Functional characterization of S cerevisiae splicing factor Ycr063w/Bud 31- Insights into its context dependent functions and biochemical interactions with the spliceosome

Abstract

Regulated gene expression in eukaryotes is a complex, multi-layered process. A critically regulated step is pre-mRNA splicing, essential for the synthesis of functional transcripts. Splicing is carried out by the spliceosome, a macromolecular complex composed of five nuclear snRNAs bound to snRNPs and ~120 protein factors.

While most budding yeast splicing factors are essential, some are non-essential and enhance splicing efficiency. Ycr063w is one such non-essential protein, reported to co-purify with splicing factors in the Cef1p complex and implicated in budding pathway regulation.

In Vivo Roles of Ycr063w Growth Characteristics: Null mutants of ycr063w exhibited growth arrest at extreme temperatures (37°C and 18°C) and budding anomalies, mild at permissive temperatures but severe at elevated temperatures.

Cell Cycle Regulation: FACS analyses revealed delayed G1/S transition in ycr063w mutants, with ~60% cells enlarged and single-budded. At 37°C, cells showed abnormal shapes and multiple buds.

Splicing of Budding Pathway Transcripts: Semi-quantitative RT-PCR showed significant splicing defects in SRC1 and ARP2 transcripts, while other tested transcripts were modestly affected.

Genetic Interactions: No genetic interaction was observed between Ycr063w and Prp17, another Cef1p complex member, suggesting non-redundant functions.

Spliceosomal Associations of Ycr063w In Vitro Splicing Assays: Using S100 extracts, accumulation of lariat intermediates at elevated temperatures indicated a role for Ycr063w in the second catalytic step of splicing.

Intronic Parameters: Ycr063w was required for splicing actin intron variants with branch site-3 splice site distances >15 nts at 37°C, but dispensable when the distance was 11 nts.

snRNA Associations: Immunoprecipitation experiments revealed salt-stable associations of Ycr063w with U5 and U6 snRNAs, suggesting involvement in active spliceosomes or post-splicing complexes.

Earliest Association: Ycr063w was detected in precatalytic complexes (A2-1 stage), associating with spliceosomes prior to Cef1p complex activation.