Biology subjects

Leger-Silvestre, I.

Publications and source records attributed to Leger-Silvestre, I..

3 recordsLinked to original sources

bioRxiv2025

New Insights into nuclear import and nucleolar localization of yeast RNA exosome subunits

The RNA exosome is a multiprotein complex essential for RNA maturation and degradation. In budding yeast, a nine-subunit protein complex (Exo9) associated with Rrp44 forms Exo10 in the cytoplasm and, in complex with Rrp6, Exo11 in the nucleus. Depending on its subcellular localization, the exosome interacts with different cofactors and RNA substrates. In the cytoplasm, Exo10 associates with the SKI complex via Ski7, while in the nucleus, Exo11 interacts with the TRAMP complex. Within the nucleolus, the exosome participates in ribosomal RNA (rRNA) processing, facilitated by Mtr4-dependent adaptors Utp18 and Nop53. In this manuscript, we have performed a comprehensive study that addresses the targeting mechanism and precise subcellular localization of all members of the Exo11 complex. We observed a high concentration of all Exo11 subunits in the nucleolus and identified the importins Srp1 () and Kap95 ({beta}) as responsible for the nuclear import of Exo9 subunits. Notably, Exo9 subunits localization was not significantly disrupted in the simultaneous absence of NLS-containing subunits Rrp6 and Rrp44, suggesting redundant nuclear import pathways for Exo9. Additionally, we show evidence that Ski7 may play a role in the Exo9 retention in the cytoplasm. To explore the exosome sub-nucleolar localization, we compared Rrp43 with nuclear exosome cofactors and show that it is enriched in the same nucleolar region as Mtr4 and Nop53. In conclusion, our findings provide a detailed characterization of Exo11 distribution, highlight the primary nuclear import mechanisms for Exo9, and reveal the specific localization of the exosome within the granular component (GC) of the yeast nucleolus, suggesting a spatial regulation of the RNA processing pathway. Highlights- Comprehensive study of the localization of all exosome subunits in yeast. - Identification of karyopherins involved in exosome nuclear import. - Ski7 dependent cytoplasmic retention of exosome. - Yeast sub-nucleolar organization of exosome and its cofactors.

molecular biology↗

bioRxiv2018

Genetic analysis of RNA polymerase I unveils new role of the Rpa12 subunit during transcription

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6{Delta} and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that wild-type RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.\n\nAuthor summaryThe nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.

molecular biology↗

bioRxiv2018

Nucleolar stress triggers the irreversible cell cycle slow down leading to cell death during replicative aging in Saccharomyces cerevisiae

The accumulation of Extrachromosomal rDNA Circles (ERCs) and their asymmetric segregation upon division have been hypothesized to be responsible for replicative senescence in mother yeasts and rejuvenation in daughter cells. However, it remains unclear by which molecular mechanisms ERCs would trigger the irreversible cell cycle slow-down leading to cell death. We show that ERCs accumulation is concomitant with a nucleolar stress, characterized by a massive accumulation of pre-rRNAs in the nucleolus, leading to a loss of nucleus-to-cytoplasm ratio, decreased growth rate and cell-cycle slow-down. This nucleolar stress, observed in old mothers, is not inherited by rejuvenated daughters. Unlike WT, in the long-lived mutant fob1{triangleup}, a majority of cells is devoid of nucleolar stress and does not experience replicative senescence before death. Our study provides a unique framework to order the successive steps that govern the transition to replicative senescence and highlights the causal role of nucleolar stress in cellular aging.

cell biology↗