Splicing Regulation

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Splicing Regulation

• The role of SR proteins in spliceosome assembly

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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. Finally, 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.
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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 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.
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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
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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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SR Proteins

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

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• Human SR Proteins

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

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• Human SR Proteins

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

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

• Activity of SR proteins in spliceosome assembly

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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
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U2AF recruitment model An exonic enhancer-bound
SR protein interacts with the RS domain of
U2AF35, thereby recruiting U2AF65 to the pre-mRNA
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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.
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Model of spliceosome assembly using different SR
proteins
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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.
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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

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SR Protein Specific Kinases

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

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SRPK1 and Clk/Sty Protein Kinases Show Distinct
Substrate Specificities for Serine/Arginine-rich
Splicing Factors
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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
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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.
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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
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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. But evidence that a specific
kinase can directly affect the splicing activity
of SR proteins has been lacking. Here, by using
purified recombinant Clk/Sty, a catalytically
inactive mutant, and individual SR proteins, we
show that Clk/Sty directly affects the activity
of SR proteins, but not other essential splicing
factors, in reconstituted splicing assays. We
also provide evidence 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.
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SR Proteins must be appropriately phosphorylated
to activate splicing
SR-P
P
-P
-
-
UN SR
b-globin
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Hyperphosphorylation of SR proteins results in
Splicing Inhibition
Clk recombinant Clk/Sty kinase ClkRcatalytically
inactive mutant Clk/Sty kinase
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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
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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

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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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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.
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RNA Polymerase II Targets Pre-mRNA Splicing
Factors to Transcription Sites In Vivo
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SR Proteins and Transcription

• SR protein ASF/SF2 is a general pre-mRNA splicing
  factor as well as a regulator of alternative
  splicing. 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
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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
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Compartmentalization of RNA Processing Factors
within Nuclear Speckles P.J. Mintz and D.L.
Spector Journal of Structural Biology 129,
241251 (2000)
Colocalization of SC35 and b-tropomyosin RNA. (A,
B, C) Double labeling of transiently transfected
HeLa cells with anti-SC35 antibody (green) and
b-tropomyosin minigene RNA (red) reveals
colocalization of the subspeckles at the sites of
transcription of this RNA (yellow).
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Biochemical and genetic data have indicated an
interaction of the CTD of the largest subunit of
RNA polymerase II with components of the pre-mRNA
splicing. Using a microscopy approach to probe
the cell biological role of this interaction in
vivo, it was found that the CTD of RNAP II
targets pre-mRNA splicing factors to
transcription sites in vivo. Truncation of the
CTD prevents the accumulation of splicing factors
at a newly formed site of transcription and
blocks pre-mRNA splicing. The inhibitory effect
on splicing factor targeting was general, since
truncation of the CTD resulted in the failure of
not only SR proteins, but also snRNPs and snRNAs
to accumulate at a transcription site.
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Quantitative analysis of colocalization. Cells
were labeled with antibodies against SC35,
U2-B99, or the m3G-cap on snRNAs. The percentage
of cells exhibiting colocalization of splicing
factors and pem RNA was determined in mock
transfected cells or cells transfected with
either RNA pol II LS-WT or -D5. Results represent
means of five experiments.
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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 IIO strongly activates splicing, whereas
hypophosphorylated RNAP IIA can inhibit the
reaction.
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The Cycle of Phosphorylation and
Dephosphorylation of RNAP II CTD
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Model for Co-transcriptional Splicing of pre-mRNA
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Exon Tethering in Transcriptionby RNA Polymerase
II

• M.J. Dye,.N. Gromak, and N.J. Proudfoot
• Molecular Cell 21, 849859, March 17, 2006

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Exon Tethering in Transcriptionby RNA Polymerase
II

• There is an emerging consensus that RNA
  polymerase II (RNA Pol II) transcription and
  pre-mRNA processing are tightly coupled events.
  Here it is shown that exons flanking an intron
  that has been engineered to be cotranscriptionally
  cleaved are accurately and efficiently spliced
  together. These data underline the close coupling
  of processes in the initial stages of protein
  coding gene expression and provide evidence for a
  molecular tether connecting emergent splice sites
  in the pre-mRNA to transcribing RNA Pol II. This
  observation suggests that for some genes a
  continuous intron transcript is not required for
  pre-mRNA splicing in vivo.

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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

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SR Proteins have Different RNA specificities
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Substrate Specificities of SR Proteins in
Constitutive Splicing Are Determined by Their RNA
Recognition Motifs
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RNA Binding Motifs in RNA Binding Proteins
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Determination of RNA-binding sequence or
structural specificity for RNA binding proteins

• In vitro SELEX
• In vivo Yeast three-hybrid approach

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Specific SR protein-dependent splicing substrates
identified through genomic SELEX S. Kim, H. Shi,
D. Lee and J. T. Lis Nucleic Acids Research,
2003, 31955-1961
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B52-binding RNA transcripts of genomic sequences
B52, a Drosophila SR protein, has two RRM
domains that can bind specific RNAs with high
affinity as demonstrated by a previous SELEX
experiment that started with a random sequence
pool. The RNA aptamer sequence identified by
that study can function as an ESE to stimulate
the splicing of a fushi tarazu (ftz)-derived
substrate in vitro. Based on this observation,
B52-binding sites in the context of genomic
sequences were sought, to identify native
B52-dependent splicing substrates.
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B52-binding RNA transcripts of genomic sequences

• Drosophila genomic DNA from adult flies was
  amplified with primers to generate a sequence
  library that contains genomic segments of 200 bp
  flanked by a pair of constant regions, one of
  which contains a promoter for the T7 RNA
  polymerase.
• An RNA pool was transcribed from this library and
  used in the selection.
• The Drosophila genome (represented in multiple copies in a pool of 1010
  segments of 200 bp.
• Sequences of 200 nt are long enough to allow a
  homology search and to serve as probes to screen
  a genomic or cDNA library.

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B52-binding RNA transcripts of genomic sequences

• Selection and amplification were carried out
  against full-length, Baculovirus-expressed B52.
  After six rounds of selection, cloned cDNAs were
  generated from the final pool of selected RNA.
• DNA of 96 clones was transcribed to generate
  corresponding RNAs for individual electrophoretic
  mobility shift assays with the B52 protein.
• Eighty-seven out of the 96 clones tested showed
  binding to B52.
• Using the genomic SELEX process, the size of the
  candidate population was narrowed down to be
  further investigated in this study from the
  13,601 genes predicted in the Drosophila genome
  to a few dozen genes.

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An overview of the process leading to the
isolation of B52-dependent pre-mRNA splicing
substrates. The number of candidates in each step
is indicated.
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Identification of a B52-binding site on a
sequence segment selected by the genomic SELEX.
(A) B52 binding sites found in two segments by
the multiple alignment program ClustalW are shown
as are their mutated derivatives. Lower case
letters in the Mut' constructs indicate sequence
differences from the original isolates. (B) A
mobility shift assay with B52 shows the binding
site of segment 1-3 is critical for B52 binding.
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A yeast three-hybrid system
Transient Transfected RNA Hybrid
Library RNA
Transient Transfected Protein Hybrid 2

SR
MS2 RNA
B42 AD
MS2 coat protein
Stably Transfected Protein Hybrid 1
LEX A DBD
Selection Markers His, Ura, Trp and LacZ