I. INTRODUCTION The RNA world pictures a time when structures catalyzed many reactions, almost certainly including splicing. self-splicing reactions of group I and II introns may be direct remnants the world. Both these catalytic can facilitate cis -splicing, where two exons are initially part contiguous RNA, trans on separate RNAs. In latter case, partial intron sequences flanking pair through sequence complementarity to form structure joining associated exons. Many have speculated that...

Selection of the nucleophile for first step nuclear pre-mRNA splicing was probed by site-specific incorporation into substrates nucleotides modified at 2' position. The differing abilities ribose, 2'-deoxyribose, and arabinose to base-pair within an RNA.RNA duplex contribute a nucleophilic 2'-OH group were exploited analyze paired/unpaired disposition branch site nucleotide. results provide direct evidence bulged model in which either two adjacent purines consensus sequence may shift...

An RNA recognition motif (RRM) of approximately 80 amino acids constitutes the core RNA-binding domains found in a large family proteins involved processing. The U1 domain A protein component human small nuclear ribonucleoprotein (RNP), which encompasses RRM sequence, was analyzed by using NMR spectroscopy. is highly stable monomer solution consisting four antiparallel beta-strands and two alpha-helices. conserved RNP1 RNP2 consensus sequences, containing residues previously suggested to be...

The association of proteins with the branch site region during pre-mRNA splicing was probed using a novel methodology to site-specifically modify photo-reagent benzophenone. Three sets were distinguished by kinetics their associations pre-mRNAs, discrete complexes, and differing factor requirements. An early U1 snRNP-dependent cross-link p80 species followed cross-links p14, p35, p150 polypeptides associated U2 snRNP-pre-mRNA complex. Concomitant formation spliceosome, rearrangement protein...

Many RNA-associated proteins contain a ribonucleoprotein (RNP) consensus octamer encompassed by conserved 80 amino acid sequence, which we have termed an RNA recognition motif (RRM). RRM family members either one (class I) or multiple II) copies of this motif. We report here that class II component the U1 small nuclear RNP (snRNP), A protein snRNP (U1snRNP-A), contains two RRMs (RRM1 and -2), yet has only binding domain (RRM1) interacts specifically with stem-loop RNA. Quantitative analysis...

The assembly of prespliceosomes is responsible for selection intron sites splicing. U1 and U2 snRNPs recognize 5' splice branch sites, respectively; although there information regarding the composition these complexes, little known about interaction among components or between two snRNPs. Here we describe protein network interactions linking with ATPase Prp5, important site recognition fidelity during first steps reaction, using fission yeast Schizosaccharomyces pombe. snRNP core U1A binds...

Although pseudouridine nucleobases are abundant in tRNAs, rRNAs, and small nuclear RNAs (snRNAs), they not known to have physiologic roles cell differentiation. We identified a residue (Ψ28) on spliceosomal U6 snRNA that is induced during filamentous growth of Saccharomyces cerevisiae. Pus1p catalyzes this modification upregulated filamentation. Several mutants strongly pseudouridylated at Ψ28. Remarkably, these activate pseudohyphal growth, dependent upon Pus1p, arguing U6-Ψ28 per se can...

Mutations in the U2 snRNP component SF3B1 are prominent myelodysplastic syndromes (MDSs) and other cancers have been shown recently to alter branch site (BS) or 3′ splice selection splicing. However, molecular mechanism of altered splicing is not known. We show here that hsh155 mutant alleles Saccharomyces cerevisiae , counterparts mutations frequently found cancers, specifically change suboptimal BS pre-mRNA substrates. Hsh155p interacts directly with Prp5p, first ATPase acts during...

Protein arginine methyltransferases (PRMTs) are required for the regulation of RNA processing factors. Type I PRMT enzymes catalyze mono- and asymmetric dimethylation; II symmetric dimethylation. To understand specific mechanisms activity in splicing regulation, we inhibited PRMTs probed their transcriptomic consequences. Using newly developed Splicing Kinetics Transcript Elongation Rates by Sequencing (SKaTER-seq) method, analysis co-transcriptional demonstrated that inhibition resulted...

Intron removal from pre-mRNA is catalyzed by the spliceosome, which comprises 5 snRNPs containing small nuclear RNAs (snRNAs). U2 snRNA makes critical RNA-RNA and RNA-protein contacts throughout splicing cycle. Mutations in snRNA, particularly at position C28, have been linked to cancers. To study gene expression changes mediated mutated snRNAs, U2-2 C28 mutants, knockout (KO), overexpression (OE) cell lines were constructed followed RNA sequencing. We observed significant over 4,000...

We have defined the nucleotide sequence of a protein-binding domain within U1 RNA that specifically recognizes and binds both to small nuclear ribonucleoprotein component (the 70K protein) previously RNA-binding protein. investigated direct interactions between purified protein by reconstitution in vitro. Thirty-one nucleotides RNA, corresponding stem-loop I, were required for this interaction. Nucleotides at 5' end are involved base pairing with splice site pre-mRNA not binding. In contrast...

Fidelity and efficiency of pre-mRNA splicing are critical for generating functional mRNAs, but how such accuracy in 5′ splice site (SS) selection is attained not fully clear. Through a series yeast genetic screens, we isolated alleles prp28 that improve suboptimal 5′SS substrates, demonstrating WT-Prp28p proofreads, consequently rejects, poor 5′SS. Prp28p thought to facilitate the disruption 5′SS–U1 snRNA pairing allow 5′SS–U6 catalytic spliceosome; unexpectedly, proofreading by dependent on...