Alternative splicing during chondrogenesis: modulation of fibronectin exon EIIIA splicing by SR proteins

The alternative exon EIIIA of the fibronectin gene is included in mRNAs produced in undifferentiated mesenchymal cells but excluded from differentiated chondrocytes. As members of the SR protein family of splicing factors have been demonstrated to be involved in the alternative splicing of other mRNAs, the role of SR proteins in chondrogenesis-associated EIIIA splicing was investigated. SR proteins interacted with chick exon EIIIA sequences that are required for exon inclusion in a gel mobility shift assay. Addition of SR proteins to in vitro splicing reactions increased the rate and extent of exon EIIIA inclusion. Co-transfection studies employing cDNAs encoding individual SR proteins revealed that SRp20 decreased mRNA accumulation in HeLa cells, which make A+ mRNA, apparently by interfering with pre-mRNA splicing. Co-transfection studies also demonstrated that SRp40 increased exon EIIIA inclusion in chondrocytes, but not in HeLa cells, suggesting the importance of cellular context for SR protein activity. Immunoblot analysis did not reveal a relative depletion of SRp40 in chondrocytic cells. Possible mechanisms for regulation of EIIIA splicing in particular, and chondrogenesis associated splicing in general, are discussed.     

A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer.

The cardiac troponin T pre-mRNA contains an exonic splicing enhancer that is required for inclusion of the alternative exon 5. Here we show that enhancer activity is exquisitely sensitive to changes in the sequence of a 9-nucleotide motif (GAGGAAGAA) even when its purine content is preserved. A series of mutations that increased or decreased the level of exon inclusion in vivo were used to correlate enhancer strength with RNA-protein interactions in vitro. Analyses involving UV cross-linking and immunoprecipitation indicated that only four (SRp30a, SRp40, SRp55, and SRp75) of six essential splicing factors known as SR proteins bind to the active enhancer RNA. Moreover, purified SRp40 and SRp55 activate splicing of exon 5 when added to a splicing-deficient S100 extract. Purified SRp30b did not stimulate splicing in S100 extracts, which is consistent with its failure to bind the enhancer RNA. In vitro competition of SR protein splicing activity and UV cross-linking demonstrated that the sequence determinants for SR protein binding were precisely coincident with the sequence determinants of enhancer strength. Thus, a subset of SR proteins interacts directly with the exonic enhancer to promote inclusion of a poorly defined alternative exon. Independent regulation of the levels of SR proteins may, therefore, contribute to the developmental regulation of exon inclusion.     

Regulation of SRp20 exon 4 splicing

SR proteins are essential splicing factors involved in the use of both constitutive and alternative exons. We previously showed that the SR proteins SRp20 and ASF/SF2 have antagonistic activities on SRp20 pre-mRNA splicing. SRp20 activates exon 4 recognition in its pre-mRNA, whereas ASF/SF2 inhibits this recognition. In experiments aimed at testing the specificity of SRp20 and ASF/SF2 for exon 4 splicing regulation, we show here that this specificity lies in the RNA binding domains of SRp20 and ASF/SF2 and not in the RS domains. Surprisingly, a deletion of 14 amino acids at the end of ASF/SF2-RBD2 converts ASF/SF2 from an inhibitor to an activator of exon 4 splicing. We found that ASF3 also inhibits exon 4 recognition, thus acting similarly to ASF/SF2, while SC35 activates a cryptic 5' splice site downstream of exon 3 and, in doing so, represses exon 4 use. In contrast, Tra2 and the SR proteins 9G8 and SRp40 do not appear to affect exon 4 splicing.     

Identification and characterization of three members of the human SR family of pre-mRNA splicing factors.

SR proteins have a characteristic C-terminal Ser/Arg-rich repeat (RS domain) of variable length and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors. We have cloned a cDNA encoding a novel human SR protein designated SRp30c, which has an unusually short RS domain. We also cloned cDNAs encoding the human homologues of Drosophila SRp55/B52 and rat SRp40/HRS. Recombinant proteins expressed from these cDNAs are active in constitutive splicing, as shown by their ability to complement a HeLa cell S100 extract deficient in SR proteins. Additional cDNA clones reflect extensive alternative splicing of SRp40 and SRp55 pre-mRNAs. The predicted protein isoforms lack the C-terminal RS domain and might be involved in feedback regulatory loops. The ability of human SRp30c, SRp40 and SRp55 to modulate alternative splicing in vivo was compared with that of other SR proteins using a transient contransfection assay. The overexpression of individual SR proteins in HeLa cells affected the choice of alternative 5' splice sites of adenovirus E1A and/or human beta-thalassemia reporters. The resulting splicing patterns were characteristic for each SR protein. Consistent with the postulated importance of SR proteins in alternative splicing in vivo, we demonstrate complex changes in the levels of mRNAs encoding the above SR proteins upon T cell activation, concomitant with changes in the expression of alternatively spliced isoforms of CD44 and CD45.     

ESEfinder: A web resource to identify exonic splicing enhancers

Point mutations frequently cause genetic diseases by disrupting the correct pattern of pre-mRNA splicing. The effect of a point mutation within a coding sequence is traditionally attributed to the deduced change in the corresponding amino acid. However, some point mutations can have much more severe effects on the structure of the encoded protein, for example when they inactivate an exonic splicing enhancer (ESE), thereby resulting in exon skipping. ESEs also appear to be especially important in exons that normally undergo alternative splicing. Different classes of ESE consensus motifs have been described, but they are not always easily identified. ESEfinder (http://exon.cshl.edu/ESE/) is a web-based resource that facilitates rapid analysis of exon sequences to identify putative ESEs responsive to the human SR proteins SF2/ASF, SC35, SRp40 and SRp55, and to predict whether exonic mutations disrupt such elements.     

Human transformer 2beta and SRp55 interact with a calcitonin-specific splice enhancer

The calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively processed in a tissue-specific manner leading to the production of calcitonin mRNA in thyroid C cells and CGRP mRNA in neurons. Sequences in the human calcitonin-specific fourth exon function as an exonic splice enhancer (ESE) which is required for incorporation of exon 4 into calcitonin mRNA. Deletion of these sequences from the rat calcitonin/CGRP gene was reported to have no effect on calcitonin splicing. We demonstrate that sequences in the rat calcitonin/CGRP fourth exon act as an ESE. In addition, we observed that three proteins in HeLa nuclear extract, of apparent molecular weights of 40, 55 and 85 kDa, specifically interact with the exon 4 ESE. The 40-kDa protein is human transformer 2beta (hTra2beta), a homolog of the Drosophila splice regulator transformer 2. hTra2beta is required for calcitonin splicing in vitro, one of the first biological functions identified for hTra2beta. The 55-kDa protein is SRp55, a member of the SR family of phosphoproteins. Binding of SRp55 to an ESE required for calcitonin mRNA splicing suggests that the different levels of SRp55 present in different cell types may regulate calcitonin/CGRP alternative splicing.     

TIA proteins are necessary but not sufficient for the tissue-specific splicing of the myosin phosphatase targeting subunit 1

We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. We previously identified a U-rich intronic enhancer flanking the 5' splice site (IE1), and a bipartite exonic enhancer/suppressor, that regulate splicing of the MYPT1 central alternative exon. Here we show that T-cell inhibitor of apoptosis (TIA-1) and T-cell inhibitor of apoptosis-related (TIAR) proteins bind to the IE1. Co-transfection of TIA expression vectors with a MYPT1 mini-gene construct increase splicing of the central alternative exon. TIA proteins do not enhance splicing when the palindromic exonic splicing enhancer (ESE) is mutated, indicating that TIAs are necessary but not sufficient for splicing. The ESE specifically binds SRp55 and SRp20 proteins, supporting a model in which both SR and TIA proteins binding to their cis-elements are required for the recruitment of the splicing complex to a weak 5' splice site. Inactivation of TIA proteins in the DT40 cell line (TIA-1(-/-)TIAR(+/-)) reduced the splicing of the central alternative exon of the endogenous MYPT1 as well as stably transfected MYPT1 minigene constructs. Splicing of the MYPT1 3' alternative exon and the MLC(17) alternative exon were unaffected, suggesting that TIA proteins regulate a subset of smooth muscle/nonmuscle alternative splicing reactions. Finally, reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.     

Alternative splicing of CD45 pre-mRNA is uniquely obedient to conditions in lymphoid cells.

The leucocyte common antigen (LCA or CD45) consists of various isoforms generated by alternative splicing of variable exons 4, 5 and 6 (or A, B and C). To follow splicing behaviour in different cell types we developed a human CD45 mini-gene and analysed its expression in transfected cell lines and transgenic mouse tissues. In Cos-1, HeLa and 3T3 cells we found distinct expression patterns which could only be modulated slightly by protein synthesis inhibitors but not by variation in culture conditions like pH, serum concentration and cell density, or by stimulation with phorbol ester (TPA). In all non-lymphoid transgenic tissues the default splicing pattern (CD45R0) was found, while the expression profile in lymphoid cells, where all eight isoforms are present, mimics that of the endogenous mouse LCA gene products. Next, to examine the factors involved in alternative exon use we analysed the expression pattern of members of the family of SR proteins, well known splicing regulators with arginine/serine-rich (R/S) domains. Cell lines expressed variable levels of SRp75, SRp30 and SRp20 and constant amounts of SRp40. Mouse tissues expressed large amounts of SRp75, SRp55 and SRp40, additional expression of SRp30s and SRp20 was restricted to lymphoid tissues. Therefore, SRp30 and SRp20 may contribute to forming the appropriate cellular conditions for alternative use of CD45 exons 4-6 in the haematopoietic compartment.     

SRp38基因研究进展

SR蛋白在前体mRNA可变剪接调控中发挥重要作用。可变剪接调节因子SRp38作为一种新近发现的具有神经及生殖组织特异性的SR蛋白,有典型的SR蛋白结构特征并能够调控GluR-B、TRK-C以及NCAML1等基因的可变剪接,但与其他SR蛋白不一致的是,SRp38可以在一定条件下(有丝分裂M期,热休克)抑制前体mRNA剪接,从而防止错误剪接的出现。SRp38的RRM结构域可以识别特殊的RNA序列并跟U1snRNP结合,而其RS结构域则参与调控前体mRNA剪接。SRp38的磷酸化状态可以影响其调控功能的发挥,在有丝分裂M期及热休克时,该蛋白质均呈去磷酸化状态。SRp38在爪蟾胚胎神经发育过程中发挥作用并且可以同TLS(translocation liposarcoma)蛋白相互作用,提示其可能通过调节前体mRNA可变剪接在神经系统的发育分化以及在肿瘤的发生中扮演角色。     

Molecular and genetic studies imply Akt-mediated signaling promotes protein kinase CbetaII alternative splicing via phosphorylation of serine/arginine-rich splicing factor SRp40

Insulin regulates alternative splicing of PKCbetaII mRNA by phosphorylation of SRp40 via a phosphatidylinositol 3-kinase pathway (Patel, N. A., Chalfant, C. E., Watson, J. E., Wyatt, J. R., Dean, N. M., Eichler, D. C., and Cooper, D. C. (2001) J. Biol. Chem. 276, 22648-22654). Transient transfection of constitutively active Akt2 kinase promotes PKCbetaII exon inclusion. Serine/arginine-rich (SR) RNA-binding proteins regulating the selection of alternatively spliced exons are potential substrates of Akt kinase because many of them contain RXRXX(S/T) motifs. Here we show that Akt2 kinase phosphorylated SRp40 in vivo and in vitro. Mutation of Ser86 on SRp40 blocked in vitro phosphorylation. In control Akt2(+/+) fibroblasts, insulin treatment increased the phosphorylation of endogenous SR proteins, but their phosphorylation state remained unaltered by insulin in fibroblasts from Akt2(-/-) mice. Levels of PKCbetaII protein were up-regulated by insulin in Akt2(+/+) cells; however, only very low levels of PKCbetaII were detected in Akt2(-/-) cells and did not change following insulin treatment. Endogenous PKCbetaI and -betaII mRNA levels in Akt2(+/+) and Akt2(-/-) gastrocnemius muscle tissues were compared using quantitative real time PCR. The results indicated a 54% decrease in the expression of PKCbetaII levels in Akt(-/-), whereas PKCbetaI levels remained unchanged in both samples. Further, transfection of Akt2(-/-) cells with a PKCbetaII splicing minigene revealed defective betaII exon inclusion. Co-transfection of the mutated SRp40 attenuated betaII exon inclusion. This study provides in vitro and in vivo evidence showing Akt2 kinase directly phosphorylated SRp40, thereby connecting the insulin, PI 3-kinase/Akt pathway with phosphorylation of a site on a nuclear splicing protein promoting exon inclusion. This model is upheld in Akt2-deficient mice with insulin resistance leading to diabetes mellitus.     

Insulin regulates alternative splicing of protein kinase C beta II through a phosphatidylinositol 3-kinase-dependent pathway involving the nuclear serine/arginine-rich splicing factor, SRp40, in skeletal muscle cells

Insulin regulates the inclusion of the exon encoding protein kinase C (PKC) betaII mRNA. In this report, we show that insulin regulates this exon inclusion (alternative splicing) via the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway through the phosphorylation state of SRp40, a factor required for insulin-regulated splice site selection for PKCbetaII mRNA. By taking advantage of a well known inhibitor of PI 3-kinase, LY294002, we demonstrated that pretreatment of L6 myotubes with LY294002 blocked insulin-induced PKCbetaII exon inclusion as well as phosphorylation of SRp40. In the absence of LY294002, overexpression of SRp40 in L6 cells mimicked insulin-induced exon inclusion. When antisense oligonucleotides targeted to a putative SRp40-binding sequence in the betaII-betaI intron were transfected into L6 cells, insulin effects on splicing and glucose uptake were blocked. Taken together, these results demonstrate a role for SRp40 in insulin-mediated alternative splicing independent of changes in SRp40 concentration but dependent on serine phosphorylation of SRp40 via a PI 3-kinase signaling pathway. This switch in PKC isozyme expression is important for increases in the glucose transport effect of insulin. Significantly, insulin regulation of PKCbetaII exon inclusion occurred in the absence of cell growth and differentiation demonstrating that insulin-induced alternative splicing of PKCbetaII mRNA in L6 cells occurs in response to a metabolic change.     

Interactions of Arabidopsis RS domain containing cyclophilins with SR proteins and U1 and U11 small nuclear ribonucleoprotein-specific proteins suggest their involvement in pre-mRNA Splicing

Ser/Arg (SR)-rich proteins are important splicing factors in both general and alternative splicing. By binding to specific sequences on pre-mRNA and interacting with other splicing factors via their RS domain they mediate different intraspliceosomal contacts, thereby helping in splice site selection and spliceosome assembly. While characterizing new members of this protein family in Arabidopsis, we have identified two proteins, termed CypRS64 and CypRS92, consisting of an N-terminal peptidyl-prolyl cis/trans isomerase domain and a C-terminal domain with many SR/SP dipeptides. Cyclophilins possess a peptidyl-prolyl cis/trans isomerase activity and are implicated in protein folding, assembly, and transport. CypRS64 interacts in vivo and in vitro with a subset of Arabidopsis SR proteins, including SRp30 and SRp34/SR1, two homologs of mammalian SF2/ASF, known to be important for 5' splice site recognition. In addition, both cyclophilins interact with U1-70K and U11-35K, which in turn are binding partners of SRp34/SR1. CypRS64 is a nucleoplasmic protein, but in most cells expressing CypRS64-GFP fusion it was also found in one to six round nuclear bodies. However, co-expression of CypRS64 with its binding partners resulted in re-localization of CypRS64 from the nuclear bodies to nuclear speckles, indicating functional interactions. These findings together with the observation that binding of SRp34/SR1 to CypRS64 is phosphorylation-dependent indicate an involvement of CypRS64 in nuclear pre-mRNA splicing, possibly by regulating phosphorylation/dephosphorylation of SR proteins and other spliceosomal components. Alternatively, binding of CypRS64 to proteins important for 5' splice site recognition suggests its involvement in the dynamics of spliceosome assembly.     

Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5′-Splice Site of Exon 3

HIV-1 non-coding exon 3 can either be spliced to exons 4, 4a, 4b, 4c, and 5 to generate tat, rev, and nef mRNAs or remain unspliced to produce the 13a7 vpr mRNA. Here we show that serine- and arginine-rich proteins 55 and 75 (SRp55 and SRp75) inhibit splicing from the 5′-splice site of exon 3 thereby causing an accumulation of the partially unspliced 13a7 vpr mRNA. In contrast, serine- and arginine-rich protein 40 (SRp40) induces splicing from exon 3 to exon 4, thereby promoting the production of the 1347 tat mRNA. We demonstrate that SRp55 stimulates vpr mRNA production by interacting with the previously identified HIV-1 splicing enhancer named GAR and inhibiting its function. This inhibition requires both serine arginine-rich and RNA-binding domains of SRp55, indicating that production of HIV-1 vpr mRNA depends on the interaction of SRp55 with an unknown factor.     

Novel role for RNA-binding protein CUGBP2 in mammalian RNA editing. CUGBP2 modulates C to U editing of apolipoprotein B mRNA by interacting with apobec-1 and ACF, the apobec-1 complementation factor

Mammalian apolipoprotein B (apoB) mRNA editing is mediated by a multicomponent holoenzyme containing apobec-1 and ACF. We have now identified CUGBP2, a 54-kDa RNA-binding protein, as a component of this holoenzyme. CUGBP2 and ACF co-fractionate in bovine liver S-100 extracts, and addition of recombinant apobec-1 leads to assembly of a holoenzyme. Immunodepletion of CUGBP2 co-precipitates ACF, and these proteins co-localize the nucleus of transfected cells, suggesting that CUGBP2 and ACF are bound in vivo. CUGBP2 binds apoB RNA, specifically an AU-rich sequence located immediately upstream of the edited cytidine. ApoB RNA from McA cells, bound to CUGBP2, was more extensively edited than the unbound fraction. However, addition of recombinant CUGBP2 to a reconstituted system demonstrated a dose-dependent inhibition of C to U RNA editing, which was rescued with either apobec-1 or ACF. Antisense CUGBP2 knockout increased endogenous apoB RNA editing, whereas antisense knockout of either apobec-1 or ACF expression eliminated apoB RNA editing, establishing the absolute requirement of these components of the core enzyme. These data suggest that CUGBP2 plays a role in apoB mRNA editing by forming a regulatory complex with the three components of the minimal editing enzyme, apobec-1, ACF, and apoB RNA.