scholarly journals Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

1996 ◽  
Vol 16 (5) ◽  
pp. 2325-2331 ◽  
Author(s):  
R R Gontarek ◽  
D Derse
Keyword(s):  
Alternative Splicing ◽  
Exon Skipping ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Exonic Splicing Enhancer ◽  
Exon Inclusion ◽  
Alternative Mrna Splicing ◽  
Splicing Enhancer

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.

scholarly journals Roles of hnRNP A1, SR Proteins, and p68 Helicase in c-H-ras Alternative Splicing Regulation

2003 ◽  
Vol 23 (8) ◽  
pp. 2927-2941 ◽  
Author(s):  
Sònia Guil ◽  
Renata Gattoni ◽  
Montserrat Carrascal ◽  
Joaquín Abián ◽  
James Stévenin ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Sr Proteins ◽  
Splicing Regulation ◽  
Hnrnp A1 ◽  
Carboxy Terminus ◽  
Extracellular Signals ◽  
Nuclear Extracts ◽  
Acting In Concert

ABSTRACT Human ras genes play central roles in coupling extracellular signals with complex intracellular networks controlling proliferation, differentiation, and apoptosis, among others processes. c-H-ras pre-mRNA can be alternatively processed into two mRNAs due to the inclusion or exclusion of the alternative exon IDX; this renders two proteins, p21H-Ras and p19H-RasIDX, which differ only at the carboxy terminus. Here, we have characterized some of the cis-acting sequences and trans-acting factors regulating IDX splicing. A downstream intronic silencer sequence (rasISS1), acting in concert with IDX, negatively regulates upstream intron splicing. This effect is mediated, at least in part, by the binding of hnRNP A1. Depletion and add-back experiments in nuclear extracts have confirmed hnRNP A1's inhibitory role in IDX splicing. Moreover, the addition of two SR proteins, SC35 and SRp40, can counteract this inhibition by strongly promoting the splicing of the upstream intron both in vivo and in vitro. Further, the RNA-dependent helicase p68 is also associated with both IDX and rasISS1 RNA, and suppression of p68 expression in HeLa cells by RNAi experiments results in a marked increase of IDX inclusion in the endogenous mRNA, suggesting a role for this protein in alternative splicing regulation.

scholarly journals Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF

1993 ◽  
Vol 13 (5) ◽  
pp. 2993-3001
Author(s):  
A Mayeda ◽  
D M Helfman ◽  
A R Krainer
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factor ◽  
Alternative Exon ◽  
Hnrnp A1 ◽  
Exon Inclusion ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

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.

scholarly journals Identification of a Bidirectional Splicing Enhancer: Differential Involvement of SR Proteins in 5′ or 3′ Splice Site Activation

1999 ◽  
Vol 19 (11) ◽  
pp. 7347-7356 ◽  
Author(s):  
Cyril F. Bourgeois ◽  
Michel Popielarz ◽  
Georges Hildwein ◽  
James Stevenin
Keyword(s):  
Splice Site ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Wild Type ◽  
Adenovirus E1a ◽  
Differential Involvement ◽  
Splicing Enhancer

ABSTRACT The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5′ splice sites and of one major or one minor 3′ splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5′ splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5′ splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3′ splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5′ splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

scholarly journals Human Autoimmune Sera as Molecular Probes for the Identification of an Autoantigen Kinase Signaling Pathway

2002 ◽  
Vol 196 (9) ◽  
pp. 1213-1226 ◽  
Author(s):  
Makoto Kamachi ◽  
Truc M. Le ◽  
Susan J. Kim ◽  
Meghan E. Geiger ◽  
Paul Anderson ◽  
...  
Keyword(s):  
Topoisomerase I ◽  
Molecular Probes ◽  
Splicing Factors ◽  
Apoptotic Cells ◽  
Sr Protein ◽  
Small Nuclear Ribonucleoprotein ◽  
Alternative Mrna Splicing ◽  
U1 Snrnp

Using human autoimmune sera as molecular probes, we previously described the association of phosphorylated serine/arginine splicing factors (SR splicing factors) with the U1-small nuclear ribonucleoprotein (U1-snRNP) and U3-small nucleolar RNP (snoRNP) in apoptotic cells. SR proteins are highly conserved autoantigens whose activity is tightly regulated by reversible phosphorylation of serine residues by at least eight different SR protein kinase kinases (SRPKs), including SRPK1, SRPK2, and the scleroderma autoantigen topoisomerase I. In this report, we demonstrate that only one of the known SRPKs, SRPK1, is associated with the U1-snRNP autoantigen complex in healthy and apoptotic cells. SRPK1 is activated early during apoptosis, followed by caspase-mediated proteolytic inactivation at later time points. SRPKs are cleaved in vivo after multiple apoptotic stimuli, and cleavage can be inhibited by overexpression of bcl-2 and bcl-xL, and by exposure to soluble peptide caspase inhibitors. Incubation of recombinant caspases with in vitro–translated SRPKs demonstrates that SRPK1 and SRPK2 are in vitro substrates for caspases-8 and -9, respectively. In contrast, topoisomerase I is cleaved by downstream caspases (-3 and -6). Since each of these SRPKs sits at a distinct checkpoint in the caspase cascade, SRPKs may serve an important role in signaling pathways governing apoptosis, alternative mRNA splicing, SR protein trafficking, RNA stability, and possibly the generation of autoantibodies directed against splicing factors.

scholarly journals CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2 and promotes growth and metastasis of pancreatic cancer

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Shi Chen ◽  
Can Yang ◽  
Zu-Wei Wang ◽  
Jian-Fei Hu ◽  
Jing-Jing Pan ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Alternative Splicing ◽  
Prognostic Value ◽  
Phosphorylation Site ◽  
Exon Skipping ◽  
Ductal Adenocarcinoma ◽  
Normal Tissues ◽  
Exon 10

Abstract Background Both aberrant alternative splicing and m6A methylation play complicated roles in the development of pancreatic cancer (PC), while the relationship between these two RNA modifications remains unclear. Methods RNA sequencing (RNA-seq) was performed using 15 pairs of pancreatic ductal adenocarcinoma (PDAC) tissues and corresponding normal tissues, and Cdc2-like kinases 1 (CLK1) was identified as a significantly upregulated alternative splicing related gene. Real-time quantitative PCR (qPCR) and western blotting were applied to determine the CLK1 levels. The prognostic value of CLK1 was elucidated by Immunohistochemistry (IHC) analyses in two independent PDAC cohorts. The functional characterizations and mechanistic insights of CLK1 in PDAC growth and metastasis were evaluated with PDAC cell lines and nude mice. SR-like splicing factors5250-Ser (SRSF5250-Ser) was identified as an important target phosphorylation site by phosphorylation mass spectrometry. Through transcriptome sequencing, Methyltransferase-like 14exon10 (METTL14exon10) and Cyclin L2exon6.3 skipping were identified as key alternative splicing events regulated by the CLK1-SRSF5 axis. RIP assays, RNA-pulldown and CLIP-qPCR were performed to confirm molecular interactions and the precise binding sites. The roles of the shift of METTL14exon 10 and Cyclin L2exon6.3 skipping were surveyed. Results CLK1 expression was significantly increased in PDAC tissues at both the mRNA and protein levels. High CLK1 expression was associated with poor prognosis. Elevated CLK1 expression promoted growth and metastasis of PC cells in vitro and in vivo. Mechanistically, CLK1 enhanced phosphorylation on SRSF5250-Ser, which inhibited METTL14exon10 skipping while promoted Cyclin L2exon6.3 skipping. In addition, aberrant METTL14exon 10 skipping enhanced the N6-methyladenosine modification level and metastasis, while aberrant Cyclin L2exon6.3 promoted proliferation of PDAC cells. Conclusions The CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2, which promotes growth and metastasis and regulates m6A methylation of PDAC cells. This study suggests the potential prognostic value and therapeutic targeting of this pathway in PDAC patients.

scholarly journals Optimization of a Weak 3′ Splice Site Counteracts the Function of a Bovine Papillomavirus Type 1 Exonic Splicing Suppressor In Vitro and In Vivo

2000 ◽  
Vol 74 (13) ◽  
pp. 5902-5910 ◽  
Author(s):  
Zhi-Ming Zheng ◽  
Jesse Quintero ◽  
Eric S. Reid ◽  
Christian Gocke ◽  
Carl C. Baker
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Splicing Factors ◽  
Bovine Papillomavirus ◽  
Splicing Pattern ◽  
In Vitro Splicing

ABSTRACT Alternative splicing is a critical component of the early to late switch in papillomavirus gene expression. In bovine papillomavirus type 1 (BPV-1), a switch in 3′ splice site utilization from an early 3′ splice site at nucleotide (nt) 3225 to a late-specific 3′ splice site at nt 3605 is essential for expression of the major capsid (L1) mRNA. Three viral splicing elements have recently been identified between the two alternative 3′ splice sites and have been shown to play an important role in this regulation. A bipartite element lies approximately 30 nt downstream of the nt 3225 3′ splice site and consists of an exonic splicing enhancer (ESE), SE1, followed immediately by a pyrimidine-rich exonic splicing suppressor (ESS). A second ESE (SE2) is located approximately 125 nt downstream of the ESS. We have previously demonstrated that the ESS inhibits use of the suboptimal nt 3225 3′ splice site in vitro through binding of cellular splicing factors. However, these in vitro studies did not address the role of the ESS in the regulation of alternative splicing. In the present study, we have analyzed the role of the ESS in the alternative splicing of a BPV-1 late pre-mRNA in vivo. Mutation or deletion of just the ESS did not significantly change the normal splicing pattern where the nt 3225 3′ splice site is already used predominantly. However, a pre-mRNA containing mutations in SE2 is spliced predominantly using the nt 3605 3′ splice site. In this context, mutation of the ESS restored preferential use of the nt 3225 3′ splice site, indicating that the ESS also functions as a splicing suppressor in vivo. Moreover, optimization of the suboptimal nt 3225 3′ splice site counteracted the in vivo function of the ESS and led to preferential selection of the nt 3225 3′ splice site even in pre-mRNAs with SE2 mutations. In vitro splicing assays also showed that the ESS is unable to suppress splicing of a pre-mRNA with an optimized nt 3225 3′ splice site. These data confirm that the function of the ESS requires a suboptimal upstream 3′ splice site. A surprising finding of our study is the observation that SE1 can stimulate both the first and the second steps of splicing.

scholarly journals Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.

1993 ◽  
Vol 13 (5) ◽  
pp. 2993-3001 ◽  
Author(s):  
A Mayeda ◽  
D M Helfman ◽  
A R Krainer
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factor ◽  
Alternative Exon ◽  
Hnrnp A1 ◽  
Exon Inclusion ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

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.

scholarly journals Phosphorylation Regulates In Vivo Interaction and Molecular Targeting of Serine/Arginine-rich Pre-mRNA Splicing Factors

1999 ◽  
Vol 145 (3) ◽  
pp. 447-455 ◽  
Author(s):  
Joanne M. Yeakley ◽  
Hélène Tronchère ◽  
James Olesen ◽  
Jacqueline A. Dyck ◽  
Huan-You Wang ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Sr Proteins ◽  
Molecular Targeting ◽  
Splicing Factors ◽  
Binding Studies ◽  
Sr Protein ◽  
Domain Interactions

The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain–mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein–specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain–mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein–specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Δ yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain–containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

scholarly journals SRp20 and CUG-BP1 Modulate Insulin Receptor Exon 11 Alternative Splicing

2008 ◽  
Vol 29 (3) ◽  
pp. 871-880 ◽  
Author(s):  
Supriya Sen ◽  
Indrani Talukdar ◽  
Nicholas J. G. Webster
Keyword(s):  
Alternative Splicing ◽  
Insulin Receptor ◽  
Exon Skipping ◽  
Regulatory Sequences ◽  
Exon Inclusion ◽  
Disease States ◽  
Splicing Silencer ◽  
Sequence Elements ◽  
Exon 11

ABSTRACT The insulin receptor (IR) exists as two isoforms, IR-A and IR-B, which result from alternative splicing of exon 11 in the primary transcript. This alternative splicing is cell specific, and the relative proportions of exon 11 isoforms also vary during development, aging, and different disease states. We have previously demonstrated that both intron 10 and exon 11 contain regulatory sequences that affect IR splicing both positively and negatively. In this study, we sought to define the precise sequence elements within exon 11 that control exon recognition and cellular factors that recognize these elements. Using minigenes carrying linker-scanning mutations within exon 11, we detected both exonic splicing enhancer and exonic splicing silencer elements. We identified binding of SRp20 and SF2/ASF to the exonic enhancers and CUG-BP1 to the exonic silencer by RNA affinity chromatography. Overexpression and knockdown studies with hepatoma and embryonic kidney cells demonstrated that SRp20 and SF2/ASF increase exon inclusion but that CUG-BP1 causes exon skipping. We found that CUG-BP1 also binds to an additional intronic splicing silencer, located at the 3′ end of intron 10, to promote exon 11 skipping. Thus, we propose that SRp20, SF2/ASF, and CUG-BP1 act antagonistically to regulate IR alternative splicing in vivo and that the relative ratios of SRp20 and SF2/ASF to CUG-BP1 in different cells determine the degree of exon inclusion.

scholarly journals Combinatorial Control of Signal-Induced Exon Repression by hnRNP L and PSF

2007 ◽  
Vol 27 (19) ◽  
pp. 6972-6984 ◽  
Author(s):  
Alexis A. Melton ◽  
Jason Jackson ◽  
Jiarong Wang ◽  
Kristen W. Lynch
Keyword(s):  
T Cells ◽  
Alternative Splicing ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Function ◽  
Exon Skipping ◽  
Dependent Manner

ABSTRACT Cells can regulate their protein repertoire in response to extracellular stimuli via alternative splicing; however, the mechanisms controlling this process are poorly understood. The CD45 gene undergoes alternative splicing in response to T-cell activation to regulate T-cell function. The ESS1 splicing silencer in CD45 exon 4 confers basal exon skipping in resting T cells through the activity of hnRNP L and confers activation-induced exon skipping in T cells via previously unknown mechanisms. Here we have developed an in vitro splicing assay that recapitulates the signal-induced alternative splicing of CD45 and demonstrate that cellular stimulation leads to two changes to the ESS1-bound splicing regulatory complex. Activation-induced posttranslational modification of hnRNP L correlates with a modest increase in the protein's repressive activity. More importantly, the splicing factor PSF is recruited to the ESS1 complex in an activation-dependent manner and accounts for the majority of the signal-regulated ESS1 activity. The associations of hnRNP L and PSF with the ESS1 complex are largely independent of each other, but together these proteins account for the total signal-regulated change in CD45 splicing observed in vitro and in vivo. Such a combinatorial effect on splicing allows for precise regulation of signal-induced alternative splicing.

Sign in / Sign up

Export Citation Format

Share Document