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PubMed1995

Intricate combinatorial patterns of exon splicing generate multiple Rh-related isoforms in human erythroid cells.

The Rhesus (Rh) blood group system shows complex polymorphisms in the human. Some of the heterogeneity may be generated by alternative RNA splicing. For a systematic analysis of Rh-related mRNA isoforms expressed in reticulocytes, we isolated mRNA, which was then reverse transcribed and amplified by the polymerase chain reaction (PCR) to give Rh-related cDNAs of two segments of 704 bp and 975 bp. The PCR amplification of the 5'-region yielded a single PCR product, whereas a complex electrophoretic pattern of PCR bands was derived from the 3'-region. A highly reproducible ladder of multiple additional bands migrated below the PCR products corresponding to the full-size cDNAs for RhPI and RhPII and encoding two different Rh polypeptides. Eleven and five truncated isoforms of the RhPI and RhPII cDNAs, respectively, were identified in the PCR products. These isoforms appear to be generated by combinatorial splicing of six RhPI and three RhPII exons. Our results suggest that the Rh-related polypeptides consist of a mixture of RhPI and RhPII polypeptide isoforms differing at the C terminus. Multiple RNA splicing pathways are thus operative in the two Rh-related genes even within a single cell lineage of human erythroid cells.

Alternative Splicing↗
PubMed2006

The splicing factor PSF is part of a large complex that assembles in the absence of pre-mRNA and contains all five snRNPs.

PSF (PTB-associated splicing factor) is a large nuclear protein that has been implicated in numerous processes including transcription and RNA splicing. It has been shown to directly associate with U5 snRNA and has also been found within numerous purified splicing complexes. Here, we show that when HeLa nuclear extracts are adjusted to splicing conditions, PSF is found as part of a large complex that contains all five snRNPs and most known splicing factors. Formation of the complex does not require addition of exogenous pre-mRNA substrate and occurs at 4 degrees C but is salt sensitive. Sedimentation experiments and identification of individual components by mass spectrometry revealed association with multiple nuclear factors, most of which overlap with spliceosome components.

Alternative Splicing↗
PubMed1996

Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells.

In mammalian cells, genetic instructions are usually revised by RNA splicing before they are translated to proteins. Here we demonstrate that a trans-splicing group I ribozyme can be employed to intentionally modify the sequence of targeted transcripts in tissue culture cells. By analyzing the ribozyme reaction products, we demonstrate that targeted trans-splicing can proceed in murine fibroblasts with high fidelity, providing direct evidence that ribozymes function as anticipated in a therapeutically relevant setting. Trans-splicing is not very specific however, and the ribozyme reacted with and tagged a variety of cellular transcripts with its 3' exon sequence. RNA tagging provides a unique approach to study RNA catalysis in mammalian cells. Such analysis should facilitate the logical development of safe, therapeutic ribozymes that can repair mutant RNAs associated with a variety of inherited diseases.

3T3 Cells↗
PubMed2001

The mitochondrial inner membrane protein Lpe10p, a homologue of Mrs2p, is essential for magnesium homeostasis and group II intron splicing in yeast.

The yeast ORF YPL060w/LPE10 encodes a homologue of the mitochondrial protein Mrs2p. These two proteins are 32% identical, and have two transmembrane domains in their C-terminal regions and a putative magnesium transporter signature, Y/F-G-M-N, at the end of one of these domains. Data presented here indicate that Lpe10p is inserted into the inner mitochondrial membrane with both termini oriented towards the matrix space. Disruption of the LPE10 gene results in a growth defect on non-fermentable substrates (petite phenotype) and a marked defect in group II intron splicing. The fact that in intron-less strains lpe10 disruptants also exhibit a petite phenotype indicates that functions other than RNA splicing are affected by the absence of Lpe10p. In the mitochondria, concentrations of magnesium, but not of several other divalent metal ions, are increased when Lpe10p is overexpressed and reduced when it is absent. Magnesium concentrations are raised to normal levels and growth on non-fermentable substrates is partially restored by the expression of CorA, the bacterial magnesium transporter, in the lpe10 disruptant. These features are similar to those previously reported for Mrs2p, suggesting that Lpe10p and Mrs2p are functional homologues. However, they cannot easily substitute for each other. Their roles in magnesium homeostasis and, possibly as a secondary effect, in RNA splicing are discussed.

Amino Acid Sequence↗
PubMed1992

Epstein-Barr virus latent gene expression in uncultured peripheral blood lymphocytes.

In this study of Epstein-Barr virus (EBV) latency, the polymerase chain reaction was used in modified form for amplification and detection of viral mRNA sequences in peripheral blood lymphocytes from healthy seropositive adults. Six known promoters for latent gene expression and eight known gene products were identified in in vitro-immortalized lymphocytes and in the cell lines established spontaneously from seropositive adults. We examined whether mRNA expression in uncultured B cells from four seropositive adults was the same as that which occurred in spontaneously established EBV-positive B-cell lines from the same individuals. A minimum of 17 polymerase chain reaction targets was required to circumscribe the known latent mRNA structures. Expression of the C promoter for the EBNA genes was detected in B-cell RNA from three of the four subjects. Transcripts initiated from the alternative W promoter for EBNA expression were not detected. The spliced transcripts detected in the B cells contained only the C2-to-W1 alternative splice, which was nonproductive for EBNA4 gene expression. None of the other EBNA open reading frames were detected spliced onto the 3' ends of the C promoter-initiated RNAs. Spliced RNA from the TP gene was detected in all four subjects. Expression of the TP gene was restricted to TP1 promoter-initiated RNAs, as no TP2 promoter-initiated transcripts were detected. Expression of RNA from the LMP gene was not detected. The F promoter which is active in the restricted expression latency that occurs in Burkitt's lymphoma cells was not detected being expressed in peripheral blood B cells. This pattern of latent gene expression is unique to uncultured B cells, indicating that there are profound differences between viral latent states in vitro and in situ and suggesting a central role for the TP gene in the latency of EBV.

Adult↗
PubMed1989

Evidence for nuclear factors involved in recognition of 5' splice sites.

To investigate soluble factors involved in pre-messenger RNA splicing we have fractionated nuclear extract by simple centrifugation to produce a supernatant pellet pair. Factors larger than 15S including U2, U4, U5, and U6 snRNPs fractionate with the pellet; U1 snRNPs distribute equally in pellet and supernatant. Each fraction is individually incompetent for splicing and spliceosome assembly; mixing restores wild type activity and assembly. The pellet fraction directs an aberrant assembly pathway in which proper 3', but improper 5' splice site recognition occurs. Complexes formed with the pellet fraction are distinguishable from wild-type complexes using native gel electrophoresis. Pellet complexes contain U1 snRNP antigens and their formation requires ATP, U1 snRNPs, U2 snRNPs, and sequences at the 3' end of the intron - properties shared with the initial steps of normal assembly and directed by sequences at the 3' end of the intron. In contrast, pellet complex assembly shows no dependence on the presence of a 5' splice junction within precursor RNA. Furthermore, binding of factors to the 5' splice junction is deficient in pellet assemblies. Thus, the pellet lacks a factor required for proper recognition of 5' splice sites. This factor can be supplied by the supernatant. Complementation occurs when supernatant U1 RNA is destroyed, suggesting that the supernatant factor recognizing 5' splice sites is not U1 snRNPs.

Adenosine Triphosphate↗
PubMed1992

Participation of the intron in the reaction catalyzed by the Xenopus tRNA splicing endonuclease.

Introns have generally been assumed to be passive in the transfer RNA splicing reaction. Experiments have now been done showing that the endonuclease is able to cut a precursor provided that a base in the single-stranded loop of the intron can pair with the base of the 5' exon situated at the position that immediately follows the anticodon stem (position 33 in the yeast tRNA isoacceptor pre-tRNA(Leu)3, position 32 in yeast pre-tRNA(Phe)). The elucidation of the role of the intron reveals that in addition to the conserved bases, there are positions in the mature domain which, although not necessarily occupied by the same base in all pre-tRNA's, nevertheless have a fundamental role in the splicing reaction. These positions are termed cardinal positions.

Animals↗
PubMed1985

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant.

The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5' splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3' splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptide IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3' splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.

Adenoviridae↗
PubMed1999

Characterization of an alternatively spliced isoform of rat vesicle associated membrane protein-2 (VAMP-2).

VAMPs are vesicle associated membrane proteins that are essential for secretion. A spliced isoform of rat VAMP-2, called VAMP-2B, is characterized in this study. The VAMP-2B transcript is the result of alternative RNA splicing in which an intron is retained. The predicted amino acid sequence of VAMP-2B differs from VAMP-2 at its carboxy-terminal end. Because recent studies have shown that VAMP's carboxy-terminal end influences the protein's sorting, the location of myc-epitope tagged VAMP-2B in PC12 cells was determined. Subcellular fractionation showed colocalization of myc-VAMP-2B and endogenous VAMP-2. Thus alternative RNA splicing does not affect VAMP-2 sorting in PC12 cells.

Alternative Splicing↗
PubMed1997

Role of tissue specific alternative pre-mRNA splicing in the differentiation of the erythrocyte membrane.

Regulated alternative pre-mRNA splicing is neither as widely appreciated as a fundamental aspect of controlled gene expression nor as thoroughly studied as transcriptional regulation. However, as exemplified by the phenomena cited in this review, alternative splicing is a fundamentally important mechanism used in the eukaryotic world to enhance the range, versatility and plasticity of the structural information contained within a gene, and to create additional strategies by which the net quantitative output of a given gene product can be controlled. Regulation of RNA splicing gives genes a modularity that adds flexibility, and, therefore, selective advantage, to eukaryotes. It is likely, though unproven, that this opportunity for refined regulation and diversification provides at least one basis for the existence of the tandem exon-intron-exon structure found in the vast majority of eukaryotic genes and many viral genes. Many examples of alternative splicing are known, but, for the majority, no obvious biological impact of the alternatively spliced proteins on known cellular functions can be appreciated. Examples by which selectively regulated splicing pathways alter both the physiology and pathology of a major cellular event, such as differentiation and mechanical function of the red cell membrane, are thus relatively rare. The protein 4.1 gene and mRNA products thus provide an instructive and unusual system in which to explore the broader issue of the role of these regulatory mechanisms in the overall scheme of gene regulation and adaptation. The fact that hereditary hemolytic anemias result from mutations that directly or indirectly disrupt the splicing system emphasized the relevance of these mechanisms to molecular medicine. The features of splicing that we have reviewed in this paper, and the specific impact that regulated splicing exerts on differentiating red cells have, we hope, convinced the reader that RNA splicing is an important, fascinating, and potentially fruitful area for future study of human disease processes.

Actins↗
PubMed2006

The E7 oncoprotein is translated from spliced E6*I transcripts in high-risk human papillomavirus type 16- or type 18-positive cervical cancer cell lines via translation reinitiation.

High-risk human papillomaviruses (HPVs) encode two viral oncoproteins, E6 and E7, from a single bicistronic pre-mRNA containing three exons and two introns. Retention of intron 1 in the E6 coding region is essential for production of the full-length E6 oncoprotein. However, splicing of intron 1 is extremely efficient in cervical cancer cells, leading to the production of a spliced transcript, E6*I, of E6. Here, we investigated whether this splicing of intron 1 might benefit E7 production. Using RNA interference as a tool, we targeted the intron 1 region using small interfering RNAs (siRNAs) in HPV-positive cell lines. At an effective low dose, the siRNAs specifically suppressed E6 expression but not E7 expression, as demonstrated by the stabilization of p53. However, at high doses the HPV18 intron 1-specific siRNA substantially and specifically reduced the level of the 18E6*I mRNA lacking the intron region in HeLa cells, implying its nuclear silencing on the pre-mRNA before RNA splicing. Two other siRNAs targeting the exon 2 regions of HPV16 and -18, which encode the E7 oncoprotein, reduced the E6*I mRNAs to a remarkable extent and preferentially suppressed expression of E7, leading to accumulation of hypophosphorylated p105Rb and cell cycle arrest, indicating that the majority of E7 proteins are the translational products of E6*I mRNAs. This was confirmed by transient transfection in 293 cells: E7 could be translated only from the E7 open reading frame (ORF) on E6*I mRNA in a distance-dependent matter of upstream E6*I ORF by translation reinitiation. The data thus provide direct evidence that the E6*I mRNAs of high-risk HPVs are responsible for E7 production.

Cell Line↗
PubMed1996

Evidence of gene deletion of p21 (WAF1/CIP1), a cyclin-dependent protein kinase inhibitor, in thyroid carcinomas.

Eukaryotic cell cycle progression is controlled by a host of cyclin/cyclin-dependent kinases (Cdks), that are themselves regulated by multiple factors, including a group of small cyclin-Cdk inhibitor proteins (p15, p16, p21 and p27). The involvement of Cdk inhibitors in carcinogenesis has been demonstrated by the studies of p16. p53 is frequently mutated in thyroid carcinomas and p21/Waf1 is a downstream effector of p53. It is conceivable that genetic defects of genes downstream in the p53 pathway could also be oncogenic. We, therefore, examined a series of 57 thyroid tumour specimens (eight follicular adenomas and 49 carcinomas) for deletion and point mutation of the p21/Waf1 gene. Three different kinds of deletions ranging from 349 to 450 bp were detected in five papillary carcinoma specimens by reverse transcription-polymerase chain reaction (RT-PCR). All the deletions were involved in the second exon of the p21/Waf1 gene. RT-PCR single strand conformational polymorphism (SSCP) analysis of remaining samples failed to reveal any point mutations in the coding region of the gene, except for a polymorphism at codon 31 (Ser to Arg). Genomic Southern blot analysis did not demonstrate any gene deletion or rearrangement in these samples, indicating abnormal RNA splicing may be involved. Analysis of intron-exon boundary and the coding region of the second exon did not reveal any mutation except for a point mutation (C to G) located 16 bp downstream from the splice donor site of the second intron in three out of five samples with p21/Waf1 deletions. Whether the mutation plays any role in aberrant RNA splicing remains to be determined. Among the five samples with p21/Waf1 gene deletions, none of them simultaneously carried a p53 or retinoblastoma (Rb) gene mutation. No p21/Waf1 abnormality was found in the benign adenomas. Thus, 12.5% (5/40) of thyroid papillary carcinoma specimens harboured p21/Waf1 gene deletions. Our data suggest that p21/Waf1 gene deletion is involved in thyroid carcinogenesis and may play an important role in thyroid cell transformation.

Adenocarcinoma, Follicular↗
PubMed1993

Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity.

Multiple RNA splicing sites exist within human immunodeficiency virus type 1 (HIV-1) genomic RNA, and these sites enable the synthesis of many mRNAs for each of several viral proteins. We evaluated the biological significance of the alternatively spliced mRNA species during productive HIV-1 infections of peripheral blood lymphocytes and human T-cell lines to determine the potential role of alternative RNA splicing in the regulation of HIV-1 replication and infection. First, we used a semiquantitative polymerase chain reaction of cDNAs that were radiolabeled for gel analysis to determine the relative abundance of the diverse array of alternatively spliced HIV-1 mRNAs. The predominant rev, tat, vpr, and env RNAs contained a minimum of noncoding sequence, but the predominant nef mRNAs were incompletely spliced and invariably included noncoding exons. Second, the effect of altered RNA processing was measured following mutagenesis of the major 5' splice donor and several cryptic, constitutive, and competing 3' splice acceptor motifs of HIV-1NL4-3. Mutations that ablated constitutive splice sites led to the activation of new cryptic sites; some of these preserved biological function. Mutations that ablated competing splice acceptor sites caused marked alterations in the pool of virus-derived mRNAs and, in some instances, in virus infectivity and/or the profile of virus proteins. The redundant RNA splicing signals in the HIV-1 genome and alternatively spliced mRNAs provides a mechanism for regulating the relative proportions of HIV-1 proteins and, in some cases, viral infectivity.

Alternative Splicing↗
PubMed2004

Effect of SNPs in protein kinase C zeta gene on gene expression in the reporter gene detection system.

AIM: To investigated the effects of the SNPs (rs411021, rs436045, rs427811, rs385039 and rs809912) on gene expression and further identify the susceptibility genes of type 2 diabetes. METHODS: Ten allele fragments (49 bp each) were synthesized according to the 5 SNPs mentioned above. These fragments were cloned into luciferase reporter gene vector and then transfected into HepG2 cells. The activity of the luciferase was assayed. Effects of the SNPs on RNA splicing were analyzed by bioinformatics. RESULTS: rs427811T allele and rs809912G allele enhanced the activity of the reporter gene expression. None of the 5 SNPs affected RNA splicing. CONCLUSION: SNPs in protein kinase Cz (PKCZ) gene probably play a role in the susceptibility to type 2 diabetes by affecting the expression level of the relevant genes.

Base Sequence↗
PubMed1996

Analysis of alternative splicing patterns in the cystic fibrosis transmembrane conductance regulator gene using mRNA derived from lymphoblastoid cells of cystic fibrosis patients.

Using in vitro amplification of cDNA by the polymerase chain reaction, we analyzed alternatively spliced events of cystic fibrosis transmembrane conductance regulator gene in lymphoblastoid cells. Ten alternatively spliced transcripts were identified using analysis of 6 overlapping segments of amplified cDNA, 4 of which have not been described previously. These include transcripts lacking exon 16, 17b, 22 and a transcript resulting from the use of a cryptic acceptor and donor splice sites. Moreover, in 2 cystic fibrosis (CF) patients bearing nonsense mutations E60X or W1282X, we observed that nonsense mutations are associated with an alteration of splice site selection in vivo resulting in exon skipping of constitutive exons or in the use of cryptic splice sites. In addition, even though lymphoblastoid cells are not the relevant tissue to address the question of the relationship between clinical respiratory phenotype and genotype, our results concerning adult CF patients (delta F508/ delta F508) suggest that individual-specific RNA splicing patterns could influence the severity of the CF pulmonary disease. If this phenomenon of alternative splicing events proves to be significant in CF and to be a common feature of disease genes, the study of RNA splicing could become an important tool for the analysis of the genotype-phenotype relationship in many inherited disorders.

Adult↗
PubMed1987

Developmentally induced, muscle-specific trans factors control the differential splicing of alternative and constitutive troponin T exons.

Alternative RNA splicing is a ubiquitous process permitting single genes to encode multiple protein isoforms. Here we report experiments in which a gene construct, containing combinatorial Troponin T (TnT) exons that manifest an exceptional diversity of alternative splicing in vivo, has been transfected into muscle and nonmuscle cells. Analyses of the spliced RNAs show that the alternative TnT exons retain their capacity for differential splicing in the modified minigene context when introduced into a variety of nonmuscle and muscle cells. The patterns of alternative splicing differ depending on cell type. Only in differentiated myotubes are the alternative exons normally incorporated during splicing, reproducing their behavior in the native gene; they are excluded in nonmuscle cells and myoblasts that do not express the endogenous TnT. These results provide proof that trans factors required for correct alternative splicing are induced during myogenesis. Surprisingly, such factors are also required for the correct splicing of constitutive TnT exons.

Animals↗
PubMed1985

Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing.

N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA molecule, the genomic RNA of Rous sarcoma virus. Specific fragments of 32P-labeled Rous sarcoma virus RNA were isolated by hybridization with complementary DNA restriction fragments spanning nucleotides 6185 to 8050. RNA was digested with RNase and finger-printed, and individual oligonucleotides were analyzed for the presence of m6A by paper electrophoresis and thin-layer chromatography. With this technique, seven sites of methylation in this region of the Rous sarcoma virus genome were localized at nucleotides 6394, 6447, 6507, 6718, 7414, 7424, and 8014. Further, m6A was observed at two additional sites whose nucleotide assignments remain ambiguous. A clustering of two or more m6A residues was seen at three positions within the RNA analyzed. Modification at certain sites was found to be heterogeneous, in that different molecules of RNA appeared to be methylated differently. Previous studies have determined that methylation occurs only in the sequences Gm6AC and Am6AC. We observed a high frequency of methylation at PuGm6ACU sequences. The possible involvement of m6A in RNA splicing events is discussed.

Adenosine↗