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PubMed2004

Serum response factor is alternatively spliced in human colon cancer.

BACKGROUND: Serum response factor (SRF) is a transcription factor that plays an important role in cellular differentiation and cell cycle regulation. SRF function is regulated in part by alternative splicing. Little is known about the expression or role of these alternatively spliced isoforms during tumorigenesis. We hypothesized that there is a change in the splice variants during intestinal tumorigenesis and that this change promotes the tumor phenotype. MATERIALS AND METHODS: SRF expression was determined by Western blotting of benign intestinal cells and human colon cancer cell lines. To determine the effect of alternative splicing of SRF on intestinal growth and proliferation, the predominant alternatively spliced isoform of SRF that we identified in colon cancer cells, SRFDelta5, was transfected into IEC-6 cells. IEC-6 and IEC-6SRFDelta5 cells were plated and cell numbers were determined at four time points. RESULTS: Western blotting demonstrates that full-length SRF is the predominant form of SRF in rat IEC-6 cells, normal human colonic mucosa, and HT-29 cells, derived from a well-differentiated human colonic adenocarcinoma. In the colon cancer cell lines derived from poorly differentiated tumors (WiDr, HCT 116, LoVo, and SW480), SRFDelta5 is the predominant isoform expressed. There was a significant increase in cell survival in IEC-6 cells transfected with SRFDelta5 compared to parental cells. CONCLUSION: Our data demonstrate that an alternatively spliced isoform of SRF, SRFDelta5, is expressed in human colon cancer cell lines. Additionally, these data demonstrate that expression of SRFDelta5 may contribute to the tumor phenotype by affecting cell survival. This is the first study to document a change in expression of the alternatively spliced isoform of SRF in human malignancy.

Alternative Splicing↗
PubMed2005

A novel role for Vpr of human immunodeficiency virus type 1 as a regulator of the splicing of cellular pre-mRNA.

Vpr, one of the accessory gene products of human immunodeficiency virus type 1 (HIV-1), affects aspects of both viral and cellular proliferation, being involved in long terminal repeat (LTR) activation, arrest of the cell cycle at the G2 phase, and apoptosis. We have discovered a novel role for Vpr as a regulator of the splicing of pre-mRNA both in vivo and in vitro. We found, by RT-PCR and RNase protection analysis, that Vpr caused the accumulation of incompletely spliced forms of alpha-globin 2 and beta-globin pre-mRNAs in cells that had been transiently transfected with a Vpr expression vector. We postulated that this novel effect of Vpr might occur via a pathway that is distinct from arrest of the cell cycle at G2. By analyzing splicing reactions in vitro, we showed that Vpr inhibited the splicing of beta-globin pre-mRNA in vitro. The splicing of intron 1 of alpha-globin 2 pre-mRNA was modestly inhibited by Vpr but the splicing of intron 2 was unaffected. Interestingly, an experimental infection system which utilizes high-titered HIV-1/vesticular stomatitis virus G protein showed that Vpr expressed from an HIV-1 provirus was sufficient to accumulate endogenous alpha-globin 2 pre-mRNA. Thus, it is likely that Vpr contributes to selective inhibition of the splicing of cellular pre-mRNA.

Blotting, Western↗
PubMed2004

Commitment to splice site pairing coincides with A complex formation.

Differential recognition of exons by the spliceosome regulates gene expression and exponentially increases the complexity of metazoan proteomes. After definition of the exons, the spliceosome is activated by a series of sequential structural rearrangements. Formation of the first ATP-independent spliceosomal complex commits the pre-mRNA to the general splicing pathway. However, the time at which a commitment to a specific splice site choice and pairing is made is unknown. Here, we demonstrate that alternative splicing patterns are irreversibly chosen at a kinetic step different from the ATP-independent commitment to splicing. Splice sites become committed at the first ATP-dependent spliceosomal complex when rearrangements lock U2 snRNP onto the pre-mRNA. Thus, commitment to the splicing pathway and commitment to splice site pairing are separate steps during spliceosomal assembly, and ATP hydrolysis drives the irreversible juxtaposition of exons within the spliceosome.

Animals↗
PubMed2005

Negative control contributes to an extensive program of meiotic splicing in fission yeast.

Despite a high frequency of introns in the fission yeast Schizosaccharomyces pombe, regulated splicing is virtually unknown. We present evidence that splicing constitutes a major mechanism for controlling gene expression during meiosis, as 12 of 96 transcripts tested, which encode known components as well as previously uncharacterized ORFs, retain introns until specific times during differentiation. The meiotically spliced pre-mRNAs include two cyclins, rem1 (discovered by Ayte and Nurse) and crs1. Consistent with the use of regulated splicing to block protein production, expression of crs1 in vegetative cells is toxic. Analyses of gene chimeras indicate that splicing is prevented in mitotically growing cells via inhibition, in contrast to the positive control of meiotic splicing in budding yeast. Most strikingly, splicing of crs1 and rem1 is regulated by sequences located outside the coding regions, far from the target introns, a phenomenon previously observed only in metazoans.

Alternative Splicing↗
PubMed2006

Comparative analysis identifies exonic splicing regulatory sequences--The complex definition of enhancers and silencers.

Exonic splicing regulatory sequences (ESRs) are cis-acting factor binding sites that regulate constitutive and alternative splicing. A computational method based on the conservation level of wobble positions and the overabundance of sequence motifs between 46,103 human and mouse orthologous exons was developed, identifying 285 putative ESRs. Alternatively spliced exons that are either short in length or contain weak splice sites show the highest conservation level of those ESRs, especially toward the edges of exons. ESRs that are abundant in those subgroups show a different distribution between constitutively and alternatively spliced exons. Representatives of these ESRs and two SR protein binding sites were shown, experimentally, to display variable regulatory effects on alternative splicing, depending on their relative locations in the exon. This finding signifies the delicate positional effect of ESRs on alternative splicing regulation.

Alternative Splicing↗
PubMed2006

Long splice variant N type calcium channels are clustered at presynaptic transmitter release sites without modular adaptor proteins.

The presynaptic N type Ca channel (CaV2.2) is associated with the transmitter release site apparatus and plays a critical role in the gating of transmitter release. It has been suggested that a distinct CaV2.2 long C terminal splice variant is targeted to the nerve terminal and is anchored at the release face by calcium/calmodulin-dependent serine protein kinase (CASK) and Munc-18-interacting protein (MINT), two modular adaptor proteins. We used the isolated chick ciliary ganglion calyx terminal together with two new antibodies (L4569, L4570) selective for CaV2.2 long C terminal splice variant to test these hypotheses. CaV2.2 long C terminal splice variant was present at the presynaptic transmitter release sites, as identified by Rab3a-interacting molecule (RIM) co-staining and quantitative immunocytochemistry. CASK was also present at the terminal both in conjunction with, and independent of its binding partner, MINT. Immunoprecipitation of CaV2.2 long C terminal splice variant from brain lysate coprecipitated CASK, confirming that these two proteins can form a complex. However, CASK was not colocalized either with CaV2.2 long C terminal splice variant or the transmitter release site marker RIM at the calyx terminal release face. Neither was MINT colocalized with CaV2.2 long C terminal splice variant. Our results show that native CaV2.2 long C terminal splice variant is targeted to the transmitter release sites at an intact presynaptic terminal. However, the lack of enrichment of CASK at the release site combined with the failure of this protein or its partner MINT to colocalize with CaV2.2 argues against the idea that these modular adaptor proteins anchor CaV2.2 at presynaptic nerve terminals.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Structural and functional diversity generated by alternative mRNA splicing.

Alternative mRNA splicing is becoming increasingly recognized as an important mechanism for the generation of structural and functional diversity in proteins. Recent estimations predict that approximately 50% of all eukaryotic proteins can be alternatively spliced. Several lines of evidence suggest that alternative mRNA splicing results in small changes in protein structure and is likely to fine-tune the function and specificity of the affected protein. However, knowledge of how alternative splicing regulates cellular processes on the molecular level is still limited. It is only recently that structures of alternatively spliced proteins have been solved. These studies have shown that alternative splicing affects the structure not only in the vicinity of the splice site but also at long distance.

Alternative Splicing↗
PubMed2005

The splicing machinery is a genetic modifier of disease severity.

Disease severity correlates with the level of correctly spliced RNA transcribed from genes carrying splicing mutations and with the ratio of alternatively spliced isoforms. Hence, a role for splicing regulation as a genetic modifier has been suggested. Here we discuss recent experiments that provide direct evidence that changes in the level of splicing factors modulate the splicing pattern of disease-associated genes. Importantly, modulation of the splicing pattern led to regulation of the protein function and modification of disease severity.

Alternative Splicing↗
PubMed2004

CIR, a corepressor of CBF1, binds to PAP-1 and effects alternative splicing.

We have reported that PAP-1, a product of a causative gene for autosomal retinitis pigmentosa, plays a role in splicing. In this study, CIR, a protein originally identified as a CBF1-interacting protein and reported to act as a transcriptional corepressor, was identified as a PAP-1 binding protein and its function as a splicing factor was investigated. In addition to a basic lysine and acidic serine-rich (BA) domain and a zinc knuckle-like motif, CIR has an arginine/serine dipeptide repeat (RS) domain in its C terminal region. The RS domain has been reported to be present in the superfamily of SR proteins, which are involved in splicing reactions. We generated CIR mutants with deletions of each BA and RS domain and studied their subcellular localizations and interactions with PAP-1 and other SR proteins, including SC35, SF2/ASF, and U2AF35. CIR was found to interact with U2AF35 through the BA domain, with SC35 and SF2/ASF through the RS domain, and with PAP-1 outside the BA domain in vivo and in vitro. CIR was found to be colocalized with SC35 and PAP-1 in nuclear speckles. Then the effect of CIR on splicing was investigated using the E1a minigene as a reporter in HeLa cells. Ectopic expression of CIR with the E1a minigene changed the ratio of spliced isoforms of E1a that were produced by alternative selection of 5'-splice sites. These results indicate that CIR is a member of the family of SR-related proteins and that CIR plays a role in splicing regulation.

Alternative Splicing↗
PubMed2005

Evolutionarily conserved coupling of transcription and alternative splicing in the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes.

Recent studies have shown that transcription and alternative splicing can be mechanistically coupled. In the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes, we showed previously that promoter/alternative first exon choice is coupled to downstream splicing events in exon 2. Here we demonstrate that this coupling is conserved among several vertebrate classes from fish to mammals. The EPB41 and EPB41L3 genes from fish, bird, amphibian, and mammal genomes exhibit shared features including alternative first exons and differential splice acceptors in exon 2. In all cases, the 5'-most exon (exon 1A) splices exclusively to a weaker internal acceptor site in exon 2, skipping a fragment designated as exon 2'. Conversely, alternative first exons 1B and 1C always splice to the stronger first acceptor site, retaining exon 2'. These correlations are independent of cell type or species of origin. Since exon 2' contains a translation initiation site, splice variants generate protein isoforms with distinct N-termini. We propose that these genes represent a physiologically relevant model system for mechanistic analysis of transcription-coupled alternative splicing.

Alternative Splicing↗
PubMed2004

Spliced spleen necrosis virus vector RNA is not encapsidated: implications for retroviral replication and vector design.

RNA splicing is a complex event in the retroviral life cycle and can involve multiple steps, as well as cis-acting sequences, to maintain a proper balance of spliced and unspliced viral RNA for translation and encapsidation. The retroviral RNA can be processed by cellular machinery and enables the removal of intronic sequences. We aimed to utilize the removal of a synthetic intron for targeted gene expression. To analyze intron removal and gene expression, we have constructed a novel self-inactivating gene-activating (SIGA) vector for potential universal gene therapy. New vectors for gene therapy are necessary for safe and effective gene delivery in humans. The SIGA vector is derived from spleen necrosis virus (SNV), which is an avian reticuloendotheliosis virus. The vector was designed so that expression of a therapeutic gene is blocked in helper cell lines due to an intervening sequence containing various blocks in transcription and translation. However, after one round of retroviral replication, the intervening sequence should be removed by the cellular machinery and the therapeutic gene will be selectively expressed in target cells. Our studies show that the intervening sequence in SIGA vector RNA is partially spliced. However, spliced vector RNA was not transduced to target cells. Previous studies showed that an infectious SNV vector enabled transduction of spliced RNA. However, yet-undefined differences in infectious and replication-deficient retroviral replication may have an effect on the transduction of spliced RNA. The results of this study present key information on spliced RNA and its encapsidation, as well as data for the construction of a new generation of SNV-derived retroviral vectors.

Animals↗
PubMed2006

Efficient and persistent splice switching by systemically delivered LNA oligonucleotides in mice.

Locked nucleic acid (LNA) oligomers were found to be very effective in their ability to modulate alternative splicing in vivo in transgenic mice that ubiquitously express a modified EGFP pre-mRNA containing an aberrantly spliced beta-globin intron (IVS2-654). Following intraperitoneal injections, the splice-switching oligonucleotide LNA SSO-654 targeted to the aberrant 5' splice site in EGFP-654 pre-mRNA corrected aberrant splicing and increased production of repaired EGFP mRNA mainly in the liver, colon, and small intestine. Little or no effect was detected in heart, lung, or kidney, the organ where most of the oligonucleotide was distributed after four consecutive daily injections. In the liver, LNA SSO-654 had an EC(50) of 3 mg/kg, approximately 17-fold more potent than its 2'-O-methyl congener. Moreover, in the liver, colon, and small intestine oral doses of 50 mg/kg resulted in detectable levels of splice switching. The effects of four daily injections at 25 mg/kg persisted for up to 29 days but did not result in liver toxicity. The results indicate that the LNA backbone confers sequence- and organ-specific functional biodistribution of the oligonucleotides and that these potent compounds have the potential to be safe and long-acting modulators of diseases treatable by splicing manipulation.

Administration, Oral↗
PubMed1986

Introns as relict retrotransposons: implications for the evolutionary origin of eukaryotic mRNA splicing mechanisms.

A model is presented for the evolutionary origin of intron sequences within eukaryotic protein-coding genes. We propose that introns are the vestiges of transposable elements and, specifically, that they represent a novel class of retrovirus-like transposons. The attraction of the retrotransposon model is that it gives the RNA splicing mechanism a central role in the evolution of introns. There is a growing body of evidence to suggest that several aspects of splicing are intron-encoded. Consequently, it is reasonable to look for evolutionary explanations of the splicing mechanism in the context of the evolution of the intron sequences themselves. According to this model the ancestral intron genomes were replicated into RNA copies simply because of their insertion within transcriptionally active regions of the host genome. Splicing was necessary not only to minimize their negative effects on host gene expression, but also, and perhaps more importantly, to generate new copies of the intron genome free of flanking exon sequences. These spliced intron copies were then available for reverse transcription and reinsertion elsewhere in the genome. Thus, splicing can be seen as an essential step in the intron replication cycle. Most modern introns have probably lost the majority of their original genetic content and may be considered as degenerate evolutionary relicts. An exception to this degeneracy is the set of splicing signals which must be retained because of its continued importance to host cell survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Evolution↗
PubMed1997

The yeast tRNA splicing endonuclease: a tetrameric enzyme with two active site subunits homologous to the archaeal tRNA endonucleases.

The splicing of tRNA precursors is essential for the production of mature tRNA in organisms from all major phyla. In yeast, the tRNA splicing endonuclease is responsible for identification and cleavage of the splice sites in pre-tRNA. We have cloned the genes encoding all four protein subunits of endonuclease. Each gene is essential. Two subunits, Sen2p and Sen34p, contain a homologous domain of approximately 130 amino acids. This domain is found in the gene encoding the archaeal tRNA splicing endonuclease of H. volcanii and in other Archaea. Our results demonstrate that the eucaryal tRNA splicing endonuclease contains two functionally independent active sites for cleavage of the 5' and 3' splice sites, encoded by SEN2 and SEN34, respectively. The presence of endonuclease in Eucarya and Archaea suggests an ancient origin for the tRNA splicing reaction.

Animals↗
PubMed1985

A multicomponent complex is involved in the splicing of messenger RNA precursors.

A multicomponent complex termed spliceosome (splicing body) is unique to the splicing of messenger RNA precursors in vitro. This 60S RNA-protein complex contains RNAs from the previously characterized bipartite splicing intermediate, the 5' exon RNA, and the lariat intervening sequence-3' exon RNA, as well as some intact 455 nucleotide precursor RNA. This complex contains snRNPs, particularly U1 RNP, as shown by immunoprecipitation with specific antisera. Formation of the 60S complex appears to be an early and essential step in splicing, because the 60S complex forms during the early stage, or lag time, of the reaction before the first covalent modification, cleavage at the 5' splice site of precursor RNA. The 60S complex forms only under conditions that permit splicing; both ATP and a precursor RNA containing authentic 5' and 3' splice sites are required for formation, while antiserum specific for U1 RNP inhibits its formation. RNA within the 60S complex, predominantly precursor RNA, was chased into products with accelerated kinetics and more complete conversion than purified precursor RNA.

Adenosine Triphosphate↗
PubMed1985

Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences.

We have investigated the early events of pre-mRNA splicing in vitro by sucrose gradient sedimentation analysis. Time course experiments revealed the assembly, in two steps, of a large (50S) pre-mRNA splicing complex, preceded by formation of two other complexes that sediment at approximately 22S and 35S. Pre-mRNA and the intermediates and products of the in vitro splicing reaction cosediment with the 50S complex, while only pre-mRNA is associated with the 22S and 35S complexes. No splicing is observed in the absence of a 50S complex. Formation of the 50S complex requires ATP, whereas formation of the 22S and 35S complexes does not. U-snRNPs are necessary for assembly of the 35S and the 50S complexes but not for assembly of the 22S complex. Analysis with mutant substrate RNAs demonstrated that a polypyrimidine stretch near the 3' splice site and an intact 5' splice site are absolutely required for splicing complex formation.

Adenosine Triphosphate↗
PubMed2000

Modification of alternative splicing pathways as a potential approach to chemotherapy.

Many cancer-associated genes are alternatively spliced; their expression leads to the production of multiple splice variants. Although the functions of most of these variants are not well-defined, some have antagonistic activities related to regulated cell death mechanisms. In a number of cancers and cancer cell lines, the ratio of the splice variants is frequently shifted so that the anti-apoptotic splice variant predominates. This observation suggests that modification of splicing, which restores the proper ratio of alternatively spliced gene products, may reverse the malignant phenotype of the cells and offer a gene-specific form of anticancer chemotherapy. Our laboratory has extensively investigated the use of antisense oligonucleotides for shifting the splicing patterns of several genes. Potential application of this method for treatment of cancers, as well as of certain genetic disorders, is discussed.

Alternative Splicing↗
PubMed2000

Differential expression of alternatively spliced isoforms of neuronal nitric oxide synthase (nNOS) and N-methyl-D-aspartate receptors (NMDAR) in knockout mice deficient in nNOS alpha (nNOS alpha(Delta/Delta) mice).

Recent data suggest that the neuronal isoform of nitric oxide synthase (nNOS) and glutamate receptors of the N-methyl-D-aspartate (NMDA) type are physically coupled and, hence, functionally interrelated. Several alternatively spliced isoforms of the N-methyl-D-aspartate receptor 1 (NMDAR1) subunit and the neuronal nitric oxide synthase (nNOS) are known, and recent studies have shown that a spliced C-terminal may be responsible for the coupling of NMDAR's to nNOS via its PDZ domain and the postsynaptic density protein PSD95. However, little is known about whether and to what extent changes in nNOS expression influence NMDA receptor density or function. We have therefore compared the localization of nNOS alpha, beta and gamma with that of two relevant NMDAR1 splice variants in wild-type mice versus knockout mice deficient in nNOS alpha, generated by homologous recombination with a targeted deletion of exon 2, containing one PDZ domain (nNOS alpha(Delta/Delta) mice). Whereas nNOS alpha was completely absent in nNOS alpha(Delta/Delta) mice, nNOS beta and gamma were expressed in both wild-type and knockout animals. nNOS gamma mRNA, though, was hardly detectable, if at all, mainly within the olfactory bulb, the cerebellum and mesencephalic nuclei of knockout animals. The expression of the NMDAR1-1 splice variant (without any short carboxy-terminal amino acid motif, recognized by PDZ domains) was remarkably decreased in striatal, cortical, hippocampal and cerebellar tissue in nNOS alpha(Delta/Delta) animals, but no changes in NMDAR1-4 (with an alternatively spliced C-terminal and thus with a PDZ binding motif) mRNA and protein levels were observed. While NMDAR1-4 may be related to receptor targeting and clustering to PSD95 and to nNOS, our data suggest that differences in nNOS expression obviously do not directly influence gene expression of this particular NMDAR splice variant. Otherwise, the observed diminution of NMDAR1-1 splice variant mRNA and protein levels may, at least partially, explain the decreased vulnerability of nNOS alpha(Delta/Delta) mice to glutamate-mediated neurotoxicity.

Alternative Splicing↗