Roles for SR proteins and hnRNP A1 in the regulation of c-src exon N1

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.     

Depleting deubiquitinating enzymes promotes apoptosis in glioma cell line via RNA binding proteins SF2/ASF1

USP5 and USP8 (Deubiquitinating enzyme) are highly overexpressed and more recognized as poor prognosis marker in various cancers. Depleting USP5 or USP8 to assess the synergism with proteasome inhibitor (Bortezomib) were measured. Furthermore, in present finding USP5 cooperates hnRNPA1 & USP8 cooperate SF2/ASF1, therefore gain in expression of either hnRNPA1 or SF2/ASF1 is sufficient to promote cell survival. On the other side, apoptosis markers were more pronounced in U87 or T98G cells devoid of either USP5 or USP8. However, apparent increase in SF2/ASF1 in absence of USP5, providing resistant factor is new. Antiapoptotic activity due to rise in SF2/ASF1 was validated after co-knock down of SF2/ASF1 in addition to USP5 induces more apoptosis comparing to individual knock down of USP5 or SF2/ASF1. This reveals SF2/ASF1 (RNA binding protein) delayed the apoptotic effect due to loss of USP5, lends ubiquitination of hnRNPA1. In presence of USP5, PI3 kinase inhibition promotes even more interaction between USP5 and hnRNPA1, thereby stabilizes hnRNPA1 in U87MG. In that way hnRNPA1 and SF2/ASF1 impart oncogenic activity. In conclusion, siRNA based strategy against USP5 is not enough to inhibit glioma, moreover targeting additionally SF2/ASF1 by knocking down USP8 is suitably more effective to deal with glioma tumour reoccurrence by indirectly targeting both SF2/ASF1 and hnRNPA1 oncogene.     

Arginine/serine-rich protein interaction domain-dependent modulation of a tau exon 10 splicing enhancer: altered interactions and mechanisms for functionally antagonistic FTDP-17 mutations Delta280K AND N279K

Tau exon 10 splicing is altered by autosomal dominant mutations that cause frontotemporal dementia with parkinsonism chromosome 17-type and by unknown mechanisms in other related neurodegenerative disorders. Identifying cis- and trans-regulators of tau exon 10 splicing is therefore crucial for understanding disease mechanisms. We previously identified several splicing enhancers and silencers within exon 10 and intron 10. Here, we show that splicing factors SF2/ASF, Tra2beta, and a 50-kDa nuclear protein bind in vitro to the polypurine enhancer at the 5' end of exon 10. Disease splicing mutations N279K and Delta280K disrupt the enhancer and alter associations with these factors. N279K targets robustly bind Tra2beta compared with the normal enhancer, which may explain why N279K enhances exon 10 splicing in vivo. In contrast, factor associations with Delta280K targets are nearly undetectable, explaining why Delta280K almost abolishes exon 10 splicing in vivo. Small interfering RNA-mediated suppression of endogenous SF2/ASF and Tra2beta significantly reduces exon 10 splicing. Exogenous SF2/ASF dramatically enhances normal exon 10 splicing and efficiently rescues the Delta280K splicing defect. Domain deletion analyses show that the C-terminal RS domains of SF2/ASF and Tra2beta are required for normal exon 10 splicing in vivo. In contrast to Tra2beta, the SF2/ASF RS domain remains essential in the presence of a strengthened enhancer or when either weak splice site is strengthened. The data suggest that SF2/ASF has both essential and regulatory roles, whereas Tra2beta has a supporting role in exon 10 splicing.     

Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins.

SR proteins recognize exonic splicing enhancer (ESE) elements and promote exon use, whereas certain hnRNP proteins bind to exonic splicing silencer (ESS) elements and block exon recognition. We investigated how ESS3 in HIV-1 tat exon 3 blocks splicing promoted by one SR protein (SC35) but not another (SF2/ASF). hnRNP A1 mediates silencing by binding initially to a required high-affinity site in ESS3, which then promotes further hnRNP A1 association with the upstream region of the exon. Both SC35 and SF2/ASF recognize upstream ESE motifs, but only SF2/ASF prevents secondary hnRNP A1 binding, presumably by blocking its cooperative propagation along the exon. The differential antagonism between a negative and two positive regulators exemplifies how inclusion of an alternative exon can be modulated.     

The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A.

Exons 6A and 6B of the chicken beta-tropomyosin gene are mutually exclusive and selected in a tissue-specific manner. Exon 6A is present in non-muscle and smooth muscle cells, while exon 6B is present in skeletal muscle cells. In this study we have investigated the mechanism underlying exon 6A recognition in non-muscle cells. Previous reports have identified a pyrimidine-rich intronic enhancer sequence (S4) downstream of exon 6A as essential for exon 6A 5'-splice site recognition. We show here that preincubation of HeLa cell extracts with an excess of RNA containing this sequence specifically inhibits exon 6A recognition by the splicing machinery. Splicing inhibition by an excess of this RNA can be rescued by addition of the SR protein ASF/SF2, but not by the SR proteins SC35 or 9G8. ASF/SF2 stimulates exon 6A splicing through specific interaction with the enhancer sequence. Surprisingly, SC35 behaves as an inhibitor of exon 6A splicing, since addition to HeLa nuclear extracts of increasing amounts of the SC35 protein completely abolish the stimulatory effect of ASF/SF2 on exon 6A splicing. We conclude that exon 6A recognition in vitro depends on the ratio of the ASF/SF2 to SC35 SR proteins. Taken together our results suggest that variations in the level or activity of these proteins could contribute to the tissue-specific choice of beta-tropomyosin exon 6A. In support of this we show that SR proteins isolated from skeletal muscle tissues are less efficient for exon 6A stimulation than SR proteins isolated from HeLa cells.     

An extended inhibitory context causes skipping of exon 7 of SMN2 in spinal muscular atrophy

SMN1 and SMN2 represent the two nearly identical copies of the survival of motor neuron gene in humans. The most frequent cause of spinal muscular atrophy (SMA) is loss of SMN1 accompanied by the inability of SMN2 to compensate due to an inhibitory mutation at position 6 in exon 7 (C6U) that causes exon 7 exclusion. How this single exonic nucleotide regulates exon 7 recognition has been of major interest. Based on score matrices and in vitro assays, abrogation of an exonic splicing enhancer (ESE) associated with SF2/ASF has been considered as the cause of exon 7 exclusion. However, a recent report supports the creation of an exonic splicing silencer (ESS) associated with hnRNP A1 as the determining factor for exon 7 exclusion. Here we show that C6U strengthens an inhibitory context that covers a larger sequence than the hnRNP A1 binding site. The inhibitory context can also be strengthened by the addition of a G residue at the first position of exon 7 in SMN1, promoting exon 7 skipping despite the presence of SF2/ASF binding site. Through in vivo selection and a series of mutations we demonstrate that the strengthening of the extended inhibitory context at the 5' end of exon 7 is exercised through overlapping sequence motifs that collaborate to regulate exon usage.     

hnRNP A1 and the SR proteins ASF/SF2 and SC35 have antagonistic functions in splicing of beta-tropomyosin exon 6B

Mutually exclusive splicing of exons 6A and 6B from the chicken beta-tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them.     

The pathological splicing mutation c.6792C>G in NF1 exon 37 causes a change of tenancy between antagonistic splicing factors

We have previously identified an ESE in NF1 exon 37 whose disruption by the pathological mutation c.6792C>G caused aberrant splicing. We now investigate the RNA-protein complexes affected by the c.6792C>G mutation observing that this concurrently decreases the affinity for the positive splicing factor YB-1 and increases the affinity for the negative splicing factors, hnRNPA1, hnRNPA2 and a new player in these type of complexes, DAZAP1. Our findings highlight the complexity of the interplay between positive and negative factors in the exon inclusion/skipping outcome. Furthermore, our observations stress the role of a wide genomic context in NF1 exon 37 definition.     

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

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.     

Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene

Ron, the tyrosine kinase receptor for the Macrophage-stimulating protein, is involved in cell dissociation, motility, and matrix invasion. DeltaRon, a constitutively active isoform that confers increased motility to expressing cells, is generated through the skipping of exon 11. We show that abnormal accumulation of DeltaRon mRNA occurs in breast and colon tumors. Skipping of exon 11 is controlled by a silencer and an enhancer of splicing located in the constitutive exon 12. The strength of the enhancer parallels the relative abundance of DeltaRon mRNA and depends on a sequence directly bound by splicing factor SF2/ASF. Overexpression and RNAi experiments demonstrate that SF2/ASF, by controlling the production of DeltaRon, activates epithelial to mesenchymal transition leading to cell locomotion. The effect of SF2/ASF overexpression is reverted by specific knockdown of DeltaRon mRNA. This demonstrates a direct link between SF2/ASF-regulated splicing and cell motility, an activity important for embryogenesis, tissue formation, and tumor metastasis.     

Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF

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 (CD200_tr) 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.     

Human splicing factor ASF/SF2 encodes for a repressor domain required for its inhibitory activity on pre-mRNA splicing.

The essential splicing factor ASF/SF2 activates or represses splicing depending on where on the pre-mRNA it binds. We have shown previously that ASF/SF2 inhibits adenovirus IIIa pre-mRNA splicing by binding to an intronic repressor element. Here we used MS2-ASF/SF2 fusion proteins to show that the second RNA binding domain (RBD2) is both necessary and sufficient for the splicing repressor function of ASF/SF2. Furthermore, we show that the completely conserved SWQDLKD motif in ASF/SF2-RBD2 is essential for splicing repression. Importantly, this heptapeptide motif is unlikely to be directly involved in RNA binding given its position within the predicted structure of RBD2. The activity of the ASF/SF2-RBD2 domain in splicing was position-dependent. Thus, tethering RBD2 to the IIIa intron resulted in splicing repression, whereas RBD2 binding at the second exon had no effect on IIIa splicing. The splicing repressor activity of RBD2 was not unique to the IIIa pre-mRNA, as binding of RBD2 at an intronic position in the rabbit beta-globin pre-mRNA also resulted in splicing inhibition. Taken together, our results suggest that ASF/SF2 encode distinct domains responsible for its function as a splicing enhancer or splicing repressor protein.