1 / 30

Chapt. 14 Eukaryotic mRNA processing I: splicing

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

Download Presentation

Chapt. 14 Eukaryotic mRNA processing I: splicing

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

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

  2. 14.1 Genes are in Pieces • Consider sequence of human b-globin gene as a sentence: This is bhgty the human b-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. 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. Splicing Outline • Primary transcript: Introns transcribed along with exons • Final mature transcript: introns removed as exons are spliced together Fig. 2

  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. 14.2 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

  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. 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)

  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. 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

  11. 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)

  12. 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

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

  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. 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

  17. Spliceosome Catalytic Activity • Catalytic center of spliceosome appears to include Mg2+ 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. 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. 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. In vivo Protein-protein interactions:Yeast Two-Hybrid AssayBased on separability of DNA binding domain (DBD) and activation domain (AD)BD-X is bait; Y-AD is preyClone test proteins as fusions to Gal4-BD or Gal4-AD on plasmids; Transform cells and ask about expression of reporterCan also screen library for interacting protein ; Fig. 32

  22. Intron-Bridging Protein-Protein Interactionsidentified 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. 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. 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. 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. 14.3 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. 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

  28. Linear Introns of group I can cyclize 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 Fig. 49

  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)

More Related