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BIOL 205
Ian D Chin- Sang

BIOL 205: week 11, lecture 32 recognize “AAUUAAA” Group I: e.g. Tetrahymena rRNA(Tom Cech) • no sequence conservation at splice junction • carry conserved sequences internally • introns self-splice (RNAcatalysis, Ribozymes) • Guanosine as cofactor • transesterifications, no external energy input Two transesterific reactions Self-splicing reaction → RNA has conserved secondary structure - pocket created to hold guanosine residue (green G?) - internal guide sequence: RNAhas an internal guide to help fold intron close to reactive guanosine companies: - good way of targeting destruction of RNA - create ribozyme, cleaves RNA, inactivating it - but didn't really work well and RNAi came in Group I introns can be converted to ribozymes and replaced all that technology Splicesome-mediated Splicing! • GU found at 5’intron-exon junction • G marks 3’intron-exon junction • Ais part of branch site (consensus longer than this) • Intron ends up as lariat (ie lassoo) • No particular order in which introns are removed Conserved sequences related to intron splicing Complex patterns of eukaryotic mRNAsplicing – tropomyosin gene Reactions in exon splicing Summary: - 2' hydroxyl of intron attacks phosphate of exon (1 transesterification) - then 3' hydroxyl attacks 5' phosphate of nd other exons (2 transesterification) - and 5'-2' linkage created in lariat intron Spliceosome assembly and function (recognize splice site in - contain U1 or U2 small internalA) nuclear RNA (snRNA) - bind with protein to form small nuclear ribonucleoprotein particles (SNPs) - RNAcomponent of U4 and U6 base pair causes U6 to replace U1 at 5’exon-intron boundary - Factors released: allows U5 to bind with U2 - forms active site - first transesterificaton takes place - U5 aids in bringing 2 exons together - second transesterification RNA catalyses nuclear pre-mRNAsplicing November 14th 2013 → uses magnesium ions to stabilize In nuclear pre-messenger RNAsplicing, introns are excised by the spliceosome, a dynamic machine composed of both proteins and small nuclear RNAs (snRNAs). Over thirty years ago, after the discovery of self-splicing group II intron RNAs, the snRNAs were proposed to catalyse splicing. However, no definitive evidence for a role of either rescue strategies in spliceosomes from budding yeast, here we show that the U6 snRNcatalyses both of the two splicing reactions by positioning divalent metals that stabilize the leaving groups during each reaction . Notably, all of the U6 catalytic metal ligands we identified correspond to the ligands observed to position catalytic, divalent metals in crystal structures of a group II intron RNA. These findings indicate that group II introns and the spliceosome share common catalytic mechanisms and probably common evolutionary origins. Our results demonstrate that RNAmediates catalysis within the spliceosome. Acascade of alternative RNAsplicing regulates sex determination in Drosophila → codon 2 prevents transformer protein creation → all need is a mutation in sex-lethal and transformer to convert females into males mRNA-binding proteins repress mRNAtranslati
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