Processing of RNA IIBiochemistry 302What’s an intron?Overview of intron excision or exon splicing mechanisms (all 2-step)Consensus sequences near 5? and 3? splice sites in vertebrate pre-mRNAsBasic chemistry of exon splicing ? phosphate ester bond exchange or transesterificationRNA splicing can be studied in vitro (from M. Green and coworkers)Excision of introns from nuclear pre-mRNAs is spliceosome-mediatedPutative mechanism of intron removal from pre-mRNAs (orienting splice sites)Model for snRNP-mediated splicing of nuclear pre-mRNA exons (U1/2-dependent)Pre-mRNA splicing is probably a co-transcriptional processMechanisms to generate alternative transcripts from a single geneAlternative splicing for cell-type specific expression of mRNAsCis-elements and other RNA-binding proteins regulate alternative splicingGroup I intron: self-splicing RNAGroup I intron from Tetrahymena is preorganized to bind substratesSelf-splicing:Group II intronsProcessing of RNA IIBiochemistry 302February 14, 2005Bob KelmWhat’s an intron?• Transcribed sequence removed during the process of mRNA maturation (non protein-coding sequence) • Discovered by P. Sharp and R. Roberts in late 1970s by EM analysis of genomic DNA-mRNA (poly A) hybrids encoding the major virion capsid protein, hexon– DNA loops correspond to introns– Same looping pattern later shown w/ ovalbumin mRNA-genomic DNA hybridsChicken ovalbuminFig. 28-4Overview of intron excision or exon splicing mechanisms (all 2-step)• Group I Introns– Cofactor guanosine (3′OH) attacks exon/5′ intron boundary.– “Linear” intervening sequence (IVS) is generated.• Group II Introns – Branch point adenosine (2′OH) attacks exon/5′ intron boundary. – Lariat-like IVS is generated.• Nuclear mRNA splicing– Branch point adenosine (2′OH) attacks exon/5′ intron boundary generating lariat IVS.– Requires assistance of snRNPS.• Nuclear tRNA “splicing”– Protein (not RNA)-dependent.– Requires endonuclease & ligase.Consensus sequences near 5′ and 3′splice sites in vertebrate pre-mRNAsTable 28-6Lodish et al. Molecular Cell Biology, 3rded.*A U A A C *Minor class: 1/300 introns but not in yeast or worms (J. Steitz)Basic chemistry of exon splicing →phosphate ester bond exchange or transesterificationLodish et al. Molecular Cell Biology, 3rded.RNA splicing can be studied in vitro (from M. Green and coworkers)ProductIntermediateIntermediateProduct158 130 209Starting materialLodish et al. Molecular Cell Biology, 3rded.Excision of introns from nuclear pre-mRNAs is spliceosome-mediated• snRNAs (U-rich)– U1…..U6 (U3 nucleolus)– Small: 107 to 210 nt w/ lots of secondary structure– Some transcribed by RNAP II, others by RNAP III• snRNPs (“snurps”)– Some common to all snRNAs (Sm binds U1, U2, U4, U5).– Some specific to individual snRNAs.• Other specialty proteins– RNA helicases (DEAD box family)– SR proteinsFig. 28-31U1 interacts w/ 5′ intron-exon boundaryU2 interacts w/ 3′ intron-exon boundary. Note how “bulge” may serve to activate adenosine 2′OH.Putative mechanism of intron removal from pre-mRNAs (orienting splice sites)Splice site consensus:5′= G:GU exon:intron3′= Py10U/CAG:C intron:exonFig. 28-3212Note:U1 and U2 snRNPs are necessary but not sufficient to mediate splicing.Model for snRNP-mediated splicing of nuclear pre-mRNA exons (U1/2-dependent) • 1: U1 snRNP binds to 5′ splice site through base pairing with 5′end of U1 RNA• 2: Auxiliary factor U2AF binds poly Pyr tract to assist binding of U2 snRNP to branch point sequence (ATP-dependent). U1 and U2 facilitate loop structure.• 3: Spliceosome assembles (60S): U4/U6 & U5 on U1 + U2 snRNPs • 4,5: Internal rearrangement and probable U1/U4 dissociation then U2/U6 and U5-dependent lariat formation and exon splicing via 5′ and 3′ consensus sites. U2:U6 interaction is crucial. U6 is the putative ribozyme.• 6-8: Spliceosome disassembly releasing ligated exons, intron lariat & U snRNPs (recycled). Fig. 28-33inactiveU1/U4Pre-mRNA splicing is probably a co-transcriptional processLehninger Principles of Biochemistry, 4th ed., Ch 26Mechanisms to generate alternative transcripts from a single gene• Why….– Economize genomic size– To allow tissue/cell-specific gene expression• How….– Alternative promoter usage: mouse amylase– Poly A site selection: Ig heavy chain class switching– Differential RNA splicing: muscle-specific genes (e.g. α-tropomyosin, troponins, SM myosin)– Alternative polyA + splicing: dictates calcitonin or CGRP hormone expression in thyroid versus brain– RNA editing: apoBLehninger Principles of Biochemistry, 4th ed., Ch 26Alternative splicing for cell-type specific expression of mRNAsdefault productknownInferred (nuclease protection)Fig. 28-34Mechanism: Cell type-specific proteins interact w/ spliceosome to alter the default choice of splice site selection.Cis-elements and other RNA-binding proteins regulate alternative splicing• Cis-elements– Exon splicing enhancers (ESEs) – purine-rich RNA sequences that stimulate splicing of adjacent 5′ introns– Intron enhancer sequences– Negative regulatory elements (found in both introns and exons)• Trans-acting proteins– SR proteins – family of RNA-binding proteins required for spliceosome assembly and the activity of distinct ESEs, regulated by SR protein kinases– Sex and tissue-specific splicing factors (Drosophila sex determination)Group I intron: self-splicing RNA• Cech and coworkers (1982)– Discovered a 414 nt self-splicing rRNA intron– Tetrahymena thermophila(ciliated protozoan, unicellular animal model) • Highly structured RNA– Self-splicing requires Mg2+(~2 mM) and a guanosine or guanylate cofactor.– RNA modified during reaction (so not a real catalyst).• Two transesterification rxns– 1st: guanosine nucleoside cofactor attacks 5′ splice site– 2nd: 3′ end (OH) of 1st exon attacks G at the 3′ splice site3′-5′Further truncation of the released IVS yields a catalytic 395 nt RNA molecule known as L-19 IVS. It can shorten or elongate some oligonucleotides in vitro.Group I intron from Tetrahymena is preorganized to bind substratesB.L. Golden et al. Science 282:259-264, 1998P1 domain containing the 5′ splice site is thought to fit here.5′ splice site3′ splice sitenon A-form helical RNAbinding site for guanosine substrateSelf-splicing:Group