Splicing Regulation

1
Splicing Regulation

• The role of SR proteins in spliceosome assembly

2
Spliceosome assembly The spliceosome assembles
onto the pre-mRNA in a stepwise manner. The E
complex contains U1 snRNP bound to the 5 splice
site, SF1 bound to the branchpoint, and U2AF65
and U2AF35 bound to the pyrimidine tract and 3
splice site AG, respectively. In the A complex,
SF1 is replaced by U2 snRNP at the branchpoint.
The U4/U6U5 tri-snRNP then enters to form the B
complex. A rearrangement occurs to form the
catalytically active C complex, in which U2 and
U6 interact, and U6 replaces U1 at the 5 splice
site.
3
Spliceosome assembly pathway. In the first step
of spliceosome assembly, U1 snRNP binds to the 5
splice site and U2 auxiliary factor (U2AF) binds
to the pyrimidine tract and 3 splice site YAG.
This complex commits the pre-mRNA to the splicing
pathway and is called the E, or early, complex.
Next, E complex is converted to A complex when
the U2 snRNP binds to the branchpoint.
Subsequently, B complex is formed when the U4,
U5, and U6 snRNPs enter the spliceosome as a
tri-snRNP particle. Finally, a massive
rearrangement occurs in which U6 replaces U1 at
the 5splice site, U6 and U2 interact, U5 bridges
the splice sites, and U1 and U4 become
destabilized. This rearranged spliceosome is
called the C complex and is catalytically active.
4
SR proteins act as molecular bridges in
spliceosome assembly
SR
SR
Branch site
Exon Enhancers
UNCURAC
5' splice site
3' splice site
A

• CAG GUAAGU
• A G

(U/C)nN AG G
Intron
Exon 2
Exon 1
Polypyrimidine Tract
5
Serine-Arginine-rich Splicing Proteins

• SR proteins are essential splicing factors that
  contain an RNA-binding domain (N-terminus) and an
  arginine/serine-rich domain (C-terminus)
• SR proteins play important roles in spliceosome
  assembly
• SR proteins also play important roles in defining
  the 5 and 3 splice sites
• SR proteins play important roles in alternative
  splicing in defining exons and weak splice sites

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7
SR Proteins

• SR proteins contain an RNA-binding domain and an
  arginine/serine-rich domain that functions to
  promote assembly of the spliceosome.

8

• Human SR Proteins

SRp20
SC35
SRp46
SRp54
9G8
Z
SRp30c
SF2/ASF
SRp40
RRM
SRp55
RRM-2
RS
SRp75
9
SR Proteins RS Domains

• SR proteins interact with each other and other
  splicing proteins by protein-protein interactions
  through the RS domains
• The phosphorylation state of the RS domains is
  critical to these interactions

10

• Human SR Proteins

SRp20
SC35
SRp46
SRp54
9G8
Z
SRp30c
SF2/ASF
SRp40
RRM
SRp55
RRM-2
RS
SRp75
11
SR Splicing Proteins

• SR proteins play important roles in spliceosome
  assembly through protein-protein interactions to
  bridge components of the spliceosome
• SR proteins also play important roles in defining
  the 5 and 3 splice sites by binding to specific
  sites in the pre-mRNA through the RNA-binding
  domain
• SR proteins play important roles in alternative
  splicing in defining exons and weak splice sites
  by binding specific sites, termed Exonic Splicing
  Enhancers (ESE) through the RNA-binding domains.

12
Schematic diagram of human SR proteins and SR
related proteins
13
RS domain-dependent activities of SR proteins

• Activity of SR proteins in spliceosome assembly

14
Recruitment of U1 snRNP to a 5 splice site An
SR protein bound to the upstream exon interacts
with U1-70K and recruits U1 snRNP to the 5
splice site
15
U2AF recruitment model An exonic enhancer-bound
SR protein interacts with the RS domain of
U2AF35, thereby recruiting U2AF65 to the pre-mRNA
16
SR proteins have been shown to participate in the
recruitment of the U4/U6U5 tri-snRNP, although
the mechanism by which they do so has not been
elucidated. Presumably this activity is mediated
by an interaction between an SR protein present
in the partially assembled spliceosome and a
component of the tri-snRNP. Two candidate
interaction targets of SR proteins are the
U5-100K and U4/U6U5-27K protein.
17
Model of spliceosome assembly using different SR
proteins
18
Model for Cross-Bridging The RS domain of
SF2/ASF may interact with the RS domains of
U1-70K and U2AF35 to facilitate cross-intron
bridging.
19
SR Proteins RS Domains

• SR proteins interact with each other and other
  splicing proteins by protein-protein interactions
  through the RS domains
• The phosphorylation state of the RS domains is
  critical to these interactions

20
SR Protein Specific Kinases

• SR Protein Kinase I- SRPK1
• Clk/Sty Kinase

21
SRPK1 and Clk/Sty Protein Kinases Show Distinct
Substrate Specificities for Serine/Arginine-rich
Splicing Factors
22
SRPK1 and Clk/Sty Protein Kinases Show Distinct
Substrate Specificities for Serine/Arginine-rich
Splicing Factors K.Colwill, L.L. Fengi, J.M.
Yeakley, G.D. Gish, J.F. Caceres, T. Pawson, and
X.-D. Fu J. BIOLOGICAL CHEMISTRY 271, 1996
23
SRPK1-mediated phosphorylation differentially
modulates the affinity of RS domain interactions
in vitro. In a GST pull-down assay,
mock-phosphorylated (-ATP) or SRPK1- treated
GST-ASF/SF2 (ATP) were bound to 35S-labeled-in
vitro translated U1-70k followed by analysis on
SDS-PAGE. Coomassie-stained bands are shown in
the top panel to illustrate the mobility shift
due to phosphorylation, and the lower panel shows
an autoradiograph of the same gel.
24
The Protein Kinase Clk/Sty Directly Modulates SR
Protein ActivityBoth Hyper- and
Hypophosphorylation Inhibit Splicing JAYENDRA
PRASAD,1 KAREN COLWILL, TONY PAWSON, AND JAMES L.
MANLEY MOLECULAR AND CELLULAR BIOLOGY,1999,
1969917000
25
The Protein Kinase Clk/Sty Directly Modulates SR
Protein Activity Both Hyper- and
Hypophosphorylation Inhibit Splicing

• The splicing of mammalian mRNA precursors
  requires both protein phosphorylation and
  dephosphorylation, involving modification of
  members of the SR protein family of splicing
  factors.
• Several kinases have been identified that can
  phosphorylate SR proteins in vitro, and
  transfection assays have provided evidence that
  at least one of these, Clk/Sty, can modulate
  splicing in vivo.
• By using purified recombinant Clk/Sty, a
  catalytically inactive mutant, and individual SR
  proteins, it was shown that Clk/Sty directly
  affects the activity of SR proteins, but not
  other essential splicing factors, in
  reconstituted splicing assays.
• Evidence is also provided that that both hyper-
  and hypophosphorylation inhibit SR protein
  splicing activity, repressing constitutive
  splicing.
• These findings indicate that Clk/Sty directly and
  specifically influences the activity of SR
  protein splicing factors and, importantly, show
  that both under- and over-phosphorylation of SR
  proteins can modulate splicing.

26
SR Proteins must be appropriately phosphorylated
to activate splicing
P
-P
27
Hyperphosphorylation of SR proteins results in
Splicing Inhibition
Clk recombinant Clk/Sty kinase ClkRcatalytically
inactive mutant Clk/Sty kinase
28
Hypophosphorylation Inhibits Splicing
Unphosphorylated GST-ASF/SF2 inhibits pre-mRNA
splicing in HeLa nuclear extracts
Unphosphorylated GST-ASF/SF2 inhibits spliceosome
assembly in HeLa nuclear extracts
29
SR Protein Localization

• Phosphorylation of SR proteins is required for
  their import into the nucleus, where they are
  stored in interchromatin granules (ICG) or
  speckles until they are required
• Phosphorylation of SR proteins in the ICG moves
  SR proteins to sites of active transcription, and
  they participate in spliceosome assembly and
  splice site definition
• Dephosphorylation by SR-specific phosphatases
  moves the SR back to the ICG

30
SR Protein Phosphorylation

• Both Hypo- and Hyper-Phosphorylation of SR
  proteins renders them inactive in splicesome
  assembly
• Inappropriately phosphorylated SR proteins remain
  in speckles (hypo-) or a diffuse distribution
  (hyper-) and do not move to sites of RNAP II
  transcription and splicing

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32
SR protein SC35 redistributes to a diffuse
nuclear localization upon expression of Clk/STY.
A-431 cells transiently transfected with
GFP-Clk/STY wild-type (A) show diffuse
localization of SC35 (B) in contrast to
untransfected cells, or cells transfected with
catalytically inactive mutant GFP-Clk/STY K190R
(C) in which SC35 is localized in nuclear
speckles.
33

• Mass Spectrometric and Kinetic Analysis of
  ASF/SF2 Phosphorylation by SRPK1 and Clk/Sty
• Adolfo Velazquez-Dones, Jonathan C. Hagopian,
  Chen-Ting Ma, Xiang-Yang Zhong, Huilin Zhou,
  Gourisankar Ghosh, Xiang-Dong Fu, and Joseph A.
  Adams
• JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, pp.
  4176141768, 2006.

34

• Assembly of the spliceosome requires the
  participation of SR proteins.
• The repeat regions (RS domains) are
  poly-phosphorylated by the SRPK and Clk/Sty
  families of kinases. The two families of kinases
  have distinct enzymatic properties, raising the
  question of how they may work to regulate the
  function of SR proteins in RNA metabolism in
  mammalian cells.
• The authors report the first mass spectral
  analysis of the RS domain of ASF/SF2, a
  prototypical SR protein.
• SRPK1 was responsible for efficient
  phosphorylation of a short stretch of amino acids
  in the N-terminal portion of the RS domain of
  ASF/SF2.
• Clk/Sty was able to transfer phosphate to all
  available serine residues in the RS domain,
  indicating that SR proteins may be phosphorylated
  by different kinases in a stepwise manner.
• Both kinases bind with high affinity and use
  fully processive catalytic mechanisms to achieve
  either restrictive or complete RS domain
  phosphorylation.
• These findings have important implications on the
  regulation of SR proteins in vivo by the SRPK and
  Clk/Sty families of kinases.

35
Ordered Multi-site Phosphorylation of the
SplicingFactor ASF/SF2 By SRPK1Ma,
Velazquez-Dones, Hagopian Ghosh, Fu and AdamsJ.
Mol. Biol. (2008) 376, 5568

• ASF/SF2, an SR protein is activated by multi-site
  phosphorylation of its C-terminal RS domain.
• The protein kinase responsible for this
  modification, SRPK1, catalyzes the selective
  phosphorylation of approximately a dozen serines
  in only the N-terminal portion of the RS domain
  (RS1).
• To unravel the nature of selective phosphate
  incorporation in ASF/SF2, region-specific
  phosphorylation in the RS domain was monitored as
  a function of reaction progress.
• Arg-to-Lys mutations were made at several
  positions to produce unique protease cleavage
  sites that separate the RS domain into
  identifiable N- and C-terminal phosphopeptides
  upon treatment with lysyl endoproteinase.
• These studies revealed that SRPK1 docks near the
  C-terminus of the RS1 segment and then moves in
  an N-terminal direction along the RS domain.
• These data suggest that SRPK1 uses a unique
  grab-and-pull mechanism to control the
  region-specific phosphorylation of its protein
  substrate.

36
WT-ASF/SF2 structure and ASF(5R1K) cleavage
pattern. (a) Domain organization of wt-ASF/SF2
and expected LysC cleavage fragments of
ASF(5R1K). LysC protease cleavage sites are shown
using open arrows.(b) Identification of
phosphorylated peptide fragments by
autoradiography. ASF(5R1K) was phosphorylated
using SRPK1 and then treated with LysC before
incubation with Ni2-Sepharose resin.
37
C-terminal phosphorylation is favored in
pulsechase experiments.Kinetic analysis of
region-specific phosphorylation.ASF(5R1K) and
SRPK1 werepre-equilibrated before the reaction
was initiated with 1 or100 µM 32PATP. Further
32P incorporation was stopped at8 s or 10 min by
the addition of 10 mM cold ATP. The reaction was
treated immediately with LysC and the products
were analyzed byautoradiography. The ratio of
32P in the N-terminal fragment to 32P in the
C-terminal fragment was plotted against the
phospho-content of uncleaved ASF(5R1K).
38
Results Conclusions

• After a reaction time of 8 s when only 2.2 sites
  are phosphorylated, approximately three times as
  much 32P is incorporated into the C-terminal
  compared to the N-terminal fragment.
• After a reaction time of 10 min when nearly all
  sites are phosphorylated, the ratio of N- to
  C-terminal fragments is close to the expected
  ratio of 0.7
• These findings suggest that phosphorylation in
  the C-terminal portion of RS1 is both
  thermodynamically and kinetically favored over
  N-terminal phosphorylation.

39
Grab-and-Pull Model
40
A Complex Signaling Pathway Regulates SRp38
Phosphorylation and Pre-mRNA Splicing in Response
to Heat ShockY. Shi and J. L. ManleyMolecular
Cell 28, 7990, October 12, 2007

• SRp38 is a member of the SR protein family and,
  when dephosphorylated, functions as a general and
  potent splicing repressor in response to heat
  shock.
• SRp38 is dephosphorylated by the phosphatase PP1,
  which is activated by dissociation of its
  inhibitors, including NIPP1.
• PP1 is targeted to SRp38 through direct
  interaction via its arginine/serine-rich (RS)
  domain.
• The specific dephosphorylation of SRp38 and not
  other SR proteins is determined largely by the
  low activities of SR protein kinases for it
  compared to other SR proteins.
• 14-3-3 proteins associate with SRp38 and protect
  it from dephosphorylation under non-stress
  conditions, but dissociate upon heat shock.
• This study delineates a complex mechanism
  involving multiple factors by which a stress
  signaling pathway regulates protein
  phosphorylation and, in turn, pre-mRNA splicing.

41
SRp38 Is Dephosphorylated by PP1 in Response to
Heat Shock
HeLa cells transfected with control, siPP1, or
siPP2A siRNAs were incubated at 37C (HS) or
heat-shocked at 44C for 40 min (HS). SRp38 was
monitored by western blotting. HeLa cells were
transfected with control, or siPP1a-, b-,
g-specific siRNAs, and the three PP1 isoforms and
actin were monitored by western blotting.
42
14-3-3 Proteins Protect SRp38 from
Dephosphorylation In Vitro and In Vivo
14-3-3 proteins protectSRp38 from
dephosphorylation in vivo. HeLa cells transfected
with wild-type or mutant SRp38 were untreated
(--) or heat-shocked (HS). Transfected SRp38 was
detected by western blotting.
43
A Complex Pathway Controls SRp38 in Response to
Heat Shock
Under normal conditions, phosphorylated SRp38 is
associated with 14-3-3 proteins, which help
protect it from dephosphorylation. PP1 activity
toward SRp38 is inhibited by its associated
protein, NIPP1. During heat shock, PP1
dissociates from NIPP1 and directly binds to and
dephosphorylates SRp38. Although ASF/SF2 and
other SR proteins can also be targeted by PP1,
the activities of SR protein kinases toward them
are high enough to maintain them in a
phosphorylated state, thereby establishing the
observed specificity for SRp38.
44
RNA Polymerase II Targets Pre-mRNA Splicing
Factors to Transcription Sites In Vivo
45
SR Proteins and Transcription

• SR protein ASF/SF2 is a general pre-mRNA splicing
  factor.
• ASF/SF2 is efficiently recruited to sites in the
  nucleus where adenovirus genes are transcribed
  and the resulting pre-mRNAs are processed.

Lindberg, M. Gama-Carvalho, M.Carmo-Fonseca and
J. Kreivi J. General Virology (2004), 85, 603608
46
very early
Cells were transfected with GFP-ASF/SF2 and
subsequently infected with Adenovirus. At
different times after infection, cells were fixed
and stained with an antibody to Ad-virus protein
that marks sites of Ad-virus transcription.
early
late
DRS
47
Phosphorylated RNA polymerase II stimulates
pre-mRNA splicing Y. Hirose, R, Tacke, and J. L.
Manley Genes Development 131234
Using reconstituted in vitro splicing assays, we
show that RNAP II functions directly in pre-mRNA
splicing by influencing very early steps in
assembly of the spliceosome. We demonstrate that
the phosphorylation status of the CTD
dramatically affects activity Hyperphosphorylated
RNAP II strongly activates splicing, whereas
hypophosphorylated RNAP II can inhibit the
reaction.
48
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49
Model for Co-transcriptional Splicing of pre-mRNA
50
SR Proteins Function in Coupling RNAP II
Transcription to Pre-mRNA SplicingR. Das, I. Yu,
Z. Zhang, M. P. Gygi, A.R. Krainer, S.P. Gygi,
and R.ReedMolecular Cell 26, 867881, 2007

• Transcription and splicing are functionally
  coupled, resulting in highly efficient splicing
  of RNAP II transcripts.
• To identify potential coupling factors, a
  comprehensive proteomic analysis of
  immuno-purified human RNAP II was carried out,
  and gt100 specifically associated proteins were
  identified by Mass Spectroscopy.
• Among these are the SR protein family of splicing
  factors and all of the components of U1 snRNP,
  but no other snRNPs or splicing factors.
• It is shown that SR proteins function in coupling
  transcription to splicing and evidence is
  provided that the mechanism involves
  cotranscriptional recruitment of SR proteins to
  RNAP II transcripts.

51
Evidence that U1 snRNP and SR Proteins Are the
Splicing Factors Associated with RNAP II
52
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53
Model for Cotranscriptional Recruitment of SR
Proteins and U1 snRNP to Nascent RNAP II
Transcripts
54
SR Proteins help to define 5 and 3 splice sites

• SR proteins bind to specific motifs within exons
  (ESE) to recruit the U1 snRNP to the 5 splice
  site and recruit U2AF35 to define the 3 splice
  site of some transcripts
• SR proteins have different RNA binding
  specificities

55
Cross-intron versus cross-exon complexes (a) SR
proteins function in a cross-intron recognition
complex by bridging between the interactions of
U1 snRNP bound to the upstream 5ss and U2AF65
and 35-kDa subunits bound to the polypyrimidine
tract (PPT) and the AG dinucleotide of the
downstream 3ss, respectively. Then, U2AF65
recruits U2 to the branch site sequence (BS).
(b) SR proteins also facilitate a cross-exon
recognition complex. The exons contain exonic
splicing enhancers (ESEs) that are binding sites
for SR proteins. When an SR protein binds to an
ESE, the SR protein recruits U1 snRNP to the
downstream 5ss, and U2AF65 and 35-kDa subunits
to the PPT and the 3 ss-AG dinucleotide,
respectively. In turn, U2AF65 recruits U2 snRNP
to the BS.
56
SR Proteins have Different RNA specificities
57
The role of SR proteins in mRNA splicing

• SR proteins bind to relatively short exonic and
  intronic sequences, usually 418 nt, which are
  generally found up to 150 bases from the
  regulated splice site.
• The RS domains of SR proteins are phosphorylated
  by several different kinases. The phosphorylation
  modulates proteinprotein interactions within the
  spliceosome, thereby contributing to dynamic
  structural reorganization during splicing.
• The binding of these proteins by means of their
  RNA recognition motif to the exonic and intronic
  sequences facilitates the recruitment of the
  basal splicing machinery to the splice junctions.

58
Substrate Specificities of SR Proteins in
Constitutive Splicing Are Determined by Their RNA
Recognition Motifs
59
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60
RNA Binding Motifs in RNA Binding Proteins
61
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62
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63
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64
Determination of RNA-binding sequence or
structural specificity for RNA binding proteins

• In vitro Functional SELEX

65
An increased specificity score matrix for the
prediction of SF2/ASF-specific exonic splicing
enhancers Philip J. Smith, Chaolin Zhang, Jinhua
Wang, Shern L. Chew, Michael Q. Zhang and Adrian
R. Krainer Human Molecular Genetics, 2006, Vol.
15, No. 16 24902508
1. Numerous disease-associated point mutations
exert their effects by disrupting the activity of
exonic splicing enhancers (ESEs). 2. A refined
functional SELEX screen for motifs that can act
as ESEs in response to the human SR protein
SF2/ASF was developed. 3. The test BRCA1 exon
under selection was internal in the test gene. A
naturally occurring heptameric ESE in BRCA1 exon
18 was replaced with two libraries of random
sequences, one seven nucleotides in length, the
other 14. 4. Following three rounds of selection
for in vitro splicing via internal exon
inclusion, new consensus motifs were derived.
5. Many winner sequences were demonstrated to be
functional ESEs in S100-extract- complementation
assays with recombinant SF2/ASF. 6. An increased
specificity score matrix was derived that
accurately predicts the exon-skipping phenotypes
of deleterious point mutations.
66
Experimental procedure for functional SELEX. The
SF2/ASF-specific BRCA1 exon 18 ESE was replaced
by 7 or 14 nt of randomized sequence
by overlap-extension PCR. In vitro-transcribed
RNA was incubated under splicing conditions in
HeLa S100 extract complemented by recombinant
SF2/ASF. Spliced mRNA molecules containing
SF2/ASF-responsive sequences (designated by the
white W in a black box) were purified from
denaturing polyacrylamide gels, and rebuilt into
full-length intron-containing constructs.
Following three rounds of selection, individual
ESE-containing clones (E) were sequenced and
consensus motifs and score matrices derived.
67
Splicing of the pre-mRNA pools following
selection (A) In vitro splicing of the n7 (lanes
79) and n14 (lanes 1012) winner pools following
three rounds of selection was performed in both
HeLa nuclear extract (NE), and S100 extract
complemented by recombinant SF2/ASF (A). As
controls, BRCA1 WT (lanes 13) and the nonsense
E1694X MT (lanes 46) were also spliced. The
structures of the precursor, intermediates and
products are indicated next to the autoradiogram.
The experiment was repeated three times and the
data normalized to BRCA1 WT levels of splicing
for exon inclusion (B) and inclusive
splicing (C). Black boxes indicate splicing in
nuclear extract, white boxes splicing in S100
complemented by recombinant SF2/ASF.
68
The SELEX winners comprise functional ESEs
69
The SELEX winners comprise functional ESEs
The winner sequences are functional
SF2/ASF-dependent ESEs. In vitro splicing of 10
n14 round-three winner sequences in HeLa nuclear
extract, S100 extract alone and S100 complemented
by recombinant SF2/ASF. The structures of the
precursor, intermediates and products are
indicated next to the autoradiograms.
70
SR Proteins Splicing

• SR proteins play essential roles in both
  constitutive and alternative splicing
• SR proteins interact with each other and other RS
  domain containing proteins by protein-protein
  interactions in the RS domains
• SR protein activity in splicing is modulated by
  phosphorylation
• SR serve as molecular bridges in splicesome
  assembly
• SR proteins bind to specific RNA sequences to
  recruit spliceosomal components to weak splice
  sites
• SR proteins couple transcription splicing