RNA processing. Splicing in stereo.
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Biological Evolution↗
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Results for “RNA Splicing”
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Interactions of small nuclear RNA's with precursor messenger RNA during in vitro splicing.
Precursor messenger RNA splicing requires multiple factors including U1, U2, U4, U5, and U6 small nuclear RNA's. The crosslinking reagent psoralen was used to analyze the interactions of these RNA's with an adenovirus precursor messenger RNA in HeLa nuclear extract. An endogenous U2-U4-U6 crosslinkable complex dissociated upon incubation with precursor messenger RNA. During splicing, U1, U2, U5, and U6 became crosslinked to precursor messenger RNA and U2, U5, and U6 became crosslinked to excised lariat intron. U2 also formed a doubly crosslinked complex with U6 and precursor messenger RNA. The U1, U5, and U6 crosslinks to the precursor messenger RNA mapped to intron sequences near the 5' splice site, whereas the U2 crosslink mapped to the branch site. The kinetics of crosslink formation and disappearance delineates a temporal pathway for the action of small RNA's in the spliceosome. Potential base pairing interactions between conserved sequences in the small nuclear RNA's and precursor messenger RNA at the sites of crosslinking suggest that the 5' splice site is defined in several steps prior to the first cleavage event.
Adenosine Triphosphate↗
E-cadherin gene mutations in signet ring cell carcinoma of the stomach.
To clarify the significance of E-cadherin gene alterations in the development of diffuse-type adenocarcinoma of the stomach, we analyzed mutations of the E-cadherin gene using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method followed by direct sequencing. Twenty-two signet ring cell carcinomas of the stomach (10 intramucosal and 12 advanced cancers) were examined. Genomic DNA was extracted separately from cancerous and non-cancerous tissues and PCR-SSCP analysis was performed on exons 5 to 9 and the adjacent 30- to 40-base-pair intron sequences of the E-cadherin gene. Mobility shifts were found in 2 of the 10 intramucosal cancers. In 2 of the 12 advanced cancers, abnormalities of the E-cadherin gene were observed in intramucosal lesions as well as in deeply invaded areas. These results indicated that E-cadherin gene mutations are an early event in the development of signet ring cell carcinoma of the stomach. Direct sequencing revealed that the locations of mutations of the E-cadherin gene included the branch point sequence in the intron which is responsible for RNA splicing. Reverse transcriptase-PCR demonstrated aberrant RNA splicing in a case with a branch point mutation, suggesting that branch point mutations play an important role in functional modifications of E-cadherin in signet ring cell carcinoma of the stomach.
Adult↗
Qualitative gene profiling: a novel tool in genomics and in pharmacogenomics that deciphers messenger RNA isoforms diversity.
RNA splicing is a tightly regulated process. It is essential for gene expression and, therefore, intervenes in every biological phenomenon in mammals. RNA splicing regulation is cell type-specific in such a way that a cellular situation can be characterised by its repertoire of spliced events, the spliceome. Comparison of the splicing repertoire of two situations identifies alternative exons and introns. This regulation involves cis-acting sequences and transacting factors. Mutations, as well as modifications of signalling pathways, can alter the accuracy of splicing. Since deletion of exons or retention of introns within coding sequences modifies the corresponding proteins and functional domains of proteins are encoded by contiguous exons, identifying changes in the spliceome pinpoints functional domains, which are specifically regulated at the level of RNA splicing. We have developed a new method of gene profiling, qualitative gene profiling, that allows the comparative study of the repertoires of spliced events that characterise distinct physiopathological situations. We present in this review the different uses of this new genomic technique that can help each step of the R&D process in the pharmaceutical industry, and that represents a short cut towards functional genomics and pharmacogenomics.
Animals↗
Characterization of a multisubunit transcription factor complex essential for spliced-leader RNA gene transcription in Trypanosoma brucei.
In the unicellular human parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., the spliced-leader (SL) RNA is a key molecule in gene expression donating its 5'-terminal region in SL addition trans splicing of nuclear pre-mRNA. While there is no evidence that this process exists in mammals, it is obligatory in mRNA maturation of trypanosomatid parasites. Hence, throughout their life cycle, these organisms crucially depend on high levels of SL RNA synthesis. As putative SL RNA gene transcription factors, a partially characterized small nuclear RNA-activating protein complex (SNAP(c)) and the TATA-binding protein related factor 4 (TRF4) have been identified thus far. Here, by tagging TRF4 with a novel epitope combination termed PTP, we tandem affinity purified from crude T. brucei extracts a stable and transcriptionally active complex of six proteins. Besides TRF4 these were identified as extremely divergent subunits of SNAP(c) and of transcription factor IIA (TFIIA). The latter finding was unexpected since genome databases of trypanosomatid parasites appeared to lack general class II transcription factors. As we demonstrate, the TRF4/SNAP(c)/TFIIA complex binds specifically to the SL RNA gene promoter upstream sequence element and is absolutely essential for SL RNA gene transcription in vitro.
5' Untranslated Regions↗
ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5' splice site.
Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site.
Adenosine Triphosphate↗
De novo synthesis of an intron by the maize transposable element Dissociation.
The mechanisms by which introns are gained or lost in the evolution of eukaryotic genes remain poorly understood. The discovery that transposable elements sometimes alter RNA splicing to allow partial or imperfect removal of the element from the primary transcripts suggests that transposons are a potential and continuing source of new introns. To date, splicing events that precisely restore the wild-type RNA sequence at the site of insertion have not been detected. Here we describe alternative RNA splicing patterns that result in precise removal of a Dissociation (Ds) insertion and one copy of its eight-nucleotide host site duplication from an exon sequence of the maize shrunken2-mutabe1 (sh2-m1) mutant. In one case, perfect splicing of Ds was associated with aberrant splicing of an intron located 32 bp upstream of the insertion site. The second transcript type was indistinguishable from wild-type mRNA, indicating that Ds was spliced like a normal intron in about 2% of the sh2-m1 transcripts. Our results suggest that the transposition of Ds into sh2 in 1968, in effect, marked the creation of a new intron in a modern eukaryotic gene. The possibility of precise intron formation by a transposable element demonstrated here may be a general phenomenon of intron formation, since consensus intron splice sites can be explained by insertions that duplicate host sequences upon integration. A model is presented.
Alleles↗
Trypanosomal TBP functions with the multisubunit transcription factor tSNAP to direct spliced-leader RNA gene expression.
Protein-coding genes of trypanosomes are mainly transcribed polycistronically and cleaved into functional mRNAs in a process that requires trans splicing of a capped 39-nucleotide RNA derived from a short transcript, the spliced-leader (SL) RNA. SL RNA genes are individually transcribed from the only identified trypanosome RNA polymerase II promoter. We have purified and characterized a sequence-specific SL RNA promoter-binding complex, tSNAP(c), from the pathogenic parasite Trypanosoma brucei, which induces robust transcriptional activity within the SL RNA gene. Two tSNAP(c) subunits resemble essential components of the metazoan transcription factor SNAP(c), which directs small nuclear RNA transcription. A third subunit is unrelated to any eukaryotic protein and identifies tSNAP(c) as a unique trypanosomal transcription factor. Intriguingly, the unusual trypanosome TATA-binding protein (TBP) tightly associates with tSNAPc and is essential for SL RNA gene transcription. These findings provide the first view of the architecture of a transcriptional complex that assembles at an RNA polymerase II-dependent gene promoter in a highly divergent eukaryote.
Amino Acid Sequence↗
Trypanosomatid biodiversity in Costa Rica: genotyping of parasites from Heteroptera using the spliced leader RNA gene.
The biodiversity of insect trypanosomes is largely unknown, resulting in significant gaps in the understanding of pathogen evolution. A culture-independent preliminary survey of trypanosomatid fauna was conducted for the parasites of Heteroptera (Hemiptera) from several localities in Costa Rica. Trypanosomatid infections were detected by light microscopy of smeared gut contents. Out of 257 insects representing 6 families, infections were found in 62 cases; cultures were obtained for 29 new isolates. Gut material from infected hosts was preserved in the field using an SDS-EDTA buffer solution for subsequent DNA extraction in the laboratory. PCR amplification of the trypanosomatid-specific spliced leader (SL) RNA gene repeats was successful for 60 field samples. Eighteen distinct SL RNA typing units were identified in a set of 28 samples analysed in detail. Cluster analysis indicated that these typing units were unique and thus could represent new species and, in some cases, new genera. This study reveals only a minor fraction of the trypanosomatid biodiversity, which is anticipated to be high.
Animals↗
RNA processing. Splicing in space.
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Cell Nucleus↗
Splicing potentiation by growth factor signals via estrogen receptor phosphorylation.
Mitogen-activated protein kinase-mediated growth factor signals are known to augment the ligand-induced transactivation function of nuclear estrogen receptor alpha (ERalpha) through phosphorylation of Ser-118 within the ERalpha N-terminal transactivation (activation function-1) domain. We identified the spliceosome component splicing factor (SF)3a p120 as a coactivator specific for human ERalpha (hERalpha) activation function-1 that physically associated with ERalpha dependent on the phosphorylation state of Ser-118. SF3a p120 potentiated hERalpha-mediated RNA splicing, and notably, the potentiation of RNA splicing by SF3a p120 depended on hER Ser-118 phosphorylation. Thus, our findings suggest a mechanism by which growth factor signaling can regulate gene expression through the modulation of RNA splicing efficiency via phosphorylation of sequence-specific activators, after association between such activators and the spliceosome.
Animals↗
Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: implications for tauopathies.
Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3' end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9-exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10(-) tau RNA into exon 10(+) tau RNA could be achieved with approximately 34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing.
Alternative Splicing↗
The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo.
RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.
Amino Acid Sequence↗
T cell receptor-beta mRNA splicing: regulation of unusual splicing intermediates.
The expression of functional T cell receptor-beta (TCR-beta) transcripts requires the activation of programmed DNA rearrangement events. It is not clear whether other mechanisms dictate TCR-beta mRNA levels during thymic ontogeny. We examined the potential role of RNA splicing as a regulatory mechanism. As a model system, we used an immature T cell clone, SL12.4, that transcribes a fully rearranged TCR-beta gene but essentially lacks mature 1.3-kb TCR-beta transcripts in the cytoplasm. Abundant TCR-beta splicing intermediates accumulate in the nucleus of this cell clone. These splicing intermediates result from inefficient or inhibited excision of four of the five TCR-beta introns; the only intron that is efficiently spliced is the most 5' intron, IVSL. The focal point for the regulation appears to be IVS1C beta 1 and IVS2C beta 1, since unusual splicing intermediates that have cleaved the 5' splice site but not the 3' splice site of these two introns accumulate in vivo. The block in 3' splice site cleavage is of interest since sequence analysis reveals that these two introns possess canonical splice sites. A repressional mechanism involving a labile repressor protein may be responsible for the inhibition of RNA splicing since treatment of SL12.4 cells with the protein synthesis inhibitor cycloheximide reversibly induces a rapid and dramatic accumulation of fully spliced TCR-beta transcripts in the cytoplasm, concomitant with a decline in TCR-beta pre-mRNAs in the nucleus. This inducible system may be useful for future studies analyzing the underlying molecular mechanisms that regulate RNA splicing.
Animals↗
Identification of a spliced leader RNA binding protein from Trypanosoma cruzi.
Nuclear mRNAs in trypanosomatids are generated by trans-splicing. Although trans-splicing resembles cis-splicing in many ways and most of the U RNA participants have been characterized, relatively few involved proteins have been identified. Herein, we employed a yeast three-hybrid system to identify a protein, XB1, which binds to the Trypanosoma cruzi SL RNA. XB1 is a approximately 45 kDa protein which is homologous to the essential pre-mRNA-splicing factor PRP31p from Saccharomyces cerevisiae. Gel shift assays and UV cross-linking experiments with recombinant XB1 confirmed that this T. cruzi protein binds the SL RNA in vitro. The binding site of XB1 on the SL RNA was mapped to stem-loop II by deletion of the SL RNA 'bait' in the three-hybrid system. Finally, UV cross-linking SL RNA with S100 extract indicated native XB1 protein and SL RNA interaction in T. cruzi extract.
Amino Acid Sequence↗
Mutually exclusive exon splicing of type III brain sodium channel alpha subunit RNA generates developmentally regulated isoforms in rat brain.
We have identified two exons of the type III rat brain sodium channel alpha subunit gene that undergo mutually exclusive alternative RNA splicing to produce mRNAs coding either for an isoform predominant in neonatal brain (IIIN) or a different isoform (IIIA) predominant in the adult. These exons are 92 base pairs in length and encode amino acids 203-232, which correspond to part of the S3 and most of the S4 transmembrane segments within domain I and the extracellular loop between them. Despite 21 nucleotide differences between the exons, only a single amino acid at position 209 is altered, specifying either aspartic acid (IIIA) or serine (IIIN). As evidence that these isoforms are generated via alternative splicing, we demonstrate that both exons are encoded within the type III gene. The nucleotide sequences of the neonatal and adult type III exons and the intervening intron as well as the developmental regulation of this splicing are nearly identical in the type II sodium channel gene. The conservation of the exon/intron structure and of the developmentally regulated patterns of expression of the type II and III sodium channel genes suggests that alternative mRNA splicing of this exon may play a substantial role in modulating sodium channel function during brain development by alteration of a single amino acid.
Animals↗
Two distinct tumor-derived, parathyroid hormone-like peptides result from alternative ribonucleic acid splicing.
A novel PTH-like peptide has recently been isolated and cloned from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. The deduced product of the initial clones to be reported is a 177 amino-acid protein, consisting of a 36 amino-acid precursor sequence followed by a 141 amino-acid mature peptide. Southern analysis of genomic DNA is compatible with a single-copy gene, but Northern analysis of mRNAs from both tumors and normal tissues consistently reveals multiple hybridizing transcripts, suggesting the possibility of alternative RNA splicing. We provide here direct evidence of alternative RNA splicing by the identification of a second cDNA clone in a human renal carcinoma cDNA library. As compared to the initial clone, this cDNA contains a foreshortened 5'-untranslated region but is otherwise identical until the distal portion of the coding region, at which point it diverges completely to encode a 166 amino-acid mature peptide with 27 amino acids of unique C-terminal sequence. The relative lengths of the primary translation products encoded by these two cDNAs were confirmed by transcription and translation in vitro, and both products were shown to be processed by added microsomes. The unique 3'-ends of these two cDNAs, as well as that of a third cDNA isolated by another laboratory, were used to identify one or more hybridizing transcripts corresponding to each cDNA in mRNA from a human renal carcinoma as well as in mRNA from normal human keratinocytes. We conclude that alternative RNA splicing results in mRNAs which encode multiple PTH-like peptides.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Cellular viral rebound after cessation of potent antiretroviral therapy predicted by levels of multiply spliced HIV-1 RNA encoding nef.
To characterize newly arising replication of human immunodeficiency virus (HIV) type 1 in vivo at the cellular level, distinct viral RNA species in peripheral blood mononuclear cells (PBMCs) from HIV-1-infected patients were monitored during 2 weeks of structured treatment interruption (STI). HIV-1 RNA encoding tat/rev and PBMC-associated virions were almost completely depleted during antiretroviral therapy and emerged simultaneously after 2 weeks of STI, thus specifically reflecting productive viral infection at the cellular level. The magnitude of these correlates of reappearing cellular viral replication was predicted by during-therapy levels of nef transcripts in PBMCs. Significant rebound of plasma viremia, representing the progeny of a broader range of anatomical compartments, preceded and predicted productive infection in PBMCs. Thus, cellular viral rebound in PBMCs likely was primed before STI by the expression of nef in HIV-1-infected PBMCs that lacked virion production and was subsequently triggered by the plasma viremia that preceded the recurrence of productively infected PBMCs.
Anti-HIV Agents↗