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Prp8 positioning of U5 snRNA is linked to 5' splice site recognition.

ABSTRACT: Prp8 is an essential protein that regulates spliceosome assembly and conformation during pre-mRNA splicing. Recent cryo-EM structures of the spliceosome model Prp8 as a scaffold for the spliceosome's catalytic U snRNA components. Using a new amino acid probing strategy, we identified a dynamic region in human Prp8 that is positioned to stabilize the pre-mRNA in the spliceosome active site through interactions with U5 snRNA. Mutagenesis of the identified Prp8 residues in yeast indicates a role in 5' splice site recognition. Genetic interactions with spliceosome proteins Isy1, which buttresses the intron branch point, and Snu114, a regulatory GTPase that directly contacts Prp8, further corroborate a role for the same Prp8 residues in substrate positioning and activation. Together the data suggest that adjustments in interactions between Prp8 and U5 snRNA help establish proper positioning of the pre-mRNA into the active site to enhance 5' splice site fidelity.

SUBMITTER: MacRae AJ

PROVIDER: S-EPMC5959246 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Prp8 positioning of U5 snRNA is linked to 5' splice site recognition.

MacRae Andrew J AJ Mayerle Megan M Hrabeta-Robinson Eva E Chalkley Robert J RJ Guthrie Christine C Burlingame Alma L AL Jurica Melissa S MS

RNA (New York, N.Y.) 20180227 6

Prp8 is an essential protein that regulates spliceosome assembly and conformation during pre-mRNA splicing. Recent cryo-EM structures of the spliceosome model Prp8 as a scaffold for the spliceosome's catalytic U snRNA components. Using a new amino acid probing strategy, we identified a dynamic region in human Prp8 that is positioned to stabilize the pre-mRNA in the spliceosome active site through interactions with U5 snRNA. Mutagenesis of the identified Prp8 residues in yeast indicates a role in ...[more]

PMID: 29487104

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Project description:BackgroundU3 snoRNA is a box C/D small nucleolar RNA (snoRNA) involved in the processing events that liberate 18S rRNA from the ribosomal RNA precursor (pre-rRNA). Although U3 snoRNA is present in all eukaryotic organisms, most investigations of it have focused on fungi (particularly yeasts), animals and plants. Relatively little is known about U3 snoRNA and its gene(s) in the phylogenetically broad assemblage of protists (mostly unicellular eukaryotes). In the euglenozoon Euglena gracilis, a distant relative of the kinetoplastid protozoa, Southern analysis had previously revealed at least 13 bands hybridizing with U3 snoRNA, suggesting the existence of multiple copies of U3 snoRNA genes.ResultsThrough screening of a lambda genomic library and PCR amplification, we recovered 14 U3 snoRNA gene variants, defined by sequence heterogeneities that are mostly located in the U3 3'-stem-loop domain. We identified three different genomic arrangements of Euglena U3 snoRNA genes: i) stand-alone, ii) linked to tRNAArg genes, and iii) linked to a U5 snRNA gene. In arrangement ii), the U3 snoRNA gene is positioned upstream of two identical tRNAArg genes that are convergently transcribed relative to the U3 gene. This scenario is reminiscent of a U3 snoRNA-tRNA gene linkage previously described in trypanosomatids. We document here twelve different U3 snoRNA-U5 snRNA gene arrangements in Euglena; in each case, the U3 gene is linked to a downstream and convergently oriented U5 gene, with the intergenic region differing in length and sequence among the variants.ConclusionThe multiple U3 snoRNA-U5 snRNA gene linkages, which cluster into distinct families based on sequence similarities within the intergenic spacer, presumably arose by genome, chromosome, and/or locus duplications. We discuss possible reasons for the existence of the unusually large number of U3 snoRNA genes in the Euglena genome. Variability in the signal intensities of the multiple Southern hybridization bands raises the possibility that Euglena contains a naturally aneuploid chromosome complement.

Project description:Imperfect conservation of human pre-mRNA splice sites is necessary to produce alternative isoforms. This flexibility is combined with the precision of the message reading frame. Apart from intron-termini GU_AG and the branchpoint A, the most conserved are the exon-end guanine and +5G of the intron start. Association between these guanines cannot be explained solely by base-pairing with U1 snRNA in the early spliceosome complex. U6 succeeds U1 and pairs +5G in the pre-catalytic spliceosome, while U5 binds the exon end. Current U5 snRNA reconstructions by CryoEM cannot explain the conservation of the exon-end G. Conversely, human mutation analyses show that guanines of both exon termini can suppress splicing mutations. Our U5 hypothesis explains the mechanism of splicing precision and the role of these conserved guanines in the pre-catalytic spliceosome. We propose: (1) optimal binding register for human exons and U5-the exon junction positioned at U5Loop1 C39|C38; (2) common mechanism for base-pairing of human U5 snRNA with diverse exons and bacterial Ll.LtrB intron with new loci in retrotransposition-guided by base pair geometry; and (3) U5 plays a significant role in specific exon recognition in the pre-catalytic spliceosome. Statistical analyses showed increased U5 Watson-Crick pairs with the 5'exon in the absence of +5G at the intron start. In 5'exon positions -3 and -5, this effect is specific to U5 snRNA rather than U1 snRNA of the early spliceosome. Increased U5 Watson-Crick pairs with 3'exon position +1 coincide with substitutions of the conserved -3C at the intron 3'end. Based on mutation and X-ray evidence, we propose that -3C pairs with U2 G31 juxtaposing the branchpoint and the 3'intron end. The intron-termini pair, formed in the pre-catalytic spliceosome to be ready for transition after branching, and the early involvement of the 3'intron end ensure that the 3'exon contacts U5 in the pre-catalytic complex. We suggest that splicing precision is safeguarded cooperatively by U5, U6, and U2 snRNAs that stabilize the pre-catalytic complex by Watson-Crick base pairing. In addition, our new U5 model explains the splicing effect of exon-start +1G mutations: U5 Watson-Crick pairs with exon +2C/+3G strongly promote exon inclusion. We discuss potential applications for snRNA therapeutics and gene repair by reverse splicing.

Dataset Information

Prp8 positioning of U5 snRNA is linked to 5' splice site recognition.

Publications

Prp8 positioning of U5 snRNA is linked to 5' splice site recognition.

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