The bI4 group I intron binds directly to both its protein splicing partners, a tRNA synthetase and maturase, to facilitate RNA splicing activity

The imported mitochondrial leucyl-tRNA synthetase (NAM2p) and a mitochondrial-expressed intron-encoded maturase protein are required for splicing the fourth intron (bI4) of the yeast cob gene, which expresses an electron transfer protein that is essential to respiration. However, the role of the tRNA synthetase, as well as the function of the bI4 maturase, remain unclear. As a first step towards elucidating the mechanistic role of these protein splicing factors in this group I intron splicing reaction, we tested the hypothesis that both leucyl-tRNA synthetase and bI4 maturase interact directly with the bI4 intron. We developed a yeast three-hybrid system and determined that both the tRNA synthetase and bI4 maturase can bind directly and independently via RNA-protein interactions to the large bI4 group I intron. We also showed, using modified two-hybrid and three-hybrid assays, that the bI4 intron bridges interactions between the two protein splicing partners. In the presence of either the bI4 maturase or the Leu-tRNA synthetase, bI4 intron transcribed recombinantly with flanking exons in the yeast nucleus exhibited splicing activity. These data combined with previous genetic results are consistent with a novel model for a ternary splicing complex (two protein: one RNA) in which both protein splicing partners bind directly to the bI4 intron and facilitate its self-splicing activity.     

Identification and characterization of Prp45p and Prp46p,essential pre-mRNA splicing factors

Through exhaustive two-hybrid screens using a budding yeast genomic library, and starting with the splicing factor and DEAH-box RNA helicase Prp22p as bait, we identified yeast Prp45p and Prp46p. We show that as well as interacting in two-hybrid screens, Prp45p and Prp46p interact with each other in vitro. We demonstrate that Prp45p and Prp46p are spliceosome associated throughout the splicing process and both are essential for pre-mRNA splicing. Under nonsplicing conditions they also associate in coprecipitation assays with low levels of the U2, U5, and U6 snRNAs that may indicate their presence in endogenous activated spliceosomes or in a postsplicing snRNP complex.     

MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1

The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families—muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins—are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5′ end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.     

Intrinsic U2AF binding is modulated by exon enhancer signals in parallel with changes in splicing activity.

A functional analysis of exon replacement mutations was performed in parallel with RNA-protein binding assays to gain insight into the role of the exon in alternative and simple splicing events. These results show that constitutive exons from unrelated genes contain strong signals that promote splicing in multiple sequence contexts by enhancing 3' splice site activity. A clue to the nature of the relationship between the exon and adjacent 3' splice site is indicated by the binding properties of exon variant RNAs when tested with different biochemical preparations of the essential splicing protein, U2AF. In the context of a complete nuclear extract, U2AF binding to the 3' splice site is stimulated by the presence of an adjacent constitutive exon. In contrast, highly purified HeLa U2AF binds equivalently to the exon variants under conditions in which differential polypyrimidine tract binding is evident. These results provide support for an assisted binding model in which positive-acting signals within exons, exon enhancers, direct the binding of accessory factors, which in turn increase the intrinsic affinity of U2AF for the adjacent 3' splice site. Further support for an assisted binding model is indicated by biochemical complementation of U2AF binding and by the localization of a novel exon enhancer, which, when introduced into a weak exon, stimulates splicing activity in parallel with U2AF binding. Immunoprecipitation analysis identifies the splicing factor, SC35, as a constituent of the exon enhancer binding complex. These results are discussed in the context of current models for functional exon-bridging interactions.     

The STAR/GSG family protein rSLM-2 regulates the selection of alternative splice sites

We identified the rat Sam68-like mammalian protein (rSLM-2), a member of the STAR (signal transduction and activation of RNA) protein family as a novel splicing regulatory protein. Using the yeast two-hybrid system, coimmunoprecipitations, and pull-down assays, we demonstrate that rSLM-2 interacts with various proteins involved in the regulation of alternative splicing, among them the serine/arginine-rich protein SRp30c, the splicing-associated factor YT521-B and the scaffold attachment factor B. rSLM-2 can influence the splicing pattern of the CD44v5, human transformer-2beta and tau minigenes in cotransfection experiments. This effect can be reversed by rSLM-2-interacting proteins. Employing rSLM-2 deletion variants, gel mobility shift assays, and linker scan mutations of the CD44 minigene, we show that the rSLM-2-dependent inclusion of exon v5 of the CD44 pre-mRNA is dependent on a short purine-rich sequence. Because the related protein of rSLM-2, Sam68, is believed to play a role as an adapter protein during signal transduction, we postulate that rSLM-2 is a link between signal transduction pathways and pre-mRNA processing.     

Identification of a cis-acting element involved in the regulation of BACE1 mRNA alternative splicing

β-site APP cleaving enzyme 1 (BACE1) is the transmembrane aspartyl protease that catalyzes the first cleavage step during proteolysis of the β-amyloid precursor protein, a process involved in the pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative splicing, and cis -acting elements important for its regulation have not been identified. We constructed and compared several BACE1 minigenes and found that BACE1 sequence from exon 3 through exon 5 was required for minigenes to undergo correct splicing. Minigene splicing was validated by showing specific splicing inhibition upon splice site mutation. Furthermore, we showed that mutation of the minigene at a predicted exonic splicing enhancer in exon 4 of BACE1 increased exon 4 skipping. Therefore, we have for the first time found evidence of a regulatory site involved in BACE1 alternative splicing, and these data indicate that minor sequence changes can dramatically alter BACE1 alternative splicing.     

hnRNP H Is a Component of a Splicing Enhancer Complex That Activates a c-src Alternative Exon in Neuronal Cells

The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5′ splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.     

Antagonistic regulation of alpha-actinin alternative splicing by CELF proteins and polypyrimidine tract binding protein

The alpha-actinin gene has a pair of alternatively spliced exons. The smooth muscle (SM) exon is repressed in most cell types by polypyrimidine tract binding protein (PTB). CELF (CUG-BP and ETR3-like factors) family proteins, splicing regulators whose activities are altered in myotonic dystrophy, were found to coordinately regulate selection of the two alpha-actinin exons. CUG-BP and ETR3 activated the SM exon, and along with CELF4 they were also able to repress splicing of the NM (nonmuscle) exon both in vivo and in vitro. Activation of SM exon splicing was associated with displacement of PTB from the polypyrimidine tract by binding of CUG-BP at adjacent sites. Our data provides direct evidence for the activity of CELF proteins as both activators and repressors of splicing within a single-model system of alternative splicing, and suggests a model whereby alpha-actinin alternative splicing is regulated by synergistic and antagonistic interactions between members of the CELF and PTB families.     

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.     

The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing

Alternative splicing of cardiac troponin T (cTNT) exon 5 undergoes a developmentally regulated switch such that exon inclusion predominates in embryonic, but not adult, striated muscle. We previously described four muscle-specific splicing enhancers (MSEs) within introns flanking exon 5 in chicken cTNT that are both necessary and sufficient for exon inclusion in embryonic muscle. We also demonstrated that CUG-binding protein (CUG-BP) binds a conserved CUG motif within a human cTNT MSE and positively regulates MSE-dependent exon inclusion. Here we report that CUG-BP is one of a novel family of developmentally regulated RNA binding proteins that includes embryonically lethal abnormal vision-type RNA binding protein 3 (ETR-3). This family, which we call CELF proteins for CUG-BP- and ETR-3-like factors, specifically bound MSE-containing RNAs in vitro and activated MSE-dependent exon inclusion of cTNT minigenes in vivo. The expression of two CELF proteins is highly restricted to brain. CUG-BP, ETR-3, and CELF4 are more broadly expressed, and expression is developmentally regulated in striated muscle and brain. Changes in the level of expression and isoforms of ETR-3 in two different developmental systems correlated with regulated changes in cTNT splicing. A switch from cTNT exon skipping to inclusion tightly correlated with induction of ETR-3 protein expression during differentiation of C2C12 myoblasts. During heart development, the switch in cTNT splicing correlated with a transition in ETR-3 protein isoforms. We propose that ETR-3 is a major regulator of cTNT alternative splicing and that the CELF family plays an important regulatory role in cell-specific alternative splicing during normal development and disease.     

Alternative splicing during chondrogenesis: cis and trans factors involved in splicing of fibronectin exon EIIIA

Primary chicken mesenchymal cells from limb buds and vertebral chondrocytes have been used to study the changes that occur in alternative mRNA splicing of fibronectin exon EIIIA during chondrogenesis. The mesenchymal cell phenotype (exon EIIIA included) and chondrocyte phenotype (exon EIIIA excluded) were preserved in culture. Both primary cell types were transfected with an EIIIA minigene and alternative splicing was monitored by S1 protection assay. Differential cell-specific splicing of the reporter was observed. The roles of two regulatory elements, an exon splicing enhancer (ESE) and an exon splicing silencer (ESS) were examined. Both elements were required for EIIIA inclusion into mRNA in mesenchymal cells. Gel mobility shift assays revealed that both chondrocyte- and mesenchymal cell-derived nuclear extracts contained exon EIIIA binding factors, but the RNA binding factors present in the two cell types appeared to be distinct. The ESE and ESS appeared to cooperate in the formation of both cell type-specific complexes. These results suggest a model in which inhibitory factors enriched in chondrocytes compete with positive factors enriched in mesenchymal cells for binding to exon EIIIA, determining whether the exon is included.     

利用酵母双杂交系统筛选与人巨细胞病毒皮层蛋白pUL23相互作用的病毒编码蛋白

人巨细胞病毒(HCMV) UL23基因编码病毒皮层蛋白,该基因缺失时,病毒在人包皮成纤维细胞(HFF)中的繁殖速度加快.为进一步阐述HCMV UL23基因编码产物 pUL23的功能及调控机制,采用鸟枪法构建了融合于GAL4活性区域的HCMV Towne株 基因组随机表达文库.利用酵母双杂交技术,以pGBKT7 -UL23为诱饵质粒,从构建 的HCMV基因组表达文库中筛选到与pUL23相互作用的病毒编码蛋白pUL24. GST-pull down实验和免疫共沉淀实验进一步确认两种病毒蛋白之间的相互作用.结果 表明,构建的HCMV基因组表达文库能够用于GAL4酵母双杂交系统筛选与诱饵蛋白相互作用的病毒自身编码蛋白.病毒蛋白pUL23和pUL24之间具有相互作用,这为进一 步阐述pUL23在HCMV感染过程中的功能提供依据.该研究为揭示HCMV病毒感染机制奠定了基础.     

Two exonic elements in the flanking constitutive exons control the alternative splicing of the alpha exon of the ZO-1 pre-mRNA

The 240-bp alpha exon of the tight junction (TJ) protein ZO-1 pre-mRNA is alternatively spliced. Expression of both ZO-1alpha+/ZO-1alpha- isoforms results in hermetic TJs, and these become leaky when ZO-1alpha- expression prevails. The alpha exon inclusion/skipping mechanism was studied by in vivo RT-PCR splicing assays in neural and epithelial cells, utilizing a canine minigene construct containing the alpha exon, and the flanking introns and exons. Inclusion of the alpha exon always occurs in wild-type MDCK cells and it is detectable in transfected HeLa cells. However, the alpha exon is skipped in transfected neural cells. Accordingly, both 5' and 3' splice sites surrounding the alpha exon appear to be suboptimal and no cis-acting splicing control elements were found in this exon. Deletion analysis revealed an 83-bp splicing enhancer in the downstream exon and a 35-bp splicing silencer at the beginning of the upstream exon. In epithelial cells all constructs rendered alpha exon inclusion. We conclude that, in neural cells, skipping of the alpha exon depends on two antagonistic exonic elements located in the flanking constitutive exons.