 The CeRep55 lncRNA signal was also detected at a region of the unpaired X chromosome where the CEC-5 and COH-3/4 signals overlapped. The pachytene chromosome showing the strongest CEC-5 enrichment was considered to be the unpaired X chromosome in males. The RNA-directed in situ hybridizations were performed with stringent conditions that detected only long RNAs.


Pachytene nuclei were analyzed . B Multicolor FISH using the 5S rDNA and LGX - R probes in the csr - 1 ( fj150 ) mutant and the csr - 1 ( fj150 ) ; CeRep55 _ X ( Q ) mutant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Pachytene nuclei were analyzed. B Multicolor FISH using the 5S rDNA and LGX-R probes in the csr-1(fj150) mutant and the csr-1(fj150); CeRep55_X(Q) mutant.


Pachytene nuclei were analyzed . D Nuclei of male testes carrying an unpaired X chromosome were immunostained with an antibody against histone H3K9me2 . H3K9me2 - positive chromosomes showing an extended state ( arrow ) were observed in the males of the CeRep55 _ X ( Q ) deletion mutant more frequently than in WT males .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Pachytene nuclei were analyzed. D Nuclei of male testes carrying an unpaired X chromosome were immunostained with an antibody against histone H3K9me2. H3K9me2-positive chromosomes showing an extended state (arrow) were observed in the males of the CeRep55_X(Q) deletion mutant more frequently than in WT males.


Pachytene nuclei were analyzed . E In male pachytene nuclei , the relative frequency of extended chromosomes in H3K9me2 - positive chromosomes was examined . H3K9me2 - positive chromosomes ≥ 2 . 5 µm were interpreted to be in an extended state . The number of nuclei counted is indicated by n . Error bars represent 95 % CIs . P - value ( two - sided ) was determined by two - proportion z - test .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Pachytene nuclei were analyzed. E In male pachytene nuclei, the relative frequency of extended chromosomes in H3K9me2-positive chromosomes was examined. H3K9me2-positive chromosomes ≥ 2.5 µm were interpreted to be in an extended state. The number of nuclei counted is indicated by n. Error bars represent 95% CIs. P-value (two-sided) was determined by two-proportion z-test.


( B ) Disintegration reaction ( 1 hour ) performed by RAG1 aa 216 - 1008 R848M / E649V , RAG2 aa 1 - 361 and full length human HMGB1 with Mg2 + or Mn2 + at the indicated temperatures . Denaturing gel displays the fluorophore - labeled DNA strand from the RAG STC before ( 16 nt band , lane 2 ) and after ( 37 nt ) the disintegration reaction . Lane 1 , fluorophore - labeled 37 nt DNA marker . Representative of 2 independent experiments .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Disintegration reaction (1 hour) performed by RAG1 aa 216-1008 R848M/E649V, RAG2 aa 1-361 and full length human HMGB1 with Mg2+ or Mn2+ at the indicated temperatures. Denaturing gel displays the fluorophore-labeled DNA strand from the RAG STC before (16 nt band, lane 2) and after (37 nt) the disintegration reaction. Lane 1, fluorophore-labeled 37 nt DNA marker. Representative of 2 independent experiments.


( D ) Results of in vivo plasmid - to - plasmid transposition assay using WT or R848M RAG1 and the indicated forms of RAG2 containing or lacking ( ∆ L ) the four amino acids at the tip of the 333 - 342 loop , performed as described in ( Zhang et al . , 2019 ) ; ( * * * * , p < 0 . 0001 ) . Three biological replicates for each condition except in the last two columns where data from 11 biological replicates are presented .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Results of in vivo plasmid-to-plasmid transposition assay using WT or R848M RAG1 and the indicated forms of RAG2 containing or lacking (∆L) the four amino acids at the tip of the 333-342 loop, performed as described in (Zhang et al., 2019); (****, p<0.0001). Three biological replicates for each condition except in the last two columns where data from 11 biological replicates are presented.


( E ) Results of in vivo plasmid - to - genome transposition assay using WT or R848M RAG1 and different forms of RAG2 as indicated . As previously demonstrated ( Zhang et al . , 2019 ) , R848 ( present in WT RAG1 ) and RAG2 acidic hinge residues 362 - 383 each potently suppress transposition . Substantial transposition is observed with the combination of RAG1 R848M and RAG2 1 - 361 , which is further increased by ∆ L ; ( * * * , p = 0 . 001 ) . Four biological replicates for each condition .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O 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) Results of in vivo plasmid-to-genome transposition assay using WT or R848M RAG1 and different forms of RAG2 as indicated. As previously demonstrated (Zhang et al., 2019), R848 (present in WT RAG1) and RAG2 acidic hinge residues 362-383 each potently suppress transposition. Substantial transposition is observed with the combination of RAG1 R848M and RAG2 1-361, which is further increased by ∆L; (***, p=0.001). Four biological replicates for each condition.


( F ) In vivo recombination activity in HEK293T cells of mouse wild - type RAG1 or RAG1 R848M with RAG2 1 - 361 or 1 - 361 ∆ L . ( p = 0 . 42 for wild - type RAG1 group , p = 0 . 41 for RAG1 R848M group ) . Three biological replicates for each condition .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

(F) In vivo recombination activity in HEK293T cells of mouse wild-type RAG1 or RAG1 R848M with RAG2 1-361 or 1-361 ∆L. (p=0.42 for wild-type RAG1 group, p=0.41 for RAG1 R848M group). Three biological replicates for each condition.


A . 3D rendering of the cerebrovascular tree in brain slices from male P90 Ctrl and Poly I : C MIA offspring stained with intravascular DiI ( red ) . Scale bar = 50 µm . B . DiI - positive blood vessels geometry analysis - quantification of blood vessel number , diameter ( mean and cumulative frequency distribution ) , length , number of branch segments and average branch length - of P90 male Ctrl and Poly I : C MIA offspring . Vessel number for Ctrl ( 20 . 93 ± 1 . 73 ) and Poly I : C ( 27 . 41 ± 1 . 99 ) MIA offspring , Mann - Whitney U test , * * p < . 01 . Mean diameter for Ctrl ( 5 . 58 ± 0 . 01 ) and Poly I : C ( 4 . 96 ± 0 . 01 ) MIA offspring , Mann - Whitney U test , * * * * p < . 0001 . Total length of vessel per image for Ctrl ( 6 . 12 ± 0 . 76 ) and Poly I : C ( 9 . 83 ± 1 . 48 ) MIA offspring , Mann - Whitney U test , * * p < . 01 . Number of branches for Ctrl ( 9 . 60 ± 0 . 54 ) and Poly I : C ( 15 . 80 ± 1 . 21 ) MIA offspring , Mann - Whitney U test , * p < . 05 . Average branch length for Ctrl ( 34 . 07 ± 1 . 35 ) and Poly I : C ( 33 . 47 ± 1 . 67 ) MIA offspring . C . 3D rendering of the cerebrovascular tree in brain slices from female P90 Ctrl and Poly I : C MIA offspring stained with intravascular DiI ( red ) . Scale bar = 50 µm . D . DiI - positive blood vessels geometry analysis - quantification of blood vessel number , diameter ( mean and cumulative frequency distribution ) , length , number of branch segments and average branch length - of P90 female Ctrl and Poly I : C MIA offspring . Vessel number for Ctrl ( 9 . 97 ± 1 . 18 ) and Poly I : C ( 8 . 61 ± 0 . 78 ) MIA offspring . Mean diameter for Ctrl ( 5 . 77 ± 0 . 03 ) and Poly I : C ( 6 . 16 ± 0 . 02 ) MIA offspring , Mann - Whitney U test , * * * * p < . 0001 . Total length of vessel per image for Ctrl ( 3 . 23 ± 0 . 50 ) and Poly I : C ( 2 . 97 ± 0 . 30 ) MIA offspring . Number of branches for Ctrl ( 10 . 50 ± 0 . 87 ) and Poly I : C ( 7 . 24 ± 0 . 44 ) MIA offspring . Average branch length for Ctrl ( 38 . 06 ± 2 . 17 ) and Poly I : C ( 46 . 93 ± 1 . 49 ) MIA offspring , Mann - Whitney U test , * * * p < . 001 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Ctrl females = white bars with purple border . Poly I : C females = purple bars with purple border . In Fig . 1 B and D black dotted lines represent the Ctrl while the blue and purple solid line represent the male Poly I : C and female Poly I : C offspring respectively . Numbers in bars indicate the number of animals ( N ) and images ( n ) . Bars represent mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 3D rendering of the cerebrovascular tree in brain slices from male P90 Ctrl and Poly I:C MIA offspring stained with intravascular DiI (red). Scale bar = 50 µm. B. DiI-positive blood vessels geometry analysis - quantification of blood vessel number, diameter (mean and cumulative frequency distribution), length, number of branch segments and average branch length - of P90 male Ctrl and Poly I:C MIA offspring. Vessel number for Ctrl (20.93 ± 1.73) and Poly I:C (27.41 ± 1.99) MIA offspring, Mann-Whitney U test, **p < .01. Mean diameter for Ctrl (5.58 ± 0.01) and Poly I:C (4.96 ± 0.01) MIA offspring, Mann-Whitney U test, ****p < .0001. Total length of vessel per image for Ctrl (6.12 ± 0.76) and Poly I:C (9.83 ± 1.48) MIA offspring, Mann-Whitney U test, **p < .01. Number of branches for Ctrl (9.60 ± 0.54) and Poly I:C (15.80 ± 1.21) MIA offspring, Mann-Whitney U test, *p < .05. Average branch length for Ctrl (34.07 ± 1.35) and Poly I:C (33.47 ± 1.67) MIA offspring. C. 3D rendering of the cerebrovascular tree in brain slices from female P90 Ctrl and Poly I:C MIA offspring stained with intravascular DiI (red). Scale bar = 50 µm. D. DiI-positive blood vessels geometry analysis - quantification of blood vessel number, diameter (mean and cumulative frequency distribution), length, number of branch segments and average branch length - of P90 female Ctrl and Poly I:C MIA offspring. Vessel number for Ctrl (9.97 ± 1.18) and Poly I:C (8.61 ± 0.78) MIA offspring. Mean diameter for Ctrl (5.77 ± 0.03) and Poly I:C (6.16 ± 0.02) MIA offspring, Mann-Whitney U test, ****p < .0001. Total length of vessel per image for Ctrl (3.23 ± 0.50) and Poly I:C (2.97 ± 0.30) MIA offspring. Number of branches for Ctrl (10.50 ± 0.87) and Poly I:C (7.24 ± 0.44) MIA offspring. Average branch length for Ctrl (38.06 ± 2.17) and Poly I:C (46.93 ± 1.49) MIA offspring, Mann-Whitney U test, ***p < .001. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. In Fig.1 B and D black dotted lines represent the Ctrl while the blue and purple solid line represent the male Poly I:C and female Poly I:C offspring respectively. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


E . Quantification of in - vivo brain permeability at P30 in male Ctrl ( 0 . 00 ± 0 . 00 ) and Poly I : C ( 3 . 68 ± 1 . 98 ) treated offspring , Mann - Whitney U test , p = 0 . 060 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and images ( n ) . Bars represent mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of in-vivo brain permeability at P30 in male Ctrl (0.00 ± 0.00) and Poly I:C (3.68 ± 1.98) treated offspring, Mann-Whitney U test, p=0.060. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


F . Quantification of Evans Blue ( EB ) analysis of in - vivo brain permeability at P90 ( top ) and representative pictures ( bottom ) of perfused brains from Male Ctrl ( left ; 0 . 00 ± 0 . 00 ) and Poly I : C ( right ; 20 . 32 ± 10 . 95 ) offspring . Mann - Whitney U test , * p < . 05 . In accompanying pseudocolor images - blue pixels are represented as a gradient of light - to - dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and images ( n ) . Bars represent mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Quantification of Evans Blue (EB) analysis of in-vivo brain permeability at P90 (top) and representative pictures (bottom) of perfused brains from Male Ctrl (left; 0.00 ± 0.00) and Poly I:C (right; 20.32 ± 10.95) offspring. Mann-Whitney U test, *p < .05. In accompanying pseudocolor images - blue pixels are represented as a gradient of light-to-dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


G . Quantitative analysis of the optical density of Claudin - 5 ( CLDN5 ) immunoreactive bands normalized to Calnexin ( CANX ) in cortices from P90 male Ctrl ( 0 . 99 ± 0 . 09 ) and Poly I : C ( 0 . 58 ± 0 . 05 ) offspring . Mann - Whitney U test , * * p < . 01 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and images ( n ) . Bars represent mean ± SEM .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Quantitative analysis of the optical density of Claudin-5 (CLDN5) immunoreactive bands normalized to Calnexin (CANX) in cortices from P90 male Ctrl (0.99 ± 0.09) and Poly I:C (0.58 ± 0.05) offspring. Mann-Whitney U test, **p < .01. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


H . Quantitative analysis of the optical density of ZO - 1 immunoreactive bands normalized by α - Tubulin ( αTUB ) in cortices from P90 male Ctrl ( 1 . 00 ± 0 . 04 ) and Poly I : C ( 0 . 75 ± 0 . 08 ) offspring . Student ' s t test , * p < . 05 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and images ( n ) . Bars represent mean ± SEM .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantitative analysis of the optical density of ZO-1 immunoreactive bands normalized by α-Tubulin (αTUB) in cortices from P90 male Ctrl (1.00 ± 0.04) and Poly I:C (0.75 ± 0.08) offspring. Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


I . Quantification of in - vivo brain permeability at P30 in female Ctrl ( 0 . 58 ± 0 . 17 ) and Poly I : C ( 0 . 31 ± 0 . 14 ) treated offspring . Data information : Ctrl females = white bars with purple border . Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) and images ( n ) . Bars represent mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. Quantification of in-vivo brain permeability at P30 in female Ctrl (0.58 ± 0.17) and Poly I:C (0.31 ± 0.14) treated offspring. Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


J . Quantification of Evans Blue ( EB ) analysis of in - vivo brain permeability at P90 ( top ) and representative pictures ( bottom ) of perfused brains from Female Ctrl ( left ; 0 . 70 ± 0 . 47 ) and Poly I : C ( right ; 0 . 10 ± 0 . 10 ) offspring . In accompanying pseudocolor images - blue pixels are represented as a gradient of light - to - dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green . Data information : Ctrl females = white bars with purple border . Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) and images ( n ) . Bars represent mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J. Quantification of Evans Blue (EB) analysis of in-vivo brain permeability at P90 (top) and representative pictures (bottom) of perfused brains from Female Ctrl (left; 0.70 ± 0.47) and Poly I:C (right; 0.10 ± 0.10) offspring. In accompanying pseudocolor images - blue pixels are represented as a gradient of light-to-dark blue depending on the intensity of the color in the original photo while the absence of blue tones is represented as green. Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


K . Quantitative analysis of the optical density of Claudin - 5 ( CLDN5 ) immunoreactive bands normalized by CANX in cortices from P90 female Ctrl ( 1 . 00 ± 0 . 16 ) and Poly I : C ( 1 . 39 ± 0 . 13 ) offspring . Data information : Ctrl females = white bars with purple border . Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) and images ( n ) . Bars represent mean ± SEM .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. Quantitative analysis of the optical density of Claudin-5 (CLDN5) immunoreactive bands normalized by CANX in cortices from P90 female Ctrl (1.00 ± 0.16) and Poly I:C (1.39 ± 0.13) offspring. Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.


L . Quantitative analysis of the optical density of ZO - 1 immunoreactive bands normalized by αTUB in cortices from P90 female Ctrl ( 1 . 00 ± 0 . 11 ) and Poly I : C ( 1 . 19 ± 0 . 19 ) offspring . Data information : Ctrl females = white bars with purple border . Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) and images ( n ) . Bars represent mean ± SEM .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L. Quantitative analysis of the optical density of ZO-1 immunoreactive bands normalized by αTUB in cortices from P90 female Ctrl (1.00 ± 0.11) and Poly I:C (1.19 ± 0.19) offspring. Data information: Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and images (n). Bars represent mean ± SEM.