CHARACTERIZATION OF THE ROLE OF THE SPLICING-FACTOR CWC24 IN pre-RNA IN Saccharomyces cerevisiae

• Grant number: 23/16763-5
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: May 06, 2024
• End date: May 05, 2025
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Carla Columbano de Oliveira
• Grantee: Moises Ricardo de Almeida Barros
• Supervisor: Manuel Ares Jr
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: University of California, Santa Cruz (UC Santa Cruz), United States
• Associated to the scholarship: 21/14137-4 - Characterization of the role to the general splicing-factor CWC24 in pre-mRNA in Saccharomyces cerevisiae, BP.DD

Abstract

The spliceosome is a large RNA-protein complex responsible for catalyzing the splicing reactions of pre-RNAs in eukaryotes. The spliceosome protein Cwc24 plays an important role in splicing regulation by binding the 5' splicing site (5'SS), and protecting this site from premature reactions. Our laboratory has shown that Cwc24 is a general splicing factor and that splicing of U3 snoRNA is the splicing event most affected by the depletion of Cwc24. Since pre-U3 has a non-consensus branch site (BS), we hypothesize that Cwc24 promotes the function of the U2 snRNP as it recognizes the BS during spliceosome assembly. To analyze the effect of the loss of Cwc24 on function of the spliceosome, we compared the protein composition of the spliceosome with and without Cwc24 by mass spectrometry at different stages of the splicing cycle. The results obtained indicate that, in the absence of Cwc24, there was an accumulation of SF3a proteins. This subcomplex is removed under the action of Prp2, so the action of this helicase must be compromised, impairing the formation of the catalytic conformation and consequently pausing the spliceosome before the first transesterification reaction. As SF3a is a component of the U2 snRNP, it is possible that this stabilization is associated with the conformational changes that occur with U2 snRNA. To confirm these observations, this project proposes to study the mechanisms that lead to the stalling of the spliceosome in the absence of Cwc24, evaluating how different conformations of U2 snRNA can influence this process.