snRNA maturation in Saccharomyces cerevisie

• Applicant: Professorin Dr. Heike Krebber
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 436315601

Spliceosomes remove intron sequences from primary transcripts to generate an open reading frame that enables correct protein expression. For this purpose, the spliceosomes assemble every time newly on mRNAs with intron sequences. They are composed of several proteins and five snRNAs: U1, U2, U4, U5 and U6. With the exception of U6, which is synthesized from RNAP III, all snRNAs are transcribed from RNAP II. Subsequently, several maturation steps occur, before the mature snRNAs are incorporated into the spliceosome. For a long time it was thought that snRNAs from bakers yeast do not shuttle out of the nucleus, but we could now show that this is not the case. Like the snRNA from human cells, yeast snRNAs leave the nucleus. Different maturation steps such as the Sm-ring assembly, which protects from degradation, occur in the cytoplasm. In our recent publication we have shown that yeast is no exception in evolution, but that nuclear snRNA export is necessary in all eukaryotes. We went further and used this excellent model organism to show why shuttling is essential. By blocking nuclear snRNA export with export mutants, we have shown that the nuclear retained immature snRNAs are in this situation incorporated into the assembling spliceosome. Their assembly leads to non-functional spliceosomes and genome-wide splicing defects. This shows that the immediate nuclear export of immature snRNAs represents a quality assurance mechanism that is essential for intact splicing.Our study revealed several new open questions, which we would like to address in this research project. We would like to take a closer look at the nuclear export and import processes. It is for example unclear if the RNA export factor Mex67 (human TAP) contacts the snRNA directly or if an adapter protein is required. Furthermore we could show that the guard proteinsNpl3, Gbp2 and Hrb1 bind to the snRNA. In continuative experiments we would like to find out, whether the guard proteins indeed are also guarding the snRNA maturation, e.g. if they have a proper 5’-end. Other quality control mechanisms might be the correct order in association of snRNA binding proteins.Moreover we have shown that U6 against the current view also traverses through the cytoplasm. We suspect that the U4/U6 di-snRNP is formed in the cytoplasm and as such is transported into the nucleus. Similar to the other snRNAs, also U6 is first synthesized as a longer precursor. We will not only analyze the co-transcriptional loading of the snRNP, but also investigate, whether processing occurs before or after the export and re-import.In summary, this study will lead to a better understanding of snRNA maturation and subsequent mRNA splicing and gene expression and it will help to recognize potential defects in these processes that can lead to neurodegenerative diseases and cancer.

• DFG Programme: Research Grants