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Chapt. 14 Eukaryotic mRNA processing I: splicing

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Describe the general mechanism of the spliceosome doing splicing of ... Describe how alternative splicing produces diversity of mRNA products; some RNA self ... – PowerPoint PPT presentation

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Title: Chapt. 14 Eukaryotic mRNA processing I: splicing


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
snRNPs
Fig. 10
  • 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
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

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
snRNP in mRNA Splicing
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 -gt 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 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
22
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

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 -gt 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)
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