La protéine Scc2 (Sister Chromatine Cohesion) de la famille des SMC (Structure Maintenance of Chromosome) favorise la biogenèse des ARNnc et la fidélité traductionnelle chez Saccharomyces cerevisae

Le complexe Scc2-Scc4 est essentiel pour l’association du complexe cohésine sur l’ADN. Les proteines Cohésine génèrent la cohésion entre les chromatides sœurs, ce qui est essentiel pour la ségrégation des chromosomes. Scc2 (également connu sous le nom NIPBL) est muté chez les patients atteints du syndrome de Cornelia de Lange, une maladie multi-organique caractérisée par des anomalies du développement du visage, de la developpement mental cardiaque et du tractus gastro-intestinal. Comment les mutations localisées au niveau du gène codant pour la proteine Scc2 conduisent à des anomalies du développement chez les patients n’a pas encore été élucidé. Une des hypothèses est que la liaison de Scc2 / cohésine à différentes régions du génome a une incidence sur la transcription. Chez la levure de bière, il a été montre que Scc2 se lie aux genes transcrits par l'ARN Pol III (les ARNt et spliceosomals) , ainsi qu‘aux gènes transcrits par l'ARN Pol II codant pour des petits ARN nucléolaires et nucléaires (snARN et snoARNs ) et des gènes de protéines ribosomiques. Nous rapportons ici que Scc2 est important pour l'expression de ces gènes. Scc2 et le régulateur transcriptionnel Paf1 collaborent pour promouvoir la production de Box H / ACA snoARNs qui guident la pseudouridylation des ARN y compris l'ARN ribosomal. Une mutation de Scc2 a été associée à des défauts dans la production d'ARN ribosomal, la biogenèse des ribosomes, et del’épissage. Alors que le mutant Scc2 n'a pas de défaut général de la synthèse protéique, il montre un déphasage accrue et une réduction de l’utilisation du site interne d'entrée ribosomale (IRES)/ coiffe-indépendante. Ces résultats suggèrent que Scc2 favorise normalement un programme d'expression génétique qui prend en charge la fidélité de la traduction. Nous émettons l'hypothèse que le dysfonctionnement de traduction peut contribuer au syndrome de Cornelia de Lange, qui est causé par des mutations dans Scc2.The Scc2-Scc4 complex is essential for loading the cohesin complex onto DNA. Cohesin generates cohesion between sister chromatids, which is critical for chromosome segregation. Scc2 (also known as NIPBL) is mutated in patients with Cornelia de Lange syndrome, a multi-organ disease characterized by developmental defects in head, limb, cognition, heart, and the gastrointestinal tract. How mutations in Scc2 lead to developmental defects in patients is yet to be elucidated. One hypothesis is that the binding of Scc2/cohesin to different regions of the genome will affect transcription. In budding yeast, Scc2 has been shown to bind to RNA Pol III transcribed genes (tRNAs, and spliceosomal), as well as RNA Pol II-transcribed genes encoding small nuclear and nucleolar RNAs (snRNAs and snoRNAs) and ribosomal protein genes. Here, we report that Scc2 is important for gene expression. Scc2 and the transcriptional regulator Paf1 collaborate to promote the production of Box H/ACA snoRNAs which guide pseudouridylation of RNAs including ribosomal RNA. Mutation of Scc2 was associated with defects in the production of ribosomal RNA, ribosome biogenesis, and splicing. While the scc2 mutant does not have a general defect in protein synthesis, it shows increased frameshifting and reduced internal ribosomal entry site (IRES) usage/cap-independent translation. These findings suggest Scc2 normally promotes a gene expression program that supports translational fidelity. We hypothesize that translational dysfunction may contribute to the human disorder Cornelia de Lange syndrome, which is caused by mutations in Scc2

La protéine Scc2 (Sister Chromatine Cohesion) de la famille des SMC (Structure Maintenance of Chromosome) favorise la biogenèse des ARNnc et la fidélité traductionnelle chez Saccharomyces cerevisae

Le complexe Scc2-Scc4 est essentiel pour l’association du complexe cohésine sur l’ADN. Les proteines Cohésine génèrent la cohésion entre les chromatides sœurs, ce qui est essentiel pour la ségrégation des chromosomes. Scc2 (également connu sous le nom NIPBL) est muté chez les patients atteints du syndrome de Cornelia de Lange, une maladie multi-organique caractérisée par des anomalies du développement du visage, de la developpement mental cardiaque et du tractus gastro-intestinal. Comment les mutations localisées au niveau du gène codant pour la proteine Scc2 conduisent à des anomalies du développement chez les patients n’a pas encore été élucidé. Une des hypothèses est que la liaison de Scc2 / cohésine à différentes régions du génome a une incidence sur la transcription. Chez la levure de bière, il a été montre que Scc2 se lie aux genes transcrits par l'ARN Pol III (les ARNt et spliceosomals) , ainsi qu‘aux gènes transcrits par l'ARN Pol II codant pour des petits ARN nucléolaires et nucléaires (snARN et snoARNs ) et des gènes de protéines ribosomiques. Nous rapportons ici que Scc2 est important pour l'expression de ces gènes. Scc2 et le régulateur transcriptionnel Paf1 collaborent pour promouvoir la production de Box H / ACA snoARNs qui guident la pseudouridylation des ARN y compris l'ARN ribosomal. Une mutation de Scc2 a été associée à des défauts dans la production d'ARN ribosomal, la biogenèse des ribosomes, et del’épissage. Alors que le mutant Scc2 n'a pas de défaut général de la synthèse protéique, il montre un déphasage accrue et une réduction de l’utilisation du site interne d'entrée ribosomale (IRES)/ coiffe-indépendante. Ces résultats suggèrent que Scc2 favorise normalement un programme d'expression génétique qui prend en charge la fidélité de la traduction. Nous émettons l'hypothèse que le dysfonctionnement de traduction peut contribuer au syndrome de Cornelia de Lange, qui est causé par des mutations dans Scc2.The Scc2-Scc4 complex is essential for loading the cohesin complex onto DNA. Cohesin generates cohesion between sister chromatids, which is critical for chromosome segregation. Scc2 (also known as NIPBL) is mutated in patients with Cornelia de Lange syndrome, a multi-organ disease characterized by developmental defects in head, limb, cognition, heart, and the gastrointestinal tract. How mutations in Scc2 lead to developmental defects in patients is yet to be elucidated. One hypothesis is that the binding of Scc2/cohesin to different regions of the genome will affect transcription. In budding yeast, Scc2 has been shown to bind to RNA Pol III transcribed genes (tRNAs, and spliceosomal), as well as RNA Pol II-transcribed genes encoding small nuclear and nucleolar RNAs (snRNAs and snoRNAs) and ribosomal protein genes. Here, we report that Scc2 is important for gene expression. Scc2 and the transcriptional regulator Paf1 collaborate to promote the production of Box H/ACA snoRNAs which guide pseudouridylation of RNAs including ribosomal RNA. Mutation of Scc2 was associated with defects in the production of ribosomal RNA, ribosome biogenesis, and splicing. While the scc2 mutant does not have a general defect in protein synthesis, it shows increased frameshifting and reduced internal ribosomal entry site (IRES) usage/cap-independent translation. These findings suggest Scc2 normally promotes a gene expression program that supports translational fidelity. We hypothesize that translational dysfunction may contribute to the human disorder Cornelia de Lange syndrome, which is caused by mutations in Scc2

The SMC Loader Scc2 Promotes ncRNA Biogenesis and Translational Fidelity

International audienceThe Scc2-Scc4 complex is essential for loading the cohesin complex onto DNA. Cohesin has important roles in chromosome segregation, DSB repair, and chromosome condensation. Here we report that Scc2 is important for gene expression in budding yeast. Scc2 and the transcriptional regulator Paf1 collaborate to promote the production of Box H/ACA snoR-NAs which guide pseudouridylation of RNAs including ribosomal RNA. Mutation of SCC2 was associated with defects in the production of ribosomal RNA, ribosome assembly, and splicing. While the scc2 mutant does not have a general defect in protein synthesis, it shows increased frameshifting and reduced cap-independent translation. These findings suggest Scc2 normally promotes a gene expression program that supports translational fidelity. We hypothesize that translational dysfunction may contribute to the human disorder Cornelia de Lange syndrome, which is caused by mutations in NIPBL, the human ortholog of SCC2

Ribosome protein distribution is compromised in the <i>scc2-4</i> mutant.

<p>Protein components of the small (Rps2) and large (Rpl25) ribosomal subunits were tagged with GFP and imaged. Representative images are shown for WT and mutant strains (A and D). Using flow cytometry, the peak GFP intensity was quantified for independent biological replicates (B and E). At least 10000 cells were examined per replicate. A KS-distance was calculated from cumulative distribution frequency curves of the fluorescence which allows us to determine statistical significance (C and F) (p<0.0001).</p

Pseudouridylation of rRNA is reduced in the <i>scc2-4</i> mutant.

<p>(A) The Box H/ACA snoRNAs that guide sequence-specific pseudouridylation are down-regulated in the <i>scc2-4</i> mutant. The error bars represent the standard deviation from triplicate samples and the asterisks indicate statistical significance at an adj p<0.05. (B) Reverse transcription with primers corresponding to residues Ψ 1003 and Ψ1123 for <i>SNR5</i> and residues Ψ2258 and Ψ2260 for <i>SNR191</i> was performed. Samples were treated with or without CMC, exposed to pH 10.4 for 4 hrs and reverse transcribed. The rectangles indicate the bands quantified to the right. Pseudouridylation assays were performed at least two times; a representative experiment is shown.</p

Ribosomal RNA production is compromised in the <i>scc2-4</i> mutant.

<p>(A) The RNA synthesis rate was examined using ³H-uridine incorporation in WT and <i>scc2-4</i> mutant strains. Strains were grown in triplicate in SD-ura medium with minimal uracil to an approximate OD₆₀₀ of 0.3. ³H-uridine was added for 5 minutes and incorporation was quantified, averaged, and expressed relative to WT. Standard error bars are indicated for n = 4. The difference between WT and mutant was significant at p<0.0001. (B). WT and <i>scc2-4</i> mutant strains were grown in SD-met at 30°C into mid-log phase. Equal numbers of cells were pulse-labeled with ³H-methylmethionine for 5 min, followed by RNA extraction and electrophoresis of the RNA on a formaldehyde gel. The gel was photographed following ethidium bromide staining (bottom). RNA was transferred to a membrane and detected by autoradiography (top). 25S and 18S rRNA were cut from the membrane, quantified with a scintillation counter, and the average is expressed relative to WT (p<0.05). p-values were calculated using a student two-tailed t-test. Standard error is indicated for n = 4.</p

Scc2 and Paf1 recruitment at snoDNAs is co-dependent.

<p>(A) WT, <i>scc2-4</i>, and <i>paf1Δ</i> mutant strains were cultured in YPD medium to mid-log phase (~ OD₆₀₀ = 0.5–0.8). Strains were crosslinked and chromatin extracted for ChIP. ChIP/qPCR analysis was carried out for Scc2-Myc and Scc2^E534K-Myc and Scc2-Myc <i>paf1Δ</i>. ChIP performed without the addition of primary antibody served as a negative control. ChIP experiments were performed at least three times. p-values were calculated using a student t-test. Standard error bars are indicated for n = 3. Values different from the WT are indicated by an asterisk (p<0.05). qPCR analysis shows reduced enrichment of indicated snoDNAs in mutant relative to WT in Paf1 ChIP (α-HA antibody, 12CA5, Roche). (B) Western blot analysis shows that the protein level of Paf1 is reduced in the <i>scc2-4</i> mutant. Scc2 levels also appear reduced in the <i>paf1Δ</i> strain. Pgk1 served as the loading control. (C) Western blot analysis shows that the protein level of Ctr9 is reduced in the <i>scc2-4</i> mutant. (D) qPCR analysis shows reduced enrichment of snoDNAs in the Scc2-Myc ChIP in the <i>paf1Δ</i> mutant strain relative to WT (α-Myc antibody, 9B11, Cell signaling). (E) A cartoon model for the co-recruitment of Scc2 and Paf1 at snoDNAs is shown. The Tbf1 transcription factor is shown in purple, RNA Pol II in yellow, and Scc2 and Paf1c in blue and green, respectively.</p

The <i>scc2-4</i> mutation compromises the association with genic regions at 30°C.

<p>WT and <i>scc2-4</i> mutant strains were cultured to mid-log phase (~OD₆₀₀ = 0.5–0.8) in YPD medium. Strains were crosslinked and chromatin extracted for ChIP. Metagene analysis was carried out for Scc2-Myc and Scc2^E534K-Myc for ChIP seq data. Two biological replicates for each are shown. (A) The <i>scc2-4</i> mutation does not affect the association of Scc2 with centromere regions. (B) The mutation reduces the association with ribosomal protein genes (132) (C) rDNA (D) snoDNAs (77) and (E) tDNAs (275).</p

Hundreds of genes are differentially expressed in the <i>scc2-4</i> mutant compared to WT at 30°C in YPD.

<p>(A) Gene expression values (mean of triplicate samples) are shown in MA plot. Differentially expressed genes (adjusted p-value <0.05) were colored in orange, and then colored red or green after a minimum fold change cut-off of 1.5 was applied. There are 2644 genes differentially expressed genes in the <i>scc2-4</i> mutant, with 1285 up-regulated and 1359 down-regulated. Applying a fold-change cutoff of 1.5 (corresponding to an absolute log₂ value of 0.6) returns a more reasonable number of genes for GO analysis and general comparison. (B) GO term analysis for the up-regulated genes shows enrichment for genes important for RNA processing/metabolism and ribosome biogenesis. (C) GO term analysis for down-regulated genes shows enrichment for genes important for biological processes such as oxidative phosphorylation, electron transport chain, and carbohydrate metabolic processes.</p