Download Chapt. 14 Eukaryotic mRNA processing I: splicing 14.1 Genes are in

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
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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
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
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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
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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)
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
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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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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
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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
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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
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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
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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
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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 -> 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
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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 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)
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