Mechanisms of substrate selectivity of the nuclear RNA exosome complex.
Final Report Abstract
RNA is the mediator between the DNA-encoded genetic information, which is stored in the nucleus in the form of chromatin, and the protein biosynthetic machinery present in the cytoplasm. A significant portion of eukaryotic genomes is transcribed into RNA, but not all of these transcripts are functional. In healthy cells, RNA produced by pervasive transcription of the genome is rapidly degraded because it is targeted by nuclear RNA surveillance pathways, which prevent the accumulation of dysfunctional RNA species, and their escape into the cytoplasm. The nuclear RNA exosome, a conserved ribonucleolytic multi-protein complex, plays a central role in RNA quality control. It targets cryptic transcripts, non-coding RNAs, and aberrant transcripts for destruction, and intersects with pathways that ensure that only correctly processed transcripts are released from chromatin for export from the nucleus. The ability of the exosome to faithfully identify its substrates is fundamental to its regulatory functions in the cell and essential for cellular well-being. In this project, we sought to identify additional factors determining the substrate selectivity of the exosome complex and regulators integrating the activity of different nuclear surveillance pathways. To tackle these questions, we employed a comparative proteomics approach based on the RNA interactome capture technique in the unicellular eukaryotic model organism fission yeast. This method allowed us to identify RNA-binding proteins that preferentially associate with RNAs targeted by nuclear surveillance pathways. Our aim was to pinpoint proteins that either guide RNA substrates towards the exosome or play a role in the retention of nuclear RNA. Among the proteins we identified was the zinc-finger protein Mub1. Further work demonstrated the role of Mub1 in maintaining low levels of a specific subset of exosome RNA substrates, particularly stress-responsive genes. Another factor that we could newly link to the turnover of exosome substrates is a chromatin remodeller of the SNF2 family, which is involved in the silencing of retrotransposon-derived gene products. Finally, we found evidence that nuclear retention of exosome substrates is linked to inefficient release of the 3′-end processing complexes after polyadenylation. Using a combination of comparative protein interactomics, chromatin interaction studies and fluorescence microscopy, we identified the DEAD-box ATPase Dbp2 as the active component of the RNP assembly checkpoint at the 3’-end of genes that mediates this release.
Publications
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From parts lists to functional significance—RNA–protein interactions in gene regulation. WIREs RNA, 11(3).
Kilchert, Cornelia; Sträßer, Katja; Kunetsky, Vladislav & Änkö, Minna‐Liisa
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RNA Exosomes and Their Cofactors. Methods in Molecular Biology, 215-235. Springer New York.
Kilchert, Cornelia
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System-wide analyses of the fission yeast poly(A) + RNA interactome reveal insights into organization and function of RNA–protein complexes. Genome Research, 30(7), 1012-1026.
Kilchert, Cornelia; Kecman, Tea; Priest, Emily; Hester, Svenja; Aydin, Ebru; Kus, Krzysztof; Rossbach, Oliver; Castello, Alfredo; Mohammed, Shabaz & Vasiljeva, Lidia
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RNA-binding protein Mub1 and the nuclear RNA exosome act to fine-tune environmental stress response. Life Science Alliance, 5(2), e202101111.
Birot, Adrien; Kus, Krzysztof; Priest, Emily; Al Alwash, Ahmad; Castello, Alfredo; Mohammed, Shabaz; Vasiljeva, Lidia & Kilchert, Cornelia
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RNA–Protein Interactomics. Protein Interactions, 271-291. Wiley.
Kilchert, Cornelia
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A census of RNA-dependent proteins in yeast.
Wäber, Nadine Bianca; Seidler, Johanna; Thelen, Fabienne; Timm, Thomas; Lochnit, Günter; Sträßer, Katja & Kilchert, Cornelia
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DEAD-box ATPase Dbp2 is the key enzyme in an mRNP assembly checkpoint at the 3’-end of genes and involved in the recycling of cleavage factors. Nature Communications, 15(1).
Aydin, Ebru; Schreiner, Silke; Böhme, Jacqueline; Keil, Birte; Weber, Jan; Žunar, Bojan; Glatter, Timo & Kilchert, Cornelia
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DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts. Nature Communications, 16(1).
Kuś, Krzysztof; Carrique, Loic; Kecman, Tea; Fournier, Marjorie; Hassanein, Sarah Sayed; Aydin, Ebru; Kilchert, Cornelia; Grimes, Jonathan M. & Vasiljeva, Lidia
