Processing of RNA IIBiochemistry 302February 13, 2006Precursor mRNA: introns and exons• Intron: Transcribed RNA sequence removed from precursor RNA during the process of maturation (for class II genes: non protein-coding sequence) • Discovered by P. Sharp & R. Roberts in late 1970s by EM analysis of genomic DNA-mRNA (polyA) 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 pre-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.Pre-mRNA splicing is an “extra step” in gene expression (eukaryotes vs prokaryotes).Consensus sequences near 5′ and 3′splice sites in vertebrate pre-mRNAsLodish et al. Molecular Cell Biology, 3rded.Table 28-6*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 trans-esterificationLodish et al. Molecular Cell Biology, 3rded.RNA splicing can be studied in vitro (from M. Green and coworkers)Lodish et al. Molecular Cell Biology, 3rded.Starting materialProductIntermediateIntermediateProduct158 130 209Excision of introns from nuclear pre-mRNAs is spliceosome-mediated• snRNAs (U-rich)– U1 → U6 (U3 nucleolus)– U11, U12, U6ATACfor minor class introns– 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/ branch point sequence. Note how “bulge” may serve to activate adenosine 2′ OH.U1, U2: orientation factorsPutative 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. This mechanism is valid for only some introns.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 an ATP-dependent co-transcriptional processLehninger Principles of Biochemistry, 4th ed., Ch 26Note the requirement of ATP hydrolysis for snRNP binding (U2, U4/U6 + U5) and conformational rearrangements in the spliceosomeso pre-mRNA splicing is not energetically neutral per se.Mechanisms 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: Igheavy 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 mRNAsMechanism: Cell type-specific proteins interact w/ spliceosome components to alter the default choice of splice site selection.knownInferred (nuclease protection)Fig. 28-34default productCis-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– Developmental control: 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)– kcat/KM~103M−1s−1• 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, 1998non A-form helical RNAbinding site for guanosine substrate5′ splice site3′ splice siteP1 domain containing the 5′ splice site is thought to fit here.Self-splicing:Group II introns• Found in mitochondrial and chloroplast pre-rRNAs.• Fold into conserved secondary structure characterized by numerous stem loops…..similar to spliceosomal U snRNAs.• Self-cleavage mechanism similar to spliceosome but only happens under conditions of elevated temperature and [Mg2+] in vitro.• U snRNAs may have evolved from group II introns.• Some can behave as mobile elements in the genome.• Encode maturases, proteins