Pillai, B. R. | bioRxiv Science

bioRxiv2025

Competing 5' splice sites of SRC1/HEH1 are marked for alternative splicing by U5 and U6 snRNAs during the B to Bact transition of the spliceosome

Alternative splicing of precursor-messenger RNA (pre-mRNA) bearing introns with competing and overlapping 5 splice sites (5SS) produces more mRNAs. We investigated the mechanism of this form of alternative splicing by monitoring RNA and protein products of the yeast SRC1/HEH1 gene. Its alternative splicing requires a sixteen-nucleotide pre-mRNA segment spanning the two 5SS. The nucleotides are decoded by U5 and U6 small nuclear RNAs (snRNA), further supported by proteins of the spliceosomal B and Bact complexes. Specific variants of the pre-mRNA segment with recalibrated binding to U5 and U6 snRNAs allowed alternative splicing independently of the trans-acting proteins. Conversely, snRNA variants specified for modified 5SS selection suppressed the splicing defects in the B complex mutants. Further data suggest that alternative splicing from HEH1-type introns containing overlapping and competing 5SS relies on pre-mRNA-snRNA interactions during B to Bact transitions of the spliceosome, supported by proteins that stabilize low-fidelity, high-efficiency conformations.

molecular biology↗

bioRxiv2022

Composition can buffer protein dynamics within liquid-like condensates.

Most non-membrane-bound compartments in cells that form via phase separation have complex composition. While phase separation of individual proteins that form these compartments is well-documented, the mechanisms that modulate dynamics of individual proteins in multicomponent systems remain unclear. Here, we used in vitro reconstitution and in vivo experiments to investigate how the dynamics of a scaffold protein PGL-3 is regulated within the liquid-like P granule compartment in C. elegans. Using mutational and biophysical perturbations, we generated PGL-3 constructs that form condensates in vitro with widely varying dynamics. Using these PGL-3 constructs, we show that introducing other P granule components buffers against change of dynamics within liquid-like condensates. This dynamics-buffering effect is mediated by weak interactions among two or more components. Such dynamics-buffering may contribute to robust functional output of cellular liquid-like compartments.

cell biology↗