Chapt. 14 Eukaryotic mRNA processing I: splicing

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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
• 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

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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
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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

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Splicing Outline

• Primary transcript Introns transcribed along
  with exons
• Final mature transcript introns removed as exons
  are spliced together

Fig. 2
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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

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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

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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
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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)
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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
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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

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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)

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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

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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

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

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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
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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
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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

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Fig. 14.27 Spliceosome cycle
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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

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In vivo Protein-protein interactionsYeast
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
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Intron-Bridging Protein-Protein
Interactionsidentified 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

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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
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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
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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
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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

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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
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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
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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)

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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)