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Chapt. 14 Eukaryotic mRNA processing I: splicing Student learning outcomes: Explain that eukaryotic mRNA precursors are spliced by a lariat, branched intermediate.

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Presentation on theme: "Chapt. 14 Eukaryotic mRNA processing I: splicing Student learning outcomes: Explain that eukaryotic mRNA precursors are spliced by a lariat, branched intermediate."— Presentation transcript:

1 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

2 14-2 14.1 Genes are in Pieces Consider sequence of human  -globin gene as a sentence: This is bhgty the human  -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

3 14-3 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

4 14-4 Splicing Outline Fig. 2 Primary transcript: Introns transcribed along with exons Final mature transcript: introns removed as exons are spliced together

5 14-5 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

6 14-6 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 1 st exon and G beginning of intron Forms loop of the lariat Separates first exon from intron –3’-OH left at end of 1 st exon attacks phosphodiester bond linking intron to 2 nd exon Forms the exon-exon phosphodiester bond Releases intron in lariat form

7 14-7 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

8 14-8 Critical signal at the Branch Branchpoint consensus sequences: Yeast sequence invariant: 5’-UACUAAC Higher eukaryote consensus variable U 47 NC 63 U 53 R 37 A 91 C 47 Branched nucleotide is final A in sequence Fig. 8 Mutant yeast genes splice aberrantly (S1 mapping)

9 14-9 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

10 14-10 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 Fig. 10

11 14-11 U1 snRNP 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) Fig. 10

12 14-12 U6 snRNP 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 Fig. 14

13 14-13 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 14-14 Yeast U2 Base Pairing with Yeast Branchpoint Sequence Mutated U2 binds mutated branchpoint sequence; Compensatory mutation suppresses lethal defect Fig. 17, 18

15 14-15 U5 snRNP and U4 snRNP 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 Fig. 10

16 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 14-16 Fig. 22 snRNP in mRNA Splicing

17 14-17 Spliceosome Catalytic Activity Catalytic center of spliceosome appears to include Mg 2+ and base- paired 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

18 14-18 Spliceosome Cycle: 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

19 Fig. 14.27 ** Spliceosome cycle

20 14-20 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

21 14-21 In vivo Protein-protein interactions: Yeast Two-Hybrid Assay B ased 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 ;

22 14-22 Intron-Bridging Protein-Protein Interactions identified by yeast two-hybrid interactions 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 Fig. 34

23 14-23 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

24 14-24 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

25 14-25 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

26 14-26 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

27 14-27 Group I Introns Fig. 48 Tetrahymena 26S rRNA 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

28 14-28 Linear Introns of group I can cyclize Fig. 49 Intron cyclizes twice, losing 15- 19 nucleotides, then linearizes a last time Last linear RNA is ribozyme that can add or subtract nucleotides from other molecules

29 14-29 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)

30 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

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