An investigation into a phenomenon that alters splicing: A tale of pseudouridines

Abstract

Pre-mRNA splicing, the removal of introns from a precursor RNA and the ligation of exons to form a mature mRNA, is carried out by the spliceosome and involves two successive trans-esterification reactions. Altered stabilities of various spliceosome conformations impact substrate specificity and are modulated by multiple spliceosomal components. A two-state model of catalytic spliceosome proposes that the spliceosome exists in kinetically competing conformations in the two catalytic steps. We postulated that classes of factors that modulate spliceosomal transitions could be identified through large-scale comprehensive techniques such as synthetic genetic array analysis in S. cerevisiae. To this end, we have identified a pseudouridine synthase - Pus3p as a novel modulator (perhaps indirectly) of the spliceosome.;Post-transcriptional modifications of the catalytic core may provide additional prospects for fine-tuning splicing, especially under non-optimum growth conditions. In Saccharomyces cerevisiae, we have identified a novel pseudouridine residue (Psi28) on U6 snRNA that is induced during filamentous growth. We identified Puslp as the pseudouridine synthase that catalyzes modification of this site and we show that it, but not other pseudouridylases, is up-regulated during filamentation. We also found several U6 snRNA mutants that are constitutively pseudouridylated; remarkably, these U6 mutants activate pseudo-hyphal growth, dependent upon the presence of Pusl p, arguing that Psi28 per se can initiate at least part of the filamentous growth program. U6-U28 pseudouridylation changes the efficiency of splicing of suboptimal introns; thus, filamentous growth-activated Pusl p-dependent pseudouridylation of U6-Psi28 alters the splicing profile of sensitive pre-mRNAs and contributes to the filamentation growth program. Collectively, our data supports a fine-tuning of splicing regulation, achieved through a post-transcriptional modification of an snRNA component, linking such a regulation to an environmental stress condition.