T-STAR的结构及生物学功能

T-STAR基因定位于染色体8q24.2,其表达产物为分子量约55kDa的Sam68样蛋白,是STAR(signaltransductionandactivatorofRNA)家族新成员,具有RNA结合蛋白特征性的结合位点和酪氨酸磷酸化功能域。可能通过酪氨酸激酶信号转导系统和pre-mRNA的选择性剪接、加工等途径,参与了精子的发生、细胞的增殖调控、转化细胞的永生化过程并可能与某些疾病有关。     

PSF and p54nrb bind a conserved stem in U5 snRNA

PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear. Here we show that PSF interacts with p54nrb, a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prpl8. We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54nrb, both individually and in combination. In all cases, iterative selection assays identified a purine-rich sequence located on the 3' side of U5 snRNA stem 1b. Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP.     

Recognition of exonic splicing enhancer sequences by the Drosophila splicing repressor RSF1.

The Drosophila repressor splicing factor 1 (RSF1) comprises an N-terminal RNA-binding region and a C-terminal domain rich in glycine, arginine and serine residues, termed the GRS domain. Recently, RSF1 has been shown to antagonize splicing factors of the serine/arginine-rich (SR) family and it is, therefore, expected to play a role in processing of a subset of Drosophila pre-mRNAs through specific interactions with RNA. To investigate the RNA-binding specificity of RSF1, we isolated RSF1-binding RNAs using an in vitro selection approach. We have identified two RNA target motifs recognized by RSF1, designated A (CAACGACGA)- and B (AAACGCGCG)-type sequences. We show here that the A-type cognate sequence behaves as an SR protein-dependent exonic splicing enhancer. Namely, three copies of the A-type ligand bind SR proteins, stimulate the efficiency of splicing of reporter pre-mRNAs several fold and lead to inclusion of a short internal exon both in vitro and in vivo. However, three copies of a B-type ligand were much less active. The finding that RSF1 acts as a potent repressor of pre-mRNA splicing in vitro led us to propose that the equilibrium between a limited number of structurally-related general splicing activators or repressors, competing for common or promiscuous binding sites, may be a major determinant of the underlying mechanisms controlling many alternative pre-mRNA process-ing events.     

RNA-binding proteins in human genetic disease

RNA-binding proteins (RBPs) are key components in RNA metabolism, regulating the temporal, spatial and functional dynamics of RNAs. Altering the expression of RBPs has profound implications for cellular physiology, affecting RNA processes from pre-mRNA splicing to protein translation. Recent genetic and proteomic data and evidence from animal models reveal that RBPs are involved in many human diseases ranging from neurologic disorders to cancer. Here we review the emerging evidence showing the involvement of RBPs in many disease networks and conclude that defects in RNA metabolism caused by aberrations in RBPs might underlie a broader spectrum of complex human disorders.     

The splicing factor crooked neck associates with the RNA-binding protein HOW to control glial cell maturation in Drosophila

In both vertebrates and invertebrates, glial cells wrap axonal processes to ensure electrical conductance. Here we report that Crooked neck (Crn), the Drosophila homolog of the yeast Clf1p splicing factor, is directing peripheral glial cell maturation. We show that crooked neck is expressed and required in glial cells to control migration and axonal wrapping. Within the cytoplasm, Crn interacts with the RNA-binding protein HOW and then translocates to the nucleus where the Crn/HOW complex controls glial differentiation by facilitating splicing of specific target genes. By using a GFP-exon trap approach, we identified some of the in vivo target genes that encode proteins localized in autocellular septate junctions. In conclusion, here we show that glial cell differentiation is controlled by a cytoplasmic assembly of splicing components, which upon translocation to the nucleus promote the splicing of genes involved in the assembly of cellular junctions.     

PTB蛋白:一种调控肿瘤代谢的RNA可变剪接调节因子

多聚嘧啶区结合蛋白(polypyrimidine tract binding protein,PTB或hnRNP I)是一种在细胞内部参与mRNA代谢过程的蛋白质。PTB蛋白可结合于核酸分子上富含嘧啶碱基的序列,对mRNA前体的剪接进行调控。如在部分肿瘤细胞中,PTB的表达量升高可对肿瘤代谢过程中关键的丙酮酸激酶M(pyruvate kinase M,PKM)基因的表达进行调控,通过抑制PKM基因的可变剪接的方式上调PKM2(pyruvate kinase M2,PKM2)的表达,进而强化肿瘤细胞的有氧糖酵解过程并促进肿瘤的发展。本文结合PTB蛋白的结构及其在PKM可变剪接过程中的调节机制,简要综述了PTB蛋白对肿瘤代谢的调控作用。     

Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon-exon junction complex.

The RNA-binding protein Y14 binds preferentially to mRNAs produced by splicing and is a component of a multiprotein complex that assembles approximately 20 nucleotides upstream of exon-exon junctions. This complex probably has important functions in post-splicing events including nuclear export and nonsense-mediated decay of mRNA. We show that Y14 binds to two previously reported components, Aly/REF and RNPS1, and to the mRNA export factor TAP. Moreover, we identified magoh, a human homolog of the Drosophila mago nashi gene product, as a novel component of the complex. Magoh binds avidly and directly to Y14 and TAP, but not to other known components of the complex, and is found in Y14-containing mRNPs in vivo. Importantly, magoh also binds to mRNAs produced by splicing upstream (approximately 20 nucleotides) of exon- exon junctions and its binding to mRNA persists after export. These experiments thus reveal specific protein-protein interactions among the proteins of the splicing-dependent mRNP complex and suggest an important role for the highly evolutionarily conserved magoh protein in this complex.     

Understanding splicing regulation through RNA splicing maps

Alternative splicing is a highly regulated process that greatly increases the proteome diversity and plays an important role in cellular differentiation and disease. Interactions between RNA-binding proteins (RBPs) and pre-mRNA are the principle regulator of splicing decisions. Findings from recent genome-wide studies of protein-RNA interactions have been combined with assays of the global effects of RBPs on splicing to create RNA splicing maps. These maps integrate information from all pre-mRNAs regulated by single RBPs to identify the global positioning principles guiding splicing regulation. Recent studies using this approach have identified a set of positional principles that are shared between diverse RBPs. Here, we discuss how insights from RNA splicing maps of different RBPs inform the mechanistic models of splicing regulation.     

Purification and molecular cloning of a novel essential component of the apolipoprotein B mRNA editing enzyme-complex

Editing of apolipoprotein B (apoB) mRNA requires the catalytic component APOBEC-1 together with "auxiliary" proteins that have not been conclusively characterized so far. Here we report the purification of these additional components of the apoB mRNA editing enzyme-complex from rat liver and the cDNA cloning of the novel APOBEC-1-stimulating protein (ASP). Two proteins copurified into the final active fraction and were characterized by peptide sequencing and mass spectrometry: KSRP, a 75-kDa protein originally described as a splicing regulating factor, and ASP, a hitherto unknown 65-kDa protein. Separation of these two proteins resulted in a reduction of APOBEC-1-stimulating activity. ASP represents a novel type of RNA-binding protein and contains three single-stranded RNA-binding domains in the amino-terminal half and a putative double-stranded RNA-binding domain at the carboxyl terminus. Purified recombinant glutathione S-transferase (GST)-ASP, but not recombinant GST-KSRP, stimulated recombinant GST-APOBEC-1 to edit apoB RNA in vitro. These data demonstrate that ASP is the second essential component of the apoB mRNA editing enzyme-complex. In rat liver, ASP is apparently associated with KSRP, which may confer stability to the editing enzyme-complex with its substrate apoB RNA serving as an additional auxiliary component.     

Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA

Pre-mRNA splicing is executed by the spliceosome, a complex of small nuclear RNAs (snRNAs) and numerous proteins. One such protein, 15.5K/Snu13p, is associated with the spliceosomal U4/U6.U5 tri-snRNP and box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which act during preribosomal RNA (rRNA) processing. As such, it is the first splicing factor to be identified in two functionally distinct particles. 15.5K binds to an internal helix-bulge-helix (K-turn) structure in the U4 snRNA and two such structures in the U3 snoRNA. Previous work has concentrated on the structural basis of the interaction of 15.5K with the RNAs and has been carried out in vitro. Here we present a functional analysis of Snu13p in vivo, using a galactose inducible SNU13 strain to investigate the basis of three lethal mutations in Saccharomyces cerevisiae. Two are point mutations that map to the RNA-binding domain, and the third is a C-terminal deletion. These mutations result in accumulation of unspliced pre-mRNA, confirming a role for Snu13p in pre-mRNA splicing. In addition, these mutants also display rRNA processing defects that are variable in nature. Analysis of one mutant in the RNA-binding domain reveals a reduction in the levels of the U4 snRNA, U6 snRNA, and box C/D snoRNAs, but not H/ACA snoRNAs, supporting a role for Snu13p in accumulation and/or maintenance of specific RNAs. The mutations in the RNA-binding domain exhibit differential binding to the U4 snRNA and U3 snoRNA in vitro, suggesting that there are differences in the mode of interaction of Snu13p with these two RNAs.     

The RNA-binding protein Staufen1 is increased in DM1 skeletal muscle and promotes alternative pre-mRNA splicing

In myotonic dystrophy type 1 (DM1), dystrophia myotonica protein kinase messenger ribonucleic acids (RNAs; mRNAs) with expanded CUG repeats (CUG(exp)) aggregate in the nucleus and become toxic to cells by sequestering and/or misregulating RNA-binding proteins, resulting in aberrant alternative splicing. In this paper, we find that the RNA-binding protein Staufen1 is markedly and specifically increased in skeletal muscle from DM1 mouse models and patients. We show that Staufen1 interacts with mutant CUG(exp) mRNAs and promotes their nuclear export and translation. This effect is critically dependent on the third double-stranded RNA-binding domain of Staufen1 and shuttling of Staufen1 into the nucleus via its nuclear localization signal. Moreover, we uncover a new role of Staufen1 in splicing regulation. Overexpression of Staufen1 rescues alternative splicing of two key pre-mRNAs known to be aberrantly spliced in DM1, suggesting its increased expression represents an adaptive response to the pathology. Altogether, our results unravel a novel function for Staufen1 in splicing regulation and indicate that it may positively modulate the complex DM1 phenotype, thereby revealing its potential as a therapeutic target.     

The Fox-1 family and SUP-12 coordinately regulate tissue-specific alternative splicing in vivo

Many pre-mRNAs are alternatively spliced in a tissue-specific manner in multicellular organisms. The Fox-1 family of RNA-binding proteins regulate alternative splicing by either activating or repressing exon inclusion through specific binding to UGCAUG stretches. However, the precise cellular contexts that determine the action of the Fox-1 family in vivo remain to be elucidated. We have recently demonstrated that ASD-1 and FOX-1, members of the Fox-1 family in Caenorhabditis elegans, regulate tissue-specific alternative splicing of the fibroblast growth factor receptor gene, egl-15, which eventually determines the ligand specificity of the receptor in vivo. Here we report that another RNA-binding protein, SUP-12, coregulates the egl-15 alternative splicing. By screening for mutants defective in the muscle-specific expression of our alternative splicing reporter, we identified the muscle-specific RNA-binding protein SUP-12. We identified juxtaposed conserved stretches as the cis elements responsible for the regulation. The Fox-1 family and the SUP-12 proteins form a stable complex with egl-15 RNA, depending on the cis elements. Furthermore, the asd-1; sup-12 double mutant is defective in sex myoblast migration, phenocopying the isoform-specific egl-15(5A) mutant. These results establish an in vivo model that coordination of the two families of RNA-binding proteins regulates tissue-specific alternative splicing of a specific target gene.     

The structure of the zinc finger domain from human splicing factor ZNF265 fold

Identification of the protein domains that are responsible for RNA recognition has lagged behind the characterization of protein-DNA interactions. However, it is now becoming clear that a range of structural motifs bind to RNA and their structures and molecular mechanisms of action are beginning to be elucidated. In this report, we have expressed and purified one of the two putative RNA-binding domains from ZNF265, a protein that has been shown to bind to the spliceosomal components U1-70K and U2AF35 and to direct alternative splicing. We show that this domain, which contains four highly conserved cysteine residues, forms a stable, monomeric structure upon the addition of 1 molar eq of Zn(II). Determination of the solution structure of this domain reveals a conformation comprising two stacked beta-hairpins oriented at approximately 80 degrees to each other and sandwiching the zinc ion; the fold resembles the zinc ribbon class of zinc-binding domains, although with one less beta-strand than most members of the class. Analysis of the structure reveals a striking resemblance to known RNA-binding motifs in terms of the distribution of key surface residues responsible for making RNA contacts, despite a complete lack of structural homology. Furthermore, we have used an RNA gel shift assay to demonstrate that a single crossed finger domain from ZNF265 is capable of binding to an RNA message. Taken together, these results define a new RNA-binding motif and should provide insight into the functions of the >100 uncharacterized proteins in the sequence data bases that contain this domain.