scholarly journals The second RNA-binding domain of the human splicing factor ASF/SF2 is the critical domain controlling adenovirus E1A alternative 5′-splice site selection

2004 ◽  
Vol 381 (2) ◽  
pp. 343-350 ◽  
Author(s):  
Vita DAUKSAITE ◽  
Göran AKUSJÄRVI
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Site Selection ◽  
Rna Binding ◽  
Splicing Factor ◽  
Rna Recognition Motif ◽  
Splice Site Selection ◽  
Critical Domain ◽  
Adenovirus E1a

The human splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2) is modular in structure with two RNA-binding domains (RBD1 and RBD2) and a C-terminal domain rich in arginine–serine dipeptide repeats. ASF/SF2 is an essential splicing factor that also functions as an important regulator of alternative splicing. In adenovirus E1A (early region 1A) alternative pre-mRNA splicing, ASF/SF2 functions as a strong inducer of proximal 5′-splice-site selection, both in vitro and in vivo. In the present study, we tested the functional role of individual domains of ASF/SF2 in alternative splicing in vitro. We show that ASF/SF2-RBD2 is the critical domain controlling E1A alternative splicing. In fact, RBD2 alone is sufficient to mimic the activity of the full-length ASF/SF2 protein as an inducer of proximal 5′-splice-site selection in vitro. The RBD2 domain induces a switch to E1A-proximal 5′-splice-site usage by repressing distal 12 S splicing and simultaneously stimulates proximal 13 S splicing. In contrast, the ASF/SF2-RBD1 domain has a more general splicing enhancer phenotype and appears to stimulate preferentially cap-proximal 5′-splice-site selection. Furthermore, the SWQDLKD motif, which is conserved in all SR proteins (serine/arginine-rich proteins) containing two RBDs, and the ribonucleoprotein-1-type RNA recognition motif were both found to be necessary for the alternative splice-site-switching activity of ASF/SF2. The RNP-1 motif was necessary for efficient RNA binding, whereas the SWQDLKD motif most probably contributes by functioning as a surface-mediating critical protein–protein contact during spliceosome assembly.

scholarly journals The anti-angiogenic isoforms of VEGF in health and disease

2009 ◽  
Vol 37 (6) ◽  
pp. 1207-1213 ◽  
Author(s):  
Yan Qiu ◽  
Coralie Hoareau-Aveilla ◽  
Sebastian Oltean ◽  
Steven J. Harper ◽  
David O. Bates
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Age Related Macular Degeneration ◽  
Splicing Factor ◽  
Splice Site Selection ◽  
Age Related ◽  
Normal Human ◽  
Radical Revision

Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys–Drash syndrome and pre-eclampsia). Administration of recombinant VEGF165b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGFxxxb isoforms to the pro-angiogenic VEGFxxx isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGFxxxb isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology.

scholarly journals Structural evolution of the tissue-specific U2AF2 paralog and alternative splicing factor LS2

2020 ◽  
Author(s):  
Ashish Ashok Kawale ◽  
J. Matthew Taliaferro ◽  
Hyun-Seo Kang ◽  
Christoph Hartmüller ◽  
Arie Geerlof ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Splicing Factor ◽  
Rna Recognition Motif ◽  
Sequencing Data ◽  
Tissue Specific ◽  
Molecular Features ◽  
G Quadruplex

AbstractThe Drosophila melanogaster LS2 protein is a tissue-specific paralog of U2AF2 that mediates testis-specific alternative splicing. In order to understand the structural mechanisms underlying the distinct RNA binding specificity we determined the solution structures of the LS2 RNA recognition motif (RRM) domains and characterized their interaction with cis-regulatory guanosine-rich RNA motifs found in intron regions upstream of alternatively spliced exons. We show that the guanosine-rich RNA adopts a G quadruplex (G4) fold in vitro. The LS2 tandem RRMs adopt canonical RRM folds that are connected by a 38-residue linker that harbors a small helical motif α0. The LS2 RRM2 domain and the α0 helix in the interdomain linker mediate interactions with the G4 RNA. The functional importance of these unique molecular features in LS2 is validated by mutational analysis in vitro and RNA splicing assays in vivo. RNA sequencing data confirm the enrichment of G4-forming LS2 target motifs near LS2-affected exons. Our data indicate a role of G quadruplex structures as cis-regulatory motifs in introns for the regulation of alternative splicing, that engage non-canonical interactions with a tandem RRM protein. These results highlight the intriguing molecular evolution of a tissue-specific splicing factor from its conserved U2AF2 paralog as a result of (retro-) gene duplication in D. melanogaster.

A novel protein factor is required for use of distal alternative 5' splice sites in vitro

1991 ◽  
Vol 11 (12) ◽  
pp. 5945-5953
Author(s):  
J E Harper ◽  
J L Manley
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Deae Cellulose ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Small Nuclear Ribonucleoprotein

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.

Factors That Influence Alternative Splice Site Selection in Vitro

1987 ◽  
pp. 97-112 ◽  
Author(s):  
JAMES L. MANLEY ◽  
JONATHAN C.S. NOBLE ◽  
XIN-YUAN FU ◽  
HUI GE
Keyword(s):  
Alternative Splice ◽  
Splice Site ◽  
Site Selection ◽  
Splice Site Selection ◽  
Alternative Splice Site

scholarly journals Epstein-Barr Virus SM Protein Utilizes Cellular Splicing Factor SRp20 To Mediate Alternative Splicing

2010 ◽  
Vol 84 (22) ◽  
pp. 11781-11789 ◽  
Author(s):  
Dinesh Verma ◽  
Swarna Bais ◽  
Melusine Gaillard ◽  
Sankar Swaminathan
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Lytic Cycle ◽  
Splicing Factor ◽  
Barr Virus ◽  
Epstein Barr ◽  
Sm Protein

ABSTRACT Epstein-Barr virus (EBV) SM protein is an essential nuclear protein produced during the lytic cycle of EBV replication. SM is an RNA-binding protein with multiple mechanisms of action. SM enhances the expression of EBV genes by stabilizing mRNA and facilitating nuclear export. SM also influences splicing of both EBV and cellular pre-mRNAs. SM modulates splice site selection of the host cell STAT1 pre-mRNA, directing utilization of a novel 5′ splice site that is used only in the presence of SM. SM activates splicing in the manner of SR proteins but does not contain the canonical RS domains typical of cellular splicing factors. Affinity purification and mass spectrometry of SM complexes from SM-transfected cells led to the identification of the cellular SR splicing factor SRp20 as an SM-interacting protein. The regions of SM and SRp20 required for interaction were mapped by in vitro and in vivo assays. The SRp20 interaction was shown to be important for the effects of SM on alternative splicing by the use of STAT1 splicing assays. Overexpression of SRp20 enhanced SM-mediated alternative splicing and knockdown of SRp20 inhibited the SM effect on splicing. These data suggest a model whereby SM, a viral protein, recruits and co-opts the function of cellular SRp20 in alternative splicing.

scholarly journals Competing Upstream 5′ Splice Sites Enhance the Rate of Proximal Splicing

2010 ◽  
Vol 30 (8) ◽  
pp. 1878-1886 ◽  
Author(s):  
Martin J. Hicks ◽  
William F. Mueller ◽  
Peter J. Shepard ◽  
Klemens J. Hertel
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Splice Sites ◽  
Base Pairing ◽  
Splice Site Selection ◽  
U1 Snrna ◽  
Pairing Potential

ABSTRACT Alternative 5′ splice site selection is one of the major pathways resulting in mRNA diversification. Regulation of this type of alternative splicing depends on the presence of regulatory elements that activate or repress the use of competing splice sites, usually leading to the preferential use of the proximal splice site. However, the mechanisms involved in proximal splice site selection and the thermodynamic advantage realized by proximal splice sites are not well understood. Here, we have carried out a systematic analysis of alternative 5′ splice site usage using in vitro splicing assays. We show that observed rates of splicing correlate well with their U1 snRNA base pairing potential. Weak U1 snRNA interactions with the 5′ splice site were significantly rescued by the proximity of the downstream exon, demonstrating that the intron definition mode of splice site recognition is highly efficient. In the context of competing splice sites, the proximity to the downstream 3′ splice site was more influential in dictating splice site selection than the actual 5′ splice site/U1 snRNA base pairing potential. Surprisingly, the kinetic analysis also demonstrated that an upstream competing 5′ splice site enhances the rate of proximal splicing. These results reveal the discovery of a new splicing regulatory element, an upstream 5′ splice site functioning as a splicing enhancer.

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 Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA.

1988 ◽  
Vol 8 (6) ◽  
pp. 2610-2619 ◽  
Author(s):  
D E Lowery ◽  
B G Van Ness
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Donor Site ◽  
Splice Donor Site ◽  
Splice Sites ◽  
Splice Donor ◽  
Splice Site Selection ◽  
Nuclear Extracts

The processing of a number of kappa-immunoglobulin primary mRNA (pre-mRNA) constructs has been examined both in vitro and in vivo. When a kappa-immunoglobulin pre-mRNA containing multiple J segment splice sites is processed in vitro, the splice sites are used with equal frequency. The presence of signal exon, S-V intron, or variable (V) region has no effect on splice site selection in vitro. Nuclear extracts prepared from a lymphoid cell line do not restore correct splice site selection. Splice site selection in vitro can be altered by changing the position or sequence of J splice donor sites. These results differ from the processing of similar pre-mRNAs expressed in vivo by transient transfection. The 5'-most J splice donor site was exclusively selected in vivo, even in nonlymphoid cells, and even in transcripts where in vitro splicing favored a 3' J splice site. The in vitro results are consistent with a model proposing that splice site selection is influenced by splice site strength and proximity; however, our in vivo results demonstrate a number of discrepancies with such a model and suggest that splice site selection may be coupled to transcription or a higher-order nuclear structure.

Exon definition may facilitate splice site selection in RNAs with multiple exons

1990 ◽  
Vol 10 (1) ◽  
pp. 84-94 ◽  
Author(s):  
B L Robberson ◽  
G J Cote ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Exon Skipping ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Spliceosome Assembly ◽  
Exon Definition ◽  
Exon 2 ◽  
Correct Orientation

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.

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