Mechanism of regulation of spliceosome activation by Brr2 and Prp8 and links to retinal disease.

摘要: Splicing is a crucial post-transcriptional processing event that entails the removal of non-coding intervening sequences (introns) from eukaryotic pre-mRNA and ligation coding (exons). It carried out in two-step reaction by spliceosome, giant highly dynamic protein-rich ribonucleoprotein (RNP) enzyme. The spliceosome consists five major subunits, U1, U2, U4/U6 U5 snRNPs multitude non-snRNP proteins. active center only develops de novo on stepwise assembly U driven several DExD/H-box ATPases/RNA helicases. Major structural compositional rearrangements are required to render catalytically competent for promoting two steps splicing. enzyme Brr2 plays role this catalytic activation process. an exceptionally large DExH-box protein (ca. 250 kDa), member Ski2-like RNA helicases stands both structurally functionally among other splicesosomal composed putative helicase cassettes fused tandem. Each cassette contains conserved dual-RecA-like domains, flanked winged helix (WH) domain Sec63 homology unit unknown function may bestow specific properties upon helicase. integral component snRNP unlike spliceosomal it preassembled with one its substrates, snRNPs, before recruitment pre-spliceosome. Furthermore, remains stably associated splicesosome again during disassembly step spliceosome. Thus, RNPase activity needs be reliably controlled facilitate multiple usages Indeed, forms stable complex proteins, scaffolding Prp8 EF-2 like GTPase Snu114, which have been implicated regulation activity. In human, mutations within C-terminal tail cause severe type retinitis pigmentosa (RP), progressive retinal dystrophy. was hitherto unclear how adopts these capabilities regulatory can lead precise timing thus unwinding Brr2. addition, molecular basis way RP-linked disease remained poorly understood. work, crystal structure solved collaboration V. Pena M. Wahl revealed first insight into similarity units DNA Hel308. Guided Hel308 structure, architecture could modeled as composite dual Hel308-like Functional roles various predicted elements were then validated mutational analysis vitro living yeast cells. results supported idea analogy β-hairpin loop RecA-2 N-terminal act strand separation device, RNAs. More recently, larger fragment human Brr2, encompassing cassettes, K. Santos Wahl, extensive interaction surface between (respectively, Brr2NCand Brr2CC), provided framework detailed structure-based Brr’s enzymatic activities. shown isolated Brr2NC harbors ATPase activities threads single-stranded through central tunnel across helix-loop-helix duplex unwinding. Although Brr2CC inactive own, strongly stimulates cassette. Mutations amino acid residues involved communication well interfere nucleotide-binding pocket Brr2CC, affected and/or while does not seem engage RNA, binds ATP acts intramolecular cofactor stimulate Using mutant constructs I also able show interacts region U4 preceding stem (the domain), translocates 3’ 5’ direction along unwind first. In second part work thesis investigated RNase H-like (RH) Jab1/MPN-like domains functions. UV-induced RNA-protein crosslinking probing methods H snRNAs vitro, where I. mass spectrometry, crosslinks mapped at base hairpin (β-finger) RH domain. Moreover, interferes Brr2-mediated sequestering Brr2’s targeting site, indicating negatively regulates keeper prevent premature These findings support platform handover 5'-splice site U1 U6 snRNA prior step. The Jab1 ubiquitin-binding comprises globular followed protruding tail, partly unstructured domain, represents hotspot leading pigmentosa. biochemical assays, inhibits preventing loading onto substrate U4/U6. Upon deletion 16 acids, Jab1Δ16 now stimulated activities, suggesting binding capacity. intact obtained observations. rests primarily IG-like motifs RecA occluding under conditions favoring binding, full-length coactivator enhancing coupling hydrolysis processivity This delicate requires dual-cassette organization observed Finally, effect stability tri-snRNP formation, cell viability splicing vivo Taken together, uncover mechanism underlying unique dual-mode superfamily 2 reveal disruption constitutes principle certain