Biology subjects

CHANFREAU, G. F.

Publications and source records attributed to CHANFREAU, G. F..

3 recordsLinked to original sources

bioRxiv2025

Transcription termination promotes splicing efficiency and fidelity in a compact genome

Splicing of terminal introns is coupled to 3'-end processing by cleavage and polyadenylation (CPA) of mRNAs in mammalian genes. Whether this functional coupling is universally conserved across eukaryotes is unclear. Here we show using long read RNA sequencing in S.cerevisiae that splicing inactivation does not result in widespread CPA impairment. We also show that inactivation of CPA has limited impact on splicing efficiency. The negative impact of CPA inactivation on splicing is mainly due to transcription termination defects that promote readthrough transcription, leading to splicing inhibition for downstream intron-containing genes. Splicing impairment due to 5' extensions is length-dependent and can be detected independently from CPA inactivation for endogenous or synthetic genes, and is likely due to an increased distance of splicing signals to the 5' cap. Finally, we found that deficient termination can promote novel intragenic and long-range intergenic splicing events. These results argue against a broad coupling between splicing and CPA in S.cerevisiae but show that efficient CPA-mediated transcription termination is critical for splicing fidelity and efficiency in a compact genome. Significance StatementAccurate gene expression requires that the enzyme that polymerizes RNA stops at the proper site (termination). In addition multiple RNA processing reactions, including removal of intervening sequences are necessary to produce mature mRNAs. How these different steps in the RNA biogenesis pathways influence each other is not well understood. In this study, we show that inactivation of termination induces mature RNA formation defects, including long RNAs that retain intervening sequences, or chimeric RNAs containing sequences from genes located next to each other on the genome. This study underscores the importance of proper termination to ensure accurate and efficient splicing of adjacent genes, which is particularly critical for compact genomes in which genes are located close to each other.

molecular biology↗

bioRxiv2024

The RNA exosome maintains cellular RNA homeostasis by controlling transcript abundance in the brain

Intracellular ribonucleases (RNases) are essential in all aspects of RNA metabolism, including maintaining accurate RNA levels. Inherited mutations in genes encoding ubiquitous RNases are associated with human diseases, primarily affecting the nervous system. Recessive mutations in genes encoding an evolutionarily conserved RNase complex, the RNA exosome, lead to syndromic neurodevelopmental disorders characterized by progressive neurodegeneration, such as Pontocerebellar Hypoplasia Type 1b (PCH1b). We establish a CRISPR/Cas9-engineered Drosophila model of PCH1b to study cell-type-specific post-transcriptional regulatory functions of the nuclear RNA exosome complex within fly head tissue. Here, we report that pathogenic RNA exosome mutations alter activity of the complex, causing widespread dysregulation of brain-enriched cellular transcriptomes, including rRNA processing defects--resulting in tissue-specific, progressive neurodegenerative effects in flies. These findings provide a comprehensive understanding of RNA exosome function within a developed animal brain and underscore the critical role of post-transcriptional regulatory machinery in maintaining cellular RNA homeostasis within the brain.

molecular biology↗

bioRxiv2024

Functional Analysis of the Zinc Finger Modules of the S. cerevisiae Splicing Factor Luc7

Identification of splice sites is a critical step in pre-mRNA splicing since definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 snRNP. This involves both base pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5 splice site/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3) which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. While we were unable to determine a function for the first zinc finger domain, humanization of the second zinc finger domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5 splice sites. In contrast, the corresponding zinc finger domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5 splice site. Our work reveals a role for the second zinc finger of Luc7 in splice site selection and suggests that different zinc finger domains may have different ATPase requirements for release by Prp28.

biochemistry↗