Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of C. elegans

BACKGROUND: SR family and SR-related proteins assemble on exonic splicing enhancer (ESE) sequences to promote both constitutive and regulated splicing. The SRm160 splicing coactivator, an SR-related nuclear matrix protein of 160 kDa, is important for the splicing of specific constitutive and ESE-dependent pre-mRNAs. RESULTS: In the present study, we show that SRm160 is required to promote pre-mRNA splicing mediated by a large population of functional ESE sequences within a randomized 18 nucleotide sequence. This suggests that it functions as a general coactivator by interacting with different SR family/SR-related proteins bound to different ESE sequences. Consistent with this, several SR family and SR-related proteins coimmunoprecipitated specifically with SRm160 in the presence of low salt. We used RNA interference (RNAi) in Caenorhabditis elegans to determine whether interactions between CeSRm160 and different CeSR family proteins are important in a whole-organism context. Previously we showed that RNAi of CeSRm160 and individual CeSR family genes other than CeSF2/ASF results in no obvious phenotype, which is indicative of gene redundancy. In the present study, we demonstrate that RNAi of CeSRm160 in combination with any CeSR family gene results in the production of unfertilized oocytes by the injected mother. CONCLUSIONS: The observation that simultaneous suppression of CeSRm160 and individual CeSR family proteins results in a distinct phenotype is indicative of critical functional interactions between these factors. Our results provide biochemical and genetic evidence indicating that interactions between SRm160 and multiple SR family proteins are important for both optimal splicing activity and for proper development.     

Identification and characterization of RED120: a conserved PWI domain protein with links to splicing and 3'-end formation

Precursor (pre)-mRNA splicing can impact the efficiency of coupled steps in gene expression. SRm160 (SR-related nuclear matrix protein of 160 kDa), is a splicing coactivator that also functions as a 3'-end cleavage-stimulatory factor. Here, we have identified an evolutionary-conserved SRm160-interacting protein, referred to as hRED120 (for human Arg/Glu/Asp-rich protein of 120 kDa). hRED120 contains a conventional RNA recognition motif and, like SRm160, a PWI nucleic acid binding domain, suggesting that it has the potential to bridge different RNP complexes. Also, similar to SRm160, hRED120 associates with snRNP components, and remains associated with mRNA after splicing. Simultaneous suppression in Caenorhabditis elegans of the ortholog of hRED120 with the orthologs of splicing and 3'-end processing factors results in aberrant growth or developmental defects. These results suggest that RED120 may function to couple splicing with mRNA 3'-end formation.     

The acute myeloid leukemia-associated protein, DEK, forms a splicing-dependent interaction with exon-product complexes

DEK is an approximately 45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.     

In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)-labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)-dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splicing speckled domains by an ATP-dependent mechanism. A second EJC protein, RNPS1, also has an ATP-dependent mobility, but SRm300, a protein that binds to SRm160 and participates with it in RNA splicing, remains immobile after ATP supplementation. This finding suggests that SRm160-containing RNA export, but not splicing, complexes have an ATP-dependent mobility. We propose that RNA export complexes have an ATP-regulated mechanism for release from binding sites at splicing speckled domains. In vitro fluorescence recovery after photobleaching is a powerful tool for identifying cofactors required for nuclear binding and mobility.     

Proteomic analysis of SRm160-containing complexes reveals a conserved association with cohesin

In this study, we describe a rapid immunoaffinity purification procedure for gel-free tandem mass spectrometry-based analysis of endogenous protein complexes and apply it to the characterization of complexes containing the SRm160 (serine/arginine repeat-related nuclear matrix protein of 160 kDa) splicing coactivator. In addition to promoting splicing, SRm160 stimulates 3'-end processing via its N-terminal PWI nucleic acid-binding domain and is found in a post-splicing exon junction complex that has been implicated in coupling splicing with mRNA turnover, export, and translation. Consistent with these known functional associations, we found that the majority of proteins identified in SRm160-containing complexes are associated with pre-mRNA processing. Interestingly, SRm160 is also associated with factors involved in chromatin regulation and sister chromatid cohesion, specifically the cohesin subunits SMC1alpha, SMC3, RAD21, and SA2. Gradient fractionation suggested that there are two predominant SRm160-containing complexes, one enriched in splicing components and the other enriched in cohesin subunits. Co-immunoprecipitation and co-localization experiments, as well as combinatorial RNA interference in Caenorhabditis elegans, support the existence of conserved and functional interactions between SRm160 and cohesin.     

A systematic characterization of Cwc21, the yeast ortholog of the human spliceosomal protein SRm300

Cwc21 (complexed with Cef1 protein 21) is a 135 amino acid yeast protein that shares homology with the N-terminal domain of human SRm300/SRRM2, a large serine/arginine-repeat protein shown previously to associate with the splicing coactivator and 3′-end processing stimulatory factor, SRm160. Proteomic analysis of spliceosomal complexes has suggested a role for Cwc21 and SRm300 at the core of the spliceosome. However, specific functions for these proteins have remained elusive. In this report, we employ quantitative genetic interaction mapping, mass spectrometry of tandem affinity-purified complexes, and microarray profiling to investigate genetic, physical, and functional interactions involving Cwc21. Combined data from these assays support multiple roles for Cwc21 in the formation and function of splicing complexes. Consistent with a role for Cwc21 at the core of the spliceosome, we observe strong genetic, physical, and functional interactions with Isy1, a protein previously implicated in the first catalytic step of splicing and splicing fidelity. Together, the results suggest multiple functions for Cwc21/SRm300 in the splicing process, including an important role in the activation of splicing in association with Isy1.     

The SR protein family

The processing of pre-mRNAs is a fundamental step required for the expression of most metazoan genes. Members of the family of serine/arginine (SR)-rich proteins are critical components of the machineries carrying out these essential processing events, highlighting their importance in maintaining efficient gene expression. SR proteins are characterized by their ability to interact simultaneously with RNA and other protein components via an RNA recognition motif (RRM) and through a domain rich in arginine and serine residues, the RS domain. Their functional roles in gene expression are surprisingly diverse, ranging from their classical involvement in constitutive and alternative pre-mRNA splicing to various post-splicing activities, including mRNA nuclear export, nonsense-mediated decay, and mRNA translation. These activities point up the importance of SR proteins during the regulation of mRNA metabolism.     

Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion

The multiple isoforms of the transmembrane glycoprotein CD44 are produced by alternative RNA splicing. Expression of CD44 isoforms containing variable 5 exon (v5) correlates with enhanced malignancy and invasiveness of some tumors. Here we demonstrate that SRm160, a splicing coactivator, regulates CD44 alternative splicing in a Ras-dependent manner. Overexpression of SRm160 stimulates inclusion of CD44 v5 when Ras is activated. Conversely, small interfering RNA (siRNA)-mediated silencing of SRm160 significantly reduces v5 inclusion. Immunoprecipitation shows association of SRm160 with Sam68, a protein that also stimulates v5 inclusion in a Ras-dependent manner, suggesting that these two proteins interact to regulate CD44 splicing. Importantly, siRNA-mediated depletion of CD44 v5 decreases tumor cell invasion. Reduction of SRm160 by siRNA transfection downregulates the endogenous levels of CD44 isoforms, including v5, and correlates with a decrease in tumor cell invasiveness.     

Over-expression of SR-cyclophilin, an interaction partner of nuclear pinin, releases SR family splicing factors from nuclear speckles

Pre-mRNA splicing takes place within a dynamic ribonucleoprotein particle called the spliceosome and occurs in an ordered pathway. Although it is known that spliceosome consists of five small nuclear RNAs and at least 50 proteins, little is known about how the interaction among the proteins changes during splicing. Here we identify that SR-cyp, a Moca family of nuclear cyclophilin, interacts and colocalizes with nuclear pinin (pnn), a SR-related protein involving in pre-mRNA splicing. Nuclear pnn interacts with SR-cyp via its C-terminal RS domain. Upon SR-cyp over-expression, however, the subnuclear distribution of nuclear pnn is altered, resulting in its redistribution from nuclear speckles to a diffuse nucleoplasmic form. The diffuse subnuclear distribution of nuclear pnn is not due to epitope masking, accelerated protein turnover or post-translational modification. Furthermore, we find that SR-cyp regulates the subnuclear distribution of other SR family proteins, including SC35 and SRm300, in a similar manner as it does on nuclear pnn. This result is significant because it suggests that SR-cyp plays a general role in modulating the distribution pattern of SR-like and SR proteins, similar to that of Clk (cdc2-like kinase)/STY on SR family splicing factors. SR-cyp might direct its effect via either alteration of protein folding/conformation or of protein-protein interaction and thus may add another control level of regulation of SR family proteins and modification of their functions.     

一种新的Srrm1/SRm160可变剪接体经历无义突变介导的mRNA降解

目的:研究基因Srrm1/SRm160的可变剪接。方法:应用RT-PCR研究Srrm1/SRm160的可变剪接,通过蛋白质的翻译抑制和RNA干扰研究剪接异构体是否经历无义突变介导的mRNA降解(NMD)过程。结果:获得Srrm1/SRm160新的可变剪接异构体,该异构体产生提前终止密码子,翻译抑制和RNA干扰证实含有提前终止密码子的剪接体经过NMD而降解。结论:Srrm1/SRm160通过可变剪接和NMD调节自身的表达水平,作为剪接因子进一步调节其他基因的可变剪接。     

A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.     

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.     

核受体辅活化子PNRC与孤儿核受体SF1相互作用位点的鉴定

为了阐明核受体辅活化子 (proline richnuclearreceptorcoactivatorprotein ,PNRC)在孤儿核受体类固醇生成因子 1(steroidogenicfactor1,SF1)基因表达调控中的作用 ,采用酵母双杂合分析、缺失突变技术和瞬时转染等研究方法鉴定了PNRC与SF1的相互作用位点 .结果显示 ,PNRC中氨基酸 2 78～ 30 0区域是与SF1相互作用的位点 .该区域富含脯氨酸 ,其中有 1个SH3结合模体 (motif) ,单独的SH3模体不足以与SF1产生有效的相互作用 .瞬时转染分析表明 ,PNRC 2 70 32 7对野生型PNRC的辅激活功能具有负显性抑制效应 .研究结果表明 ,含SH3结合模体的PNRC 2 78 30 0区域是与SF1相互作用的位点     

Pre-mRNA splicing by the essential Drosophila protein B52: tissue and target specificity

B52, an essential SR protein of Drosophila melanogaster, stimulates pre-mRNA splicing in splicing-deficient mammalian S100 extracts. Surprisingly, mutant larvae depleted of B52 were found to be capable of splicing at least several pre-mRNAs tested (H. Z. Ring and J. T. Lis, Mol. Cell. Biol. 14:7499-7506, 1994). In a homologous in vitro system, we demonstrated that B52 complements a Drosophila S100 extract to allow splicing of a Drosophila fushi tarazu (ftz) mini-pre-mRNA. Moreover, Kc cell nuclear extracts that were immunodepleted of B52 lost their ability to splice this ftz pre-mRNA. In contrast, splicing of this same ftz pre-mRNA occurred in whole larvae homozygous for the B52 deletion. Other SR protein family members isolated from these larvae could substitute for B52 splicing activity in vitro. We also observed that SR proteins are expressed variably in different larval tissues. B52 is the predominant SR protein in specific tissues, including the brain. Tissues in which B52 is normally the major SR protein, such as larval brain tissue, failed to produce ftz mRNA in the B52 deletion line. These observations support a model in which the lethality of the B52 deletion strain is a consequence of splicing defects in tissues in which B52 is normally the major SR protein.