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1.  Distinctive Features of Drosophila Alternative Splicing Factor RS Domain: Implication for Specific Phosphorylation, Shuttling, and Splicing Activation 
Molecular and Cellular Biology  2001;21(4):1345-1359.
The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5′ alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.
doi:10.1128/MCB.21.4.1345-1359.2001
PMCID: PMC99587  PMID: 11158320
2.  Exonic Splicing Enhancer-Dependent Selection of the Bovine Papillomavirus Type 1 Nucleotide 3225 3′ Splice Site Can Be Rescued in a Cell Lacking Splicing Factor ASF/SF2 through Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway 
Journal of Virology  2003;77(3):2105-2115.
Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5′ splice site alternatively splices to a proximal 3′ splice site (at nucleotide 3225) to express L2 or to a distal 3′ splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3′ splice sites and have been identified as regulating alternative 3′ splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3′ splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3′ splice site while increasing use of the distal 3′ splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3′ splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3′ splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.
doi:10.1128/JVI.77.3.2105-2115.2003
PMCID: PMC140879  PMID: 12525645
3.  Identification of Nuclear and Cytoplasmic mRNA Targets for the Shuttling Protein SF2/ASF 
PLoS ONE  2008;3(10):e3369.
The serine and arginine-rich protein family (SR proteins) are highly conserved regulators of pre-mRNA splicing. SF2/ASF, a prototype member of the SR protein family, is a multifunctional RNA binding protein with roles in pre-mRNA splicing, mRNA export and mRNA translation. These observations suggest the intriguing hypothesis that SF2/ASF may couple splicing and translation of specific mRNA targets in vivo. Unfortunately the paucity of endogenous mRNA targets for SF2/ASF has hindered testing of this hypothesis. Here, we identify endogenous mRNAs directly cross-linked to SF2/ASF in different sub-cellular compartments. Cross-Linking Immunoprecipitation (CLIP) captures the in situ specificity of protein-RNA interaction and allows for the simultaneous identification of endogenous RNA targets as well as the locations of binding sites within the RNA transcript. Using the CLIP method we identified 326 binding sites for SF2/ASF in RNA transcripts from 180 protein coding genes. A purine-rich consensus motif was identified in binding sites located within exon sequences but not introns. Furthermore, 72 binding sites were occupied by SF2/ASF in different sub-cellular fractions suggesting that these binding sites may influence the splicing or translational control of endogenous mRNA targets. We demonstrate that ectopic expression of SF2/ASF regulates the splicing and polysome association of transcripts derived from the SFRS1, PABC1, NETO2 and ENSA genes. Taken together the data presented here indicate that SF2/ASF has the capacity to co-regulate the nuclear and cytoplasmic processing of specific mRNAs and provide further evidence that the nuclear history of an mRNA may influence its cytoplasmic fate.
doi:10.1371/journal.pone.0003369
PMCID: PMC2556390  PMID: 18841201
4.  Roles for SR Proteins and hnRNP A1 in the Regulation of c-src Exon N1 
Molecular and Cellular Biology  2003;23(6):1874-1884.
The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5′ half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.
doi:10.1128/MCB.23.6.1874-1884.2003
PMCID: PMC149473  PMID: 12612063
5.  Overexpression of Essential Splicing Factor ASF/SF2 Blocks the Temporal Shift in Adenovirus Pre-mRNA Splicing and Reduces Virus Progeny Formation 
Journal of Virology  2000;74(19):9002-9009.
Expression of cytoplasmic mRNA from most adenovirus transcription units is subjected to a temporal regulation at the level of alternative pre-mRNA splicing. The general tendency is that splice site selection changes from proximal to distal late after infection. Interestingly, ASF/SF2, which is a prototypical member of the SR family of splicing factors, has the opposite effect on splice site selection, inducing an increase in proximal splice site usage. We have previously shown that SR proteins late during an adenovirus infection become partially inactivated as splicing regulatory proteins. A prediction from these results is that overexpression of an SR protein, such as ASF/SF2, during virus growth will interfere with virus replication by disturbing the balance of functional and nonfunctional ASF/SF2 in the infected cell. To test this hypothesis, we reconstructed a recombinant adenovirus expressing ASF/SF2 under the transcriptional control of a regulated promoter. The results show that, as predicted, induction of ASF/SF2 during lytic virus growth prevents the early to late shift in mRNA expression from both early (E1A and E1B) and late (L1) transcription units. Furthermore, ASF/SF2 overexpression blocks viral DNA replication and reduces selectively cytoplasmic accumulation of major late mRNA, resulting in a lower virus yield. Collectively, our results provide additional support for the hypothesis that viral control of SR protein function is important for the proper expression of viral proteins during lytic virus growth.
PMCID: PMC102096  PMID: 10982344
6.  Sam68 regulates EMT through alternative splicing–activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene 
The Journal of Cell Biology  2010;191(1):87-99.
Expression levels of SF2/ASF are controlled by Sam68 mediated activation of splicing-induced mRNA decay.
Epithelial-to-mesenchymal transition (EMT) and its reversal (MET) are crucial cell plasticity programs that act during development and tumor metastasis. We have previously shown that the splicing factor and proto-oncogene SF2/ASF impacts EMT/MET through production of a constitutively active splice variant of the Ron proto-oncogene. Using an in vitro model, we now show that SF2/ASF is also regulated during EMT/MET by alternative splicing associated with the nonsense-mediated mRNA decay pathway (AS-NMD). Overexpression and small interfering RNA experiments implicate the splicing regulator Sam68 in AS-NMD of SF2/ASF transcripts and in the choice between EMT/MET programs. Moreover, Sam68 modulation of SF2/ASF splicing appears to be controlled by epithelial cell–derived soluble factors that act through the ERK1/2 signaling pathway to regulate Sam68 phosphorylation. Collectively, our results reveal a hierarchy of splicing factors that integrate splicing decisions into EMT/MET programs in response to extracellular stimuli.
doi:10.1083/jcb.201001073
PMCID: PMC2953442  PMID: 20876280
7.  Multiple ASF/SF2 Sites in the Human Papillomavirus Type 16 (HPV-16) E4-Coding Region Promote Splicing to the Most Commonly Used 3′-Splice Site on the HPV-16 Genome▿  
Journal of Virology  2010;84(16):8219-8230.
Our results presented here demonstrate that the most abundant human papillomavirus type 16 (HPV-16) mRNAs expressing the viral oncogenes E6 and E7 are regulated by cellular ASF/SF2, itself defined as a proto-oncogene and overexpressed in cervical cancer cells. We show that the most frequently used 3′-splice site on the HPV-16 genome, site SA3358, which is used to produce primarily E4, E6, and E7 mRNAs, is regulated by ASF/SF2. Splice site SA3358 is immediately followed by 15 potential binding sites for the splicing factor ASF/SF2. Recombinant ASF/SF2 binds to the cluster of ASF/SF2 sites. Mutational inactivation of all 15 sites abolished splicing to SA3358 and redirected splicing to the downstream-located, late 3′-splice site SA5639. Overexpression of a mutant ASF/SF2 protein that lacks the RS domain, also totally inhibited the usage of SA3358 and redirected splicing to the late 3′-splice site SA5639. The 15 ASF/SF2 binding sites could be replaced by an ASF/SF2-dependent, HIV-1-derived splicing enhancer named GAR. This enhancer was also inhibited by the mutant ASF/SF2 protein that lacks the RS domain. Finally, silencer RNA (siRNA)-mediated knockdown of ASF/SF2 caused a reduction in spliced HPV-16 mRNA levels. Taken together, our results demonstrate that the major HPV-16 3′-splice site SA3358 is dependent on ASF/SF2. SA3358 is used by the most abundantly expressed HPV-16 mRNAs, including those encoding E6 and E7. High levels of ASF/SF2 may therefore be a requirement for progression to cervical cancer. This is supported by our earlier findings that ASF/SF2 is overexpressed in high-grade cervical lesions and cervical cancer.
doi:10.1128/JVI.00462-10
PMCID: PMC2916536  PMID: 20519389
8.  Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity 
The Journal of Cell Biology  1997;138(2):225-238.
SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.
PMCID: PMC2138183  PMID: 9230067
9.  Deletion of the N-terminus of SF2/ASF Permits RS-Domain-Independent Pre-mRNA Splicing 
PLoS ONE  2007;2(9):e854.
Serine/arginine-rich (SR) proteins are essential splicing factors with one or two RNA-recognition motifs (RRMs) and a C-terminal arginine- and serine-rich (RS) domain. SR proteins bind to exonic splicing enhancers via their RRM(s), and from this position are thought to promote splicing by antagonizing splicing silencers, recruiting other components of the splicing machinery through RS-RS domain interactions, and/or promoting RNA base-pairing through their RS domains. An RS domain tethered at an exonic splicing enhancer can function as a splicing activator, and RS domains play prominent roles in current models of SR protein functions. However, we previously reported that the RS domain of the SR protein SF2/ASF is dispensable for in vitro splicing of some pre-mRNAs. We have now extended these findings via the identification of a short inhibitory domain at the SF2/ASF N-terminus; deletion of this segment permits splicing in the absence of this SR protein's RS domain of an IgM pre-mRNA substrate previously classified as RS-domain-dependent. Deletion of the N-terminal inhibitory domain increases the splicing activity of SF2/ASF lacking its RS domain, and enhances its ability to bind pre-mRNA. Splicing of the IgM pre-mRNA in S100 complementation with SF2/ASF lacking its RS domain still requires an exonic splicing enhancer, suggesting that an SR protein RS domain is not always required for ESE-dependent splicing activation. Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing.
doi:10.1371/journal.pone.0000854
PMCID: PMC1952110  PMID: 17786225
10.  A Splicing-Independent Function of SF2/ASF in MicroRNA Processing 
Molecular Cell  2010;38(1):67-77.
SUMMARY
Both splicing factors and microRNAs are important regulatory molecules that play key roles in post-transcriptional gene regulation. By miRNA deep sequencing, we identified 40 miRNAs that are differentially expressed upon ectopic overexpression of the splicing factor SF2/ASF. Here we show that SF2/ASF and one of its upregulated microRNAs (miR-7) can form a negative feedback loop: SF2/ASF promotes miR-7 maturation, and mature miR-7 in turn targets the 3′UTR of SF2/ASF to repress its translation. Enhanced microRNA expression is mediated by direct interaction between SF2/ASF and the primary miR-7 transcript to facilitate Drosha cleavage and is independent of SF2/ASF’s function in splicing. Other miRNAs, including miR-221 and miR-222, may also be regulated by SF2/ASF through a similar mechanism. These results underscore a function of SF2/ASF in pri-miRNA processing and highlight the potential coordination between splicing control and miRNA-mediated gene repression in gene regulatory networks.
doi:10.1016/j.molcel.2010.02.021
PMCID: PMC3395997  PMID: 20385090
11.  Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF 
Nucleic Acids Research  2010;38(19):6684-6696.
CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200tr) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.
doi:10.1093/nar/gkq554
PMCID: PMC2965252  PMID: 20558599
12.  Nuclear Export and Retention Signals in the RS Domain of SR Proteins 
Molecular and Cellular Biology  2002;22(19):6871-6882.
Splicing factors of the SR protein family share a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RS domain, which is extensively phosphorylated, promotes protein-protein interactions and directs subcellular localization and—in certain situations—nucleocytoplasmic shuttling of individual SR proteins. We analyzed mutant versions of human SF2/ASF in which the natural RS repeats were replaced by RD or RE repeats and compared the splicing and subcellular localization properties of these proteins to those of SF2/ASF lacking the entire RS domain or possessing a minimal RS domain consisting of 10 consecutive RS dipeptides (RS10). In vitro splicing of a pre-mRNA that requires an RS domain could take place when the mutant RD, RE, or RS10 domain replaced the natural domain. The RS10 version of SF2/ASF shuttled between the nucleus and the cytoplasm in the same manner as the wild-type protein, suggesting that a tract of consecutive RS dipeptides, in conjunction with the RRMs of SF2/ASF, is necessary and sufficient to direct nucleocytoplasmic shuttling. However, the SR protein SC35 has two long stretches of RS repeats, yet it is not a shuttling protein. We demonstrate the presence of a dominant nuclear retention signal in the RS domain of SC35.
doi:10.1128/MCB.22.19.6871-6882.2002
PMCID: PMC134038  PMID: 12215544
13.  SF2/ASF Binds the Human Papillomavirus Type 16 Late RNA Control Element and Is Regulated during Differentiation of Virus-Infected Epithelial Cells 
Journal of Virology  2004;78(19):10598-10605.
Pre-mRNA splicing occurs in the spliceosome, which is composed of small ribonucleoprotein particles (snRNPs) and many non-snRNP components. SR proteins, so called because of their C-terminal arginine- and serine-rich domains (RS domains), are essential members of this class. Recruitment of snRNPs to 5′ and 3′ splice sites is mediated and promoted by SR proteins. SR proteins also bridge splicing factors across exons to help to define these units and have a central role in alternative and enhancer-dependent splicing. Here, we show that the SR protein SF2/ASF is part of a complex that forms upon the 79-nucleotide negative regulatory element (NRE) that is thought to be pivotal in posttranscriptional regulation of late gene expression in human papillomavirus type 16 (HPV-16). However, the NRE does not contain any active splice sites, is located in the viral late 3′ untranslated region, and regulates RNA-processing events other than splicing. The level of expression and extent of phosphorylation of SF2/ASF are upregulated with epithelial differentiation, as is subcellular distribution, specifically in HPV-16-infected epithelial cells, and expression levels are controlled, at least in part, by the virus transcription regulator E2.
doi:10.1128/JVI.78.19.10598-10605.2004
PMCID: PMC516382  PMID: 15367627
14.  Asf1 Can Promote Trimethylation of H3 K36 by Set2▿  
Molecular and Cellular Biology  2010;30(5):1116-1129.
Asf1 is a conserved histone H3/H4 chaperone that can assemble and disassemble nucleosomes and promote histone acetylation. Set2 is an H3 K36 methyltransferase. The functions of these proteins intersect in the context of transcription elongation by RNA polymerase II: both contribute to the establishment of repressive chromatin structures that inhibit spurious intragenic transcription. Here we characterize further interactions between budding yeast (Saccharomyces cerevisiae) Asf1 and Set2 using assays of intragenic transcription, H3/H4 posttranslational modification, coding region cross-linking of Asf1 and Set2, and cooccurrence of Asf1 and Set2 in protein complexes. We find that at some genes Asf1 and Set2 control chromatin metabolism as components of separate pathways. However, the existence of a low-abundance complex containing both proteins suggests that Asf1 and Set2 can more directly collaborate in chromatin regulation. Consistent with this possibility, we show that Asf1 stimulates Set2 occupancy of the coding region of a highly transcribed gene by a mechanism that depends on Asf1 binding to H3/H4. This function of Asf1 promotes the switch from di- to trimethylation of H3 K36 at that gene. These results support the view that Set2 function in chromatin metabolism can intimately involve histone chaperone Asf1.
doi:10.1128/MCB.01229-09
PMCID: PMC2820888  PMID: 20048053
15.  Disturbed Expression of Splicing Factors in Renal Cancer Affects Alternative Splicing of Apoptosis Regulators, Oncogenes, and Tumor Suppressors 
PLoS ONE  2010;5(10):e13690.
Background
Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions.
Methodology/Principal Findings
Using real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1.
Conclusions/Significance
We found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis.
doi:10.1371/journal.pone.0013690
PMCID: PMC2972751  PMID: 21082031
16.  Substrate Specificities of SR Proteins in Constitutive Splicing Are Determined by Their RNA Recognition Motifs and Composite Pre-mRNA Exonic Elements 
Molecular and Cellular Biology  1999;19(3):1853-1863.
We report striking differences in the substrate specificities of two human SR proteins, SF2/ASF and SC35, in constitutive splicing. β-Globin pre-mRNA (exons 1 and 2) is spliced indiscriminately with either SR protein. Human immunodeficiency virus tat pre-mRNA (exons 2 and 3) and immunoglobulin μ-chain (IgM) pre-mRNA (exons C3 and C4) are preferentially spliced with SF2/ASF and SC35, respectively. Using in vitro splicing with mutated or chimeric derivatives of the tat and IgM pre-mRNAs, we defined specific combinations of segments in the downstream exons, which mediate either positive or negative effects to confer SR protein specificity. A series of recombinant chimeric proteins consisting of domains of SF2/ASF and SC35 in various combinations was used to localize trans-acting domains responsible for substrate specificity. The RS domains of SF2/ASF and SC35 can be exchanged without effect on substrate specificity. The RNA recognition motifs (RRMs) of SF2/ASF are active only in the context of a two-RRM structure, and RRM2 has a dominant role in substrate specificity. In contrast, the single RRM of SC35 can function alone, but its substrate specificity can be influenced by the presence of an additional RRM. The RRMs behave as modules that, when present in different combinations, can have positive, neutral, or negative effects on splicing, depending upon the specific substrate. We conclude that SR protein-specific recognition of specific positive and negative pre-mRNA exonic elements via one or more RRMs is a crucial determinant of the substrate specificity of SR proteins in constitutive splicing.
PMCID: PMC83978  PMID: 10022872
17.  Structural, functional, and protein binding analyses of bovine papillomavirus type 1 exonic splicing enhancers. 
Journal of Virology  1997;71(12):9096-9107.
Alternative splicing plays an important role in regulation of bovine papillomavirus type 1 (BPV-1) gene expression. We have recently identified in BPV-1 late pre-mRNAs two purine-rich exonic splicing enhancers (SE1 and SE2) which also stimulate splicing of a Drosophila doublesex (dsx) pre-mRNA containing a suboptimal 3' splice site. In vivo studies now demonstrate that both SE1 and SE2 are required for preferential use of the BPV-1 nucleotide (nt) 3225 3' splice site in nonpermissive cells. Deletion or mutation of either element in a BPV-1 late pre-mRNA switches splicing to the late-specific alternative 3' splice site at nt 3605. To investigate the sequence specificity of these exonic splicing enhancers, various mutant SE1 or SE2 elements were connected to dsx pre-mRNAs and tested for their stimulatory effects on dsx pre-mRNA splicing in vitro. Substitution of U residues for either A or G residues in and around potential ASF/SF2 binding sites in SE1 or SE2 resulted in a significant reduction of splicing enhancer activity. However, the G-to-U substitutions in both enhancers had the largest effect, reducing splicing to near control levels. Further in vitro analyses showed that splicing enhancement by SE2 could be competed with excess unlabeled SE2 RNA, indicating that SE2 activity in HeLa nuclear extracts is mediated by trans-acting factors. UV cross-linking plus immunoprecipitation assays showed that both wild-type SE1 and SE2 RNAs could bind directly to purified HeLa SR proteins SRp30a (ASF/SF2), SRp55, and SRp75. UV cross-linking experiments also identified a 23-kDa protein which binds to SE2 but not SE1. This protein is present in both HeLa nuclear extracts and S100 extracts but absent from SR protein preparations, suggesting that it is not a classical SR protein. Mutant SE elements (containing G- to U-mutations) which had minimal splicing enhancer activity also had very weak binding capacity for these proteins, strongly suggesting that the binding of these proteins is required for splicing enhancer function.
PMCID: PMC230210  PMID: 9371566
18.  SF2/ASF Autoregulation Involves Multiple Layers of Post-transcriptional and Translational Control 
SF2/ASF is a prototypical SR protein, with important roles in splicing and other aspects of mRNA metabolism. SFRS1 (SF2/ASF) is a potent proto-oncogene with abnormal expression in many tumors. We found that SF2/ASF negatively autoregulates its expression to maintain homeostatic levels. We characterized six SF2/ASF alternatively spliced mRNA isoforms: the major isoform encodes full-length protein, whereas the others are either retained in the nucleus or degraded by NMD. Unproductive splicing accounts for only part of the autoregulation, which occurs primarily at the translational level. The effect is specific to SF2/ASF and requires RRM2. The ultraconserved 3′UTR is necessary and sufficient for downregulation. SF2/ASF overexpression shifts the distribution of target mRNA towards mono-ribosomes, and translational repression is partly independent of Dicer and a 5′ cap. Thus, multiple post-transcriptional and translational mechanisms are involved in fine-tuning the expression of SF2/ASF.
doi:10.1038/nsmb.1750
PMCID: PMC2921916  PMID: 20139984
19.  XE7: A novel splicing factor that interacts with ASF/SF2 and ZNF265 
Nucleic Acids Research  2006;34(17):4976-4986.
Pre-mRNA splicing is performed by the spliceosome. SR proteins in this macromolecular complex are essential for both constitutive and alternative splicing. By using the SR-related protein ZNF265 as bait in a yeast two-hybrid screen, we pulled out the uncharacterized human protein XE7, which is encoded by a pseudoautosomal gene. XE7 had been identified in a large-scale proteomic analysis of the human spliceosome. It consists of two different isoforms produced by alternative splicing. The arginine/serine (RS)-rich region in the larger of these suggests a role in mRNA processing. Herein we show for the first time that XE7 is an alternative splicing regulator. XE7 interacts with ZNF265, as well as with the essential SR protein ASF/SF2. The RS-rich region of XE7 dictates both interactions. We show that XE7 localizes in the nucleus of human cells, where it colocalizes with both ZNF265 and ASF/SF2, as well as with other SR proteins, in speckles. We also demonstrate that XE7 influences alternative splice site selection of pre-mRNAs from CD44, Tra2-β1 and SRp20 minigenes. We have thus shown that the spliceosomal component XE7 resembles an SR-related splicing protein, and can influence alternative splicing.
doi:10.1093/nar/gkl660
PMCID: PMC1635291  PMID: 16982639
20.  RNA splicing factors regulated by HPV16 during cervical tumour progression 
The Journal of pathology  2009;219(3):383-391.
The most prevalent human papillomaviruses (HPVs) causing cervical disease are the “high risk” HPV types 16 and 18. All papillomaviruses express a transcription factor, E2 that can regulate viral and cellular gene expression. Recently, we demonstrated high risk HPV E2-mediated transcriptional transactivation of SF2/ASF. This essential oncoprotein is a key member of a family of proteins, the SR proteins that regulate constitutive and alternative splicing. Tight control of RNA splicing is necessary for production of wild type proteins. So, aberrant expression of SR proteins is involved in the aetiology of a range of human diseases, including cancer. Here we demonstrate epithelial differentiation-specific control of SF2/ASF in HPV16 infected keratinocytes in organotypic raft culture and in low grade cervical lesions (CIN1). Further, we demonstrate HPV16 infection/differentiation-induced up-regulation of a specific subset of SR proteins and present evidence that HPV16 E2 controls expression of SRp20, SC35 and SRp75. Using a series of cell lines that model cervical tumour progression we show SF2/ASF, SRp20 and SC35 are specifically up-regulated in a model of cervical tumour progression. These SR proteins are also overexpressed in high grade cervical lesions, indicating that they may all have oncogenic functions. SR proteins could be useful biomarkers for HPV-associated disease.
doi:10.1002/path.2608
PMCID: PMC2779514  PMID: 19718710
human papillomavirus type 16; cervix; CIN; immunohistochemistry; SR proteins; SF2/ASF; HPV16 E2
21.  Regulation of Vascular Endothelial Growth Factor (VEGF) Splicing from Pro-angiogenic to Anti-angiogenic Isoforms 
The Journal of Biological Chemistry  2009;285(8):5532-5540.
Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF165, and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF165b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF165 and less VEGF165b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.
doi:10.1074/jbc.M109.074930
PMCID: PMC2820781  PMID: 19906640
Growth Factors; RNA/Splicing; ASF/SF2; Angiogenesis; SRPK1; VEGF
22.  Specific combinations of SR proteins associate with single pre-messenger RNAs in vivo and contribute different functions 
The Journal of Cell Biology  2009;184(4):555-568.
Serine/arginine-rich (SR) proteins are required for messenger RNA (mRNA) processing, export, surveillance, and translation. We show that in Chironomus tentans, nascent transcripts associate with multiple types of SR proteins in specific combinations. Alternative splicing factor (ASF)/SF2, SC35, 9G8, and hrp45/SRp55 are all present in Balbiani ring (BR) pre-messenger ribonucleoproteins (mRNPs) preferentially when introns appear in the pre-mRNA and when cotranscriptional splicing takes place. However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences. All four SR proteins are associated with the BR mRNPs during export to the cytoplasm. Interference with SC35 indicates that SC35 is important for the coordination of splicing, transcription, and 3′ end processing and also for nucleocytoplasmic export. ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with monoribosomes. Thus, individual endogenous pre-mRNPs/mRNPs bind multiple types of SR proteins during transcription, and these SR proteins accompany the mRNA and play different roles during the gene expression pathway in vivo.
doi:10.1083/jcb.200806156
PMCID: PMC2654125  PMID: 19221196
23.  NSrp70 is a novel nuclear speckle-related protein that modulates alternative pre-mRNA splicing in vivo 
Nucleic Acids Research  2011;39(10):4300-4314.
Nuclear speckles are known to be the storage sites of mRNA splicing regulators. We report here the identification and characterization of a novel speckle protein, referred to as NSrp70, based on its subcellular localization and apparent molecular weight. This protein was first identified as CCDC55 by the National Institutes of Health Mammalian Gene Collection, although its function has not been assigned. NSrp70 was colocalized and physically interacted with SC35 and ASF/SF2 in speckles. NSrp70 has a putative RNA recognition motif, the RS-like region, and two coiled-coil domains, suggesting a role in RNA processing. Accordingly, using CD44, Tra2β1 and Fas constructs as splicing reporter minigenes, we found that NSrp70 modulated alternative splice site selection in vivo. The C-terminal 10 amino acids (531–540), including 536RD537, were identified as a novel nuclear localization signal, and the region spanning 290–471 amino acids was critical for speckle localization and binding to SC35 and ASF/SF2. The N-terminal region (107–161) was essential for the pre-mRNA splicing activity. Finally, we found that knockout of NSrp70 gene in mice led to a lack of progeny, including fetal embryos. Collectively, we demonstrate that NSrp70 is a novel splicing regulator and essentially required early stage of embryonic development.
doi:10.1093/nar/gkq1267
PMCID: PMC3105421  PMID: 21296756
24.  Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF. 
Molecular and Cellular Biology  1993;13(5):2993-3001.
The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.
Images
PMCID: PMC359692  PMID: 8474457
25.  An RNA Splicing Enhancer-Like Sequence Is a Component of a Splicing Inhibitor Element from Rous Sarcoma Virus 
Molecular and Cellular Biology  1998;18(6):3103-3111.
The accumulation in infected cells of large amounts of unspliced viral RNA for use as mRNA and genomic RNA is a hallmark of retrovirus replication. The negative regulator of splicing (NRS) is a long cis-acting RNA element in Rous sarcoma virus that contributes to unspliced RNA accumulation through splicing inhibition. One of two critical sequences located in the NRS 3′ region resembles a minor class 5′ splice site and is required for U11 small nuclear ribonucleoprotein (snRNP) binding to the NRS. The second is a purine-rich region in the 5′ half that interacts with the splicing factor SF2/ASF. In this study we investigated the possibility that this purine-rich region provides an RNA splicing enhancer function required for splicing inhibition. In vitro, the NRS acted as a potent, orientation-dependent enhancer of Drosophila doublesex pre-mRNA splicing, and enhancer activity mapped to the purine-rich domain. Analysis of a number of site-directed and deletion mutants indicated that enhancer activity was diffusely located throughout a 60-nucleotide area but only the activity associated with a short region previously shown to bind SF2/ASF correlated with efficient splicing inhibition. The significance of the enhancer activity to splicing inhibition was demonstrated by using chimeras in which two authentic enhancers (ASLV and FP) were substituted for the native NRS purine region. In each case, splicing inhibition in transfected cells was restored to levels approaching that observed for the NRS. The observation that a nonfunctional version of the FP enhancer (FPD) that does not bind SF2/ASF also fails to block splicing when paired with the NRS 3′ region supports the notion that SF2/ASF binding to the NRS is relevant, but other SR proteins may substitute if an appropriate binding site is supplied. Our results are consistent with a role for the purine region in facilitated snRNP binding to the NRS via SF2/ASF.
PMCID: PMC108892  PMID: 9584151

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