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Results for “RNA Splicing”

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PubMed1990

Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes.

Trypanosomatid protozoan parasites cause several important tropical diseases and have been a fertile ground for the discovery of molecular paradigms such as trans-splicing and RNA editing. Transfection-based methods for the study of these organisms have recently been developed, and we have now designed an expression vector, pX, which contains only 2.3 kilobases of Leishmania DNA and can be stably transfected with high efficiency. Genes encoding Escherichia coli beta-galactosidase or a Leishmania amazonensis protective membrane glycoprotein (GP46A/M-2) were inserted into the pX expression site and transfected into Leishmania major, where they directed the synthesis of high levels of mRNAs formed by 5' and 3' processing events occurring predominantly at the sites used by the normal transcripts. Colony assays and immunoblot analysis showed that both proteins were produced; enzymatically active beta-galactosidase comprised approximately 1% of total protein. Sizes of the GP46A protein synthesized in transfected L. major or L. amazonensis were similar and differed from the predominant L. amazonensis GP46, suggesting that the GP46A gene may encode a variant GP46 family member. Because these vectors function efficiently in pathogenic species of Leishmania, pX will facilitate the genetic analyses of parasite proteins crucial for infectivity as well as the identification of cis-acting elements mediating transcription and replication.

Animals↗

PubMed1991

Alternative splicing contributes to K+ channel diversity in the mammalian central nervous system.

In an attempt to define the molecular basis of the functional diversity of K+ channels, we have isolated overlapping rat brain cDNAs that encoded a neuronal delayed rectifier K+ channel, K,4, that is structurally related to the Drosophila Shaw protein. Unlike previously characterized mammalian K+ channel genes, which each contain a single protein-coding exon, K,4 arises from alternative exon usage at a locus that also encodes another mammalian Shaw homolog, NGK2. Thus, the enormous diversity of K+ channels in mammals can be generated not just through gene duplication and divergence but also through alternative splicing of RNA.

Amino Acid Sequence↗

PubMed2003

An evolutionarily conserved role for SRm160 in 3'-end processing that functions independently of exon junction complex formation.

SRm160 (the SR-related nuclear matrix protein of 160 kDa) functions as a splicing coactivator and 3'-end cleavage-stimulatory factor. It is also a component of the splicing-dependent exon-junction complex (EJC), which has been implicated in coupling of pre-mRNA splicing with mRNA turnover and mRNA export. We have investigated whether the association of SRm160 with the EJC is important for efficient 3'-end cleavage. The EJC components RNPS1, REF, UAP56, and Y14 interact with SRm160. However, when these factors were tethered to transcripts, only SRm160 and RNPS1 stimulated 3'-end cleavage. Whereas SRm160 stimulated cleavage to a similar extent in the presence or absence of an active intron, stimulation of 3'-end cleavage by tethered RNPS1 is dependent on an active intron. Assembly of an EJC adjacent to the cleavage and polyadenylation signal in vitro did not significantly affect cleavage efficiency. These results suggest that SRm160 stimulates cleavage independently of its association with EJC components and that the cleavage-stimulatory activity of RNPS1 may be an indirect consequence of its ability to stimulate splicing. Using RNA interference (RNAi) in Caenorhabditis elegans, we determined whether interactions between SRm160 and the cleavage machinery are important in a whole organism context. Simultaneous RNAi of SRm160 and the cleavage factor CstF-50 (Cleavage stimulation factor 50-kDa subunit) resulted in late embryonic developmental arrest. In contrast, RNAi of CstF-50 in combination with RNPS1 or REFs did not result in an apparent phenotype. Our combined results provide evidence for an evolutionarily conserved interaction between SRm160 and the 3'-end cleavage machinery that functions independently of EJC formation.

Animals↗

PubMed2004

Productive infection of primary murine astrocytes, lymphocytes, and macrophages by human immunodeficiency virus type 1 in culture.

A mouse model of human immunodeficiency virus type 1 (HIV-1) infection would be extremely valuable for evaluation of therapies and vaccines; however, multiple blocks to productive infection of NIH 3T3 and other mouse cell lines have been reported. The authors investigated the replication of HIV-1 in primary mouse astrocytes, lymphocytes, and macrophages in culture by infection with intact HIV-1 pseudotyped with the vesicular stomatitis virus G envelope glycoprotein (VSV-G) or with the envelope glycoprotein of amphotropic murine leukemia virus. Astrocytes, lymphocytes, and macrophages were susceptible to productive infection as variously assayed by detection of p24 and Tat proteins, viral protease-mediated processing of Gag, appropriately spliced viral RNA, and infectious progeny virus. As expected, NIH 3T3 cells were not susceptible to productive infection by VSV/NL4. Susceptibility mapped neither to the Fv locus nor to a possible polymorphism in cyclin T1. This study indicates that there are no intrinsic intracellular barriers to HIV-1 replication in primary mouse cells when virus entry is efficient.

Animals↗

PubMed1989

Cyclic oxyphosphoranes as model intermediates during splicing and cleavage of RNA: ab initio molecular orbital calculations on the conformational analysis.

Ab initio molecular orbital calculations have been carried out on hydrated adducts of methyl ethylene phosphate as a model intermediate during cleavage of RNA. Upon rotating the apical methoxyl group two kinds of stable conformers and two kinds of rotational transition states are located, the most stable conformation being gs-G where the dihedral angle between the apical methyl group and the basal ring oxygen is calculated to be 76 degrees. In this gs-G conformation one of the lone pairs on the apical oxygen is oriented antiperiplanar to the basal ring ester bond. The torsional energy required to rotate the methyl group about the phosphorus-apical oxygen bond leading to ts-C conformation, where the methyl group is eclipsed with the ring oxygen, is calculated to be 5.2 kcal/mol. Judging from the published substrate's coordinates in the RNase environment, the expected pentacoordinate-intermediate/transition state during the cleavage of RNA appears to be, in fact, the most stable gs-G conformation.

Indicators and Reagents↗

PubMed2004

Crystallization and preliminary diffraction analysis of a group I ribozyme from bacteriophage Twort.

Group I introns are catalytic RNAs that are capable of performing a variety of phosphotransesterification reactions including self-splicing and RNA cleavage. The reactions are efficient, accurate and dependent only on the presence of guanosine-nucleotide substrate and sufficient magnesium ion to stabilize the structure of the RNA. To understand how the group I intron active-site facilitates catalysis, crystals of a 242-nucleotide ribozyme bound to a four-nucleotide product RNA have been produced that diffract to 3.6 A resolution. The space group of these crystals is I2(1)2(1)2(1) and the unit-cell parameters are a = 94.6, b = 141.0, c = 210.9 A. A single heavy-atom derivative has been synthesized by covalent modification of the product RNA with iodine.

Base Sequence↗

PubMed1992

Alternative forms of Max as enhancers or suppressors of Myc-ras cotransformation.

Max is a basic-helix-loop-helix-leucine zipper protein capable of forming sequence-specific DNA binding complexes with Myc proteins. An alternatively spliced messenger RNA has been identified that encodes a form of Max truncated at the COOH-terminus. This delta Max protein retained the ability to bind to the CACGTG motif in a complex with c-Myc but lacks the nuclear localization signal and the putative regulatory domain of Max. When tested in a myc-ras cotransformation assay in rat embryo fibroblasts, Max suppressed, whereas delta Max enhanced, transformation. Thus, the max gene may encode both a negative and a positive regulator of c-Myc function.

Amino Acid Sequence↗

PubMed1996

Human foamy virus replication: a pathway distinct from that of retroviruses and hepadnaviruses.

Human foamy virus (HFV) is the prototype of the Spumavirus genus of Retroviridae. In all other retroviruses, the pol gene products, including reverse transcriptase, are synthesized as Gag-Pol fusion proteins and are cleaved to functional enzymes during viral budding or release. In contrast, the Pol protein of HFV is translated from a spliced messenger RNA and lacks Gag domains. Infectious HFV particles contain double-stranded DNA similar in size to full-length provirus, suggesting that reverse transcription has taken place in viral particles before new rounds of infection, reminiscent of hepadnaviruses. These data suggest that foamy viruses possess a replication pathway containing features of both retroviruses and hepadnaviruses but distinct from both.

Amino Acid Sequence↗

PubMed2003

Messenger RNA reprogramming by spliceosome-mediated RNA trans-splicing.

In the human genome, the majority of protein-encoding genes are interrupted by introns, which are removed from primary transcripts by a macromolecular enzyme known as the spliceosome. Spliceosomes can constitutively remove all the introns in a primary transcript to yield a fully spliced mRNA or alternatively splice primary transcripts leading to the production of many different mRNAs from one gene. This review examines how spliceosomes can recombine two primary transcripts in trans to reprogram messenger RNAs.

Alternative Splicing↗

PubMed1992

Growth hormone rapidly activates insulin-like growth factor I gene transcription in vivo.

Many of the growth-promoting properties of GH are mediated by insulin-like growth factor I (IGF-I), a highly conserved circulating 70-amino acid peptide. Recent studies have shown that multiple mechanisms influence IGF-I gene expression, including transcription from two promoters, alternative RNA splicing, and variable polyadenylation. In order to determine how GH regulates IGF-I gene expression we have analyzed the response of hypophysectomized rats to a single ip injection of recombinant GH. A rise in hepatic IGF-I mRNA was detected within 2 h of GH treatment, with peak values of more than 15-fold above untreated animals by 4 h, and a decline by 16 h. A coordinate increase was seen in all IGF-I mRNA splicing and polyadenylation variants, indicating that neither alternative RNA processing nor differential poly A addition were altered by GH. Transcription run-on experiments using isolated hepatic nuclei and direct analysis of nuclear RNA demonstrated a rise in nascent IGF-I mRNA within 30 min of GH treatment, with peak levels reaching more than 10-fold above background by 2 h and declining by 6 h. As determined by RNase protection assays, transcripts directed by each promoter were coordinately and equivalently activated after GH. A single GH-responsive DNase I hypersensitive site was mapped in chromatin to the second IGF-I intron. This site exhibited rapid kinetics of induction which mirrored the pattern of transcriptional stimulation after GH treatment. These experiments show that GH enhances IGF-I expression in vivo by predominantly transcriptional mechanisms. The rapid kinetics of IGF-I gene activation and the temporally associated chromatin changes demonstrate a direct link between a GH-dependent signal transduction pathway and nuclear events.

Animals↗

PubMed1995

Inhibition of splicing of wild-type and mutated luciferase-adenovirus pre-mRNAs by antisense oligonucleotides.

We report the construction, characterization, and use of luciferase reporters to test the ability of antisense oligonucleotides to inhibit RNA splicing. beta-Globin and adenovirus introns were inserted into a luciferase cDNA, and luciferase expression was analyzed in transiently transfected cells. The adenovirus reporter expressed large amounts of luciferase, but two beta-globin constructs were inactive. RNA analyses determined that the beta-globin pre-mRNAs were not spliced. Mutagenesis of the beta-globin 5' splice site, branchpoint, and 3' splice site sequences to the adenovirus intron sequences promoted maximal splicing and luciferase activity; reciprocal changes in all three elements of the adenovirus intron eliminated luciferase activity. Wild-type and 3' splice site mutated adenovirus reporters were used to determine the ability of phosphorothioate deoxy and 2' methoxy oligonucleotides to inhibit splicing. RNase H activating oligodeoxynucleotides were better inhibitors of wild-type adenovirus expression than were 2' methoxy analogues. However, 2' methoxy oligonucleotides specific for the branchpoint were more effective inhibitors of splicing of adenovirus transcript containing the beta-globin branchpoint and 3' splice site. We suggest that pre-mRNAs with weak splice sites are potential targets for oligonucleotides that inhibit splicing by occupancy rather than cleavage of the transcripts.

Adenoviridae↗

PubMed2003

Kinetic and thermodynamic framework for assembly of the six-component bI3 group I intron ribonucleoprotein catalyst.

The yeast mitochondrial bI3 group I intron RNA splices in vitro as a six-component ribonucleoprotein complex with the bI3 maturase and Mrs1 proteins. We report a comprehensive framework for assembly of the catalytically active bI3 ribonucleoprotein. (1) In the absence of Mg(2+), two Mrs1 dimers bind independently to the bI3 RNA. The ratio of dissociation to association rate constants, k(off)/k(on), is approximately equal to the observed equilibrium K(1/2) of 0.12 nM. (2) At magnesium ion concentrations optimal for splicing (20 mM), two Mrs1 dimers bind with strong cooperativity to the bI3 RNA. k(off)/k(on) is 15-fold lower than the observed K(1/2) of 11 nM, which reflects formation of an obligate intermediate involving one Mrs1 dimer and the RNA in cooperative assembly of the Mrs1-RNA complex. (3) The bI3 maturase monomer binds to the bI3 RNA at almost the diffusion-controlled limit and dissociates with a half-life of 1 h. k(off)/k(on) is approximately equal to the equilibrium K(D) of 2.8 pM. The bI3 maturase thus represents a rare example of a group I intron protein cofactor whose binding is adequately characterized by a one-step mechanism under conditions that promote splicing. (4) Maturase and Mrs1 proteins each bind the bI3 RNA tightly, but with only modest coupling (approximately 1 kcal/mol), suggesting that the proteins interact at independent RNA binding sites. Maturase binding functions to slow dissociation of Mrs1; whereas prior Mrs1 binding increases the bI3 maturase k(on) right to the diffusion limit. (5) At effective concentrations plausibly present in yeast mitochondria, a predominant assembly pathway emerges involving rapid, tight binding by the bI3 maturase, followed by slower, cooperative assembly of two Mrs1 dimers. In the absence of other factors, disassembly of all protein subunits will occur in a single apparent step, governed by dissociation of the bI3 maturase.

Catalysis↗

PubMed2000

Different site, different splice.

The MKK3/6-p38 pathway has been found to induce the relocalization of premessenger-RNA splicing factors from the nucleus to the cytoplasm. This represents the first physiological mechanism that alters the nuclear ratios of splicing factors and modulates alternative splice-site choice in vivo.

Alternative Splicing↗

PubMed1992

STP1, a gene involved in pre-tRNA processing, encodes a nuclear protein containing zinc finger motifs.

STP1 is an unessential yeast gene involved in the removal of intervening sequences from some, but not all, families of intervening sequence-containing pre-tRNAs. Previously, we proposed that STP1 might encode a product that generates pre-tRNA conformations efficiently recognized by tRNA-splicing endonuclease. To test the predictions of this model, we have undertaken a molecular analysis of the STP1 gene and its products. The STP1 locus is located on chromosome IV close to at least two other genes involved in RNA splicing: PRP3 and SPP41. The STP1 open reading frame (ORF) could encode a peptide of 64,827 Da; however, inspection of putative transcriptional and translational regulatory signals and mapping of the 5' ends of mRNA provide evidence that translation of the STP1 ORF usually initiates at a second AUG to generate a protein of 58,081 Da. The STP1 ORF contains three putative zinc fingers. The first of these closely resembles both the DNA transcription factor consensus and the Xenopus laevis p43 RNA-binding protein consensus. The third motif more closely resembles the fingers found in spliceosomal proteins. Employing antisera to the endogenous STP1 protein and to STP1-LacZ fusion proteins, we show that the STP1 protein is localized to nuclei. The presence of zinc finger motifs and the nuclear location of the STP1 protein support the model that this gene product is involved directly in pre-tRNA splicing.

Amino Acid Sequence↗

PubMed2006

The SF3b155 N-terminal domain is a scaffold important for splicing.

Recruitment of U2 snRNP to the branch point sequence of introns is a necessary step in pre-mRNA splicing. In the current model, U2AF65, bound at the polypyrimidine tract of the intron, recruits U2 snRNP to the branch point sequence by interacting with the U2 snRNP protein SF3b155. We demonstrate that the N-terminal domain of SF3b155 contains multiple U2AF65 binding sites that are distinct from the binding site for the U2 snRNP protein p14, mapped to amino acids 396-424 of SF3b155. The N-terminal domain of SF3b155 appears to adopt a primarily unfolded structure but is functional to inhibit splicing in vitro. RNA binding studies show that the N-terminal domain of SF3b155 binds RNA nonspecifically and that the sites for U2AF65 binding and RNA binding are overlapping (or the same) within SF3b155. We propose that the N-terminal domain of SF3b155 adopts a primarily unfolded structure that functions as a scaffold to facilitate SF3b155's multiple protein-protein and protein-RNA interactions. The multiple U2AF65 binding sites on SF3b155 suggest a model in which multiple U2AF65 molecules bound to the intron could enhance U2 snRNP recruitment to the branch point sequence.

Amino Acid Sequence↗

PubMed1984

Apolipoprotein II messenger RNA. Transcriptional and splicing heterogeneity yields six 5'-untranslated leader sequences.

The structure of the mRNA for apolipoprotein II (apo-II), a major avian estrogen-responsive yolk protein, has been investigated. Primer extension using a cDNA probe-primer revealed six forms of mature apo-II mRNA. S1 and primer extension analysis with intron probes showed that only three of these forms arise due to multiple sites of transcription initiation on the apo-II gene. To investigate the basis for the additional forms of apo-II mRNA, we examined their nucleotide sequence. The sequence obtained between -10 and -42, relative to translation initiation, is identical to that reported by Wieringa et al. (Wieringa, B., AB, G., and Gruber, M. (1981) Nucleic Acids Res. 9, 469-499). At position -43, however, sequence heterogeneity appears. The minor form of the sequence starting at -43 and extending in a 5' direction corresponds exactly to the published mRNA sequence. The major form of the sequence has the insertion, 5'-CAG-3', at this position. The apparent basis for this heterogeneity is an unusual intron-exon border which violates the consensus sequence found in comparable positions in most eukaryotic genes by the existence of two adjacent 5'-CAG-3' triplets after the pyrimidine (Y)-rich track. The processing ratio between intron removal at the upstream and downstream AG dinucleotide is approximately 2.5:1. This result demonstrates that the splicing mechanism allows spatial flexibility in the positioning of the highly conserved YAG triplet within the consensus sequence at the 3' splice site.

Animals↗

PubMed1995

Interaction of the yeast splicing factor PRP8 with substrate RNA during both steps of splicing.

PRP8 protein of Saccharomyces cerevisiae interacts directly with pre-mRNA in spliceosomes, shown previously by UV-crosslinking. To analyse at which steps of splicing and with which precursor-derived RNA species the interaction(s) take place, UV-crosslinking was combined with PRP8-specific immunoprecipitation and the coprecipitated RNA species were analysed. Specific precipitation of intron-exon 2 and excised intron species was observed. PRP8 protein could be UV-crosslinked to pre-mRNA in PRP2-depleted spliceosomes stalled before initiation of the splicing reaction. Thus, the interaction of PRP8 protein with substrate RNA is established prior to the first transesterification reaction, is maintained during both steps of splicing and continues with the excised intron after completion of the splicing reaction. RNase T1 treatment of spliceosomes revealed that substrate RNA fragments of the 5' splice site region and the branchpoint-3' splice site region could be coimmunoprecipitated with PRP8 specific antibodies, indicating that these are potential sites of interaction for PRP8 protein with substrate RNA. Protection of the branch-point-3' splice site region was detected only after step 1 of splicing. The results allow a first glimpse at the pattern of PRP8 protein-RNA interactions during splicing and provide a fundamental basis for future analysis of these interactions.

Base Sequence↗

PubMed1998

Peroxisome proliferator activated receptor-alpha expression in human liver.

The peroxisome proliferator activated receptor alpha (PPAR) is a member of the steroid/hormone receptor superfamily that mediates the peroxisome proliferator-dependent transcriptional activation of genes encoding several peroxisomal and microsomal enzymes as well as peroxisome proliferation. Human liver is refractory to the pathological effects of peroxisome proliferators that are seen in mice. With the use of RNase protection assays, the ratio of hepatic PPAR alpha mRNA to beta-actin mRNA was found to be 1 order of magnitude lower in humans than that observed in mice. In addition, the isolation of human cDNA for PPAR alpha that does not encode a functional PPAR because it lacks exon 6 as a result of alternate RNA splicing suggested that this process might also diminish the expression of PPAR alpha. RNase protection analysis of total RNA revealed the presence of splice variants lacking exon 6 at significant levels in all 10 human liver samples examined. Supershift analysis using the CYP4A6-Z peroxisome proliferator response element and antisera specific for PPAR alpha revealed easily detectable amounts of PPAR alpha DNA binding activity in mouse liver lysates, whereas human liver lysates contained > 10-fold lower amounts of PPAR alpha DNA binding activity. In contrast to mouse lysates, the amount of PPAR alpha binding in human lysates was generally less than that of other unidentified proteins. These results suggest that although humans retain the coding potential for a functional receptor, the low levels of PPAR alpha expression in liver may be insufficient to compete effectively with other proteins that bind to peroxisome proliferator response elements.

Alternative Splicing↗