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3/18/2011 Chapt. 14 Eukaryotic mRNA processing I: splicing Student learning outcomes: • Explain that eukaryotic mRNA precursors are spliced by a lariat, branched intermediate • Describe the general mechanism of the spliceosome doing splicing of mRNA precursors • Appreciate that the CTD of Rpb1 of Pol II coordinates splicing, capping, polyA addition • Describe how alternative splicing produces diversity of mRNA products; some RNA self-splice Impt. Figs: 1*, 2*, 3, 4*, 8, 10*, 27*, 32*, 34, 37, 41, 46, 48 Review problems: 1, 2, 6, 15, 23, 27, 28, 30, 37; AQ 1, 3, 4, 5 14-1 14.1 Genes are in Pieces • Consider sequence of human β-globin gene as a sentence: This is bhgty the human β-globin qwtzptlrbn gene. • Italicized regions make no sense – Sequences unrelated to adjacent globin coding sequences – Intervening sequences, IVSs; introns • Parts of gene making sense – Coding regions = Exons Phil Sharp 1977 studying Adenovirus; infected cells isolated mRNA, hybridized and see mRNA smaller – surprise - must be pieces cut out Fig. 1 Ad ML mRNA hybridized to cloned genomic DNA 14-2 RNA Splicing • Some ‘lower eukaryotic’ genes have no introns • Most ‘higher eukaryotic’ genes coding for mRNA and tRNA (some rRNA) are interrupted by introns • Exons surround introns: contain sequences that finally appear in the mature RNA product – Genes for mRNAs have 0 to 362 exons (titin) – tRNA genes have either 0 or 1 exon Introns present in genes, not mature RNA RNA splicing: cuts introns out of immature RNAs, stitches together exons 14-3 1 3/18/2011 Splicing Outline • Primary transcript: Introns transcribed along with exons • Final mature transcript: introns removed as exons are spliced together Fig. 2 14-4 Splicing Signals • Splicing signals in mRNA precursors (hnRNAs) remarkably uniform: – First 2 bases of introns are GU; last 2 are AG – exon/GU- intron- AG/exon • 5’- and 3’-splice sites have consensus sequences extending beyond GU and AG motifs • Consensus sequences important to proper splicing: Abnormal splicing can occur if mutated consensus 14-5 14.2 Essential Mechanism of Splicing of Nuclear mRNA Precursors • Branched intermediate in nuclear mRNA precursor splicing - looks like a lariat • 2-step model – 2’-OH group of A in middle of intron attacks phosphodiester bond between 1st exon and G beginning of intron • Forms loop of the lariat • Separates first exon from intron – 3’-OH left at end of 1st exon attacks phosphodiester bond linking intron to 2nd exon • Forms the exon-exon phosphodiester bond • Releases intron in lariat form 14-6 2 3/18/2011 Simplified 2-step Mechanism of Splicing • Excised intron has 3’-OH • P between 2 exons in spliced product comes from 3’-splice site • Intermediate and spliced intron contain branched nucleotide • Branch involves 5’-end of intron (G) binding to A within intron Fig. 4 Figs. 5, 6 Sharp experiments of nature of products, linkages 14-7 Critical signal at the Branch • • • • Branchpoint consensus sequences: Yeast sequence invariant: 5’-UACUAAC Higher eukaryote consensus variable U47NC63U53R37A91C47 Branched nucleotide is final A in sequence Fig. 8 Mutant yeast genes splice aberrantly (S1 mapping) 14-8 Spliceosomes • Splicing takes place on particles • Yeast spliceosomes and mammalian spliceosomes are 40S and 60S, respectively • Spliceosomes: – contain pre-mRNA – plus snRNPs, and protein splicing factors – recognize splicing signals, orchestrate splice process Fig. 9 yeast pre-mRNA with splicing extract; or mutated splice site 14-9 3 3/18/2011 Fig. 10 snRNPs • Small nuclear ribonucleoproteins: small nuclear RNAs coupled to proteins (pronounced Snurps) • 5 snRNAs (small nuclear RNAs): – U1, U2, U4, U5, U6 – all are critical – Ordered addition (details Fig. 27): • U1, U6; U2 to branch; U2AF 3’, U5 + proteins 14-10 U1 snRNP Fig. 10 • U1 snRNA sequence complementary to both 5’- and 3’-splice site consensus sequences – U1 snRNA first binds to 5’ site – Does not simply brings sites together for splicing Base pairing between U1 snRNA and 5’-splice site of precursor is necessary, not sufficient for splicing (Figs. 11-13, evidence from WT, mutant U1, E1A gene of Adenovirus: Compensatory mutations do not always restore splicing) 14-11 U6 snRNP Fig. 14 • U6 snRNP associates with 5’-end of intron by base pairing of U6 snRNA invariant ACA (nt 47-49) pairs with UGU of intron • Occurs prior to formation of lariat intermediate • Association between U6 and substrate is essential • U6 snRNA also associates with U2 snRNA (at branchpoint) during splicing 14-12 4 3/18/2011 U2 snRNP • U2 snRNA base-pairs with conserved sequence at splicing branchpoint • Essential for splicing • U2 also forms base pairs with U6 – Helps orient snRNPs for splicing • 5’-end of U2 interacts with 3’-end of U6 – important in splicing in mammalian cells, not yeast 14-13 Yeast U2 Base Pairing with Yeast Branchpoint Sequence Fig. 17, 18 Mutated U2 binds mutated branchpoint sequence; Compensatory mutation suppresses lethal defect 14-14 U5 snRNP and U4 snRNP Fig. 10 • U5 snRNA associates with last nucleotide in one exon and first nucleotide of next exon – two exons line up for splicing (evidence from cross-link) • U4 base-pairs with U6, sequesters U6 – When U6 is needed in splicing reaction U4 is removed 14-15 5 3/18/2011 snRNP in mRNA Splicing Spliceosomal snRNPs substitute for elements at center of catalytic activity of group II introns (self-splicing) at same stage of splicing: U2, U5, U6 and substrate; RNA are catalytic Fig. 22 14-16 Spliceosome Catalytic Activity • Catalytic center of spliceosome appears to include Mg2+ and basepaired complex of 3 RNAs: – U2 snRNA – U6 snRNA – Branchpoint region of intron • Protein-free fragments of these RNAs can catalyze a reaction related to this first step in splicing Fig. 23 Spliceosome Cycle: 14-17 assembly, splicing, disassembly • Assembly begins with U1 binding splicing substrate commitment complex (Fig. 27) • U2 joins complex, followed by others – U2 binding requires ATP • U6/U4 and U5 join complex • U6 dissociates from U4, displaces U1 at 5’-splice site – ATP-dependent; activates spliceosome; U1 and U4 released U5 is at splice site U6 base pairs U2; 2 ATP -> 2 splice steps Controlling assembly of spliceosome regulates quality and quantity of splicing, regulate expression 14-18 6 3/18/2011 Fig. 14.27 ** Spliceosome cycle snRNP Structure All have same set of 7 Sm proteins Common targets of antibodies in patients with systemic autoimmune diseases (e.g. lupus) • Joan Steitz used Ab to find snRNPs Sm proteins bind to common Sm site on snRNAs: AAUUUGUGG • U1 snRNP has 3 other unique proteins (70K, A + C) • Sm proteins form doughnut-shaped structure with hole through the middle, like flattened funnel Other splicing factors help snRNPs bind 14-20 In vivo Protein-protein interactions: Yeast Two-Hybrid Assay Based on separability of DNA binding domain (DBD) and activation domain (AD) BD-X is bait; Y-AD is prey ; Clone test proteins as fusions to Gal4-BD or Gal4-AD on plasmids; Transform cells and ask about expression of reporter Can also screen library for interacting protein Fig. 32 14-21 7 3/18/2011 Intron-Bridging Protein-Protein Interactions identified by yeast two-hybrid interactions Fig. 34 • Branchpoint bridging protein (BBP) binds to U1 snRNP protein at 5’ end; binds RNA near 3’; binds other protein Mud2 at 3’ end • Similarity of yeast and mammalian complexes 14-22 CTD of Pol II defines exons • CTD of Pol II Rpb1 stimulates splicing of substrates • CTD binds to splicing factors; could assemble factors at end of exons to set them off for splicing Fig. 37 See Figs. 35, 36 for data 14-23 Alternative Splicing • Many eukaryotic transcripts have alternative splicing – can have profound effects on protein products: • Secreted or membrane-bound protein • Activity and inactivity Fig. 38 mouse Ig heavy chain 14-24 8 3/18/2011 Alternative splicing increases diversity • • • • • • Alternative promoters Some exons are ignored, (deletion of exon) Alternative 5’-splice sites (deletion, addition of exons) Alternative 3’-splice sites (deletion, addition of exons) Intron retained in mRNA if not recognized as intron Polyadenylation -> cleavage of pre-mRNA, loss of downstream exons Fig. 41; 2 of 64 possible products 14-25 14.3 Self-Splicing RNAs • Some RNAs splice themselves without aid from spliceosome or any other protein (1980s) • Ribozyme – catalytic RNA molecules • ProtozoanTetrahymena 26S rRNA gene has an intron, splices itself in vitro (Tom Cech, Nobel Prize) – Group I introns are self-splicing RNAs • Linear product, which can circularize, • Can catalyze reactions, addition or deletion nucleotides – Group II introns also have some self-splicing members • Lariat structure intermediate 14-26 Group I Introns • Can be removed in vitro without protein • Reaction begins with attack by free G nucleotide on 5’-splice site – Adds G to 5’-end of intron – Releases first exon • Second step: first exon attacks 3’-splice site – Ligates 2 exons together – Releases linear intron Fig. 48 Tetrahymena 26S rRNA 14-27 9 3/18/2011 Linear Introns of group I can cyclize Intron cyclizes twice, losing 1519 nucleotides, then linearizes a last time Fig. 49 Last linear RNA is ribozyme that can add or subtract nucleotides from other molecules 14-28 Group II Introns • RNAs containing group II introns self-splice by a pathway using an A-branched lariat intermediate, like spliceosome lariats (Fig. 22) • Secondary structures of splicing complexes involving spliceosomal systems and group II introns are very similar • Found in fungal mitochondrial, chloroplasts, also Archaea, Bacteria (cyanobacteria, purple bacteria) 14-29 Review questions 2. Diagram the lariat mechanism of splicing. 6. Describe results of experiment showing sequence UACUAAC within yeast intron is critical for splicing 27. Describe yeast two-hybrid assay for interaction between two known proteins (ex. Fos and Jun) 28. Describe yeast two-hybrid experiment to identify unknown protein that binds known protein (Fos) 14-30 10