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Results for “Splicing Life”

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PubMed1991

An alternatively spliced miniexon alters the subcellular fate of the human asialoglycoprotein receptor H2 subunit. Endoplasmic reticulum retention and degradation or cell surface expression.

Two types of cDNAs encoding the H2 subunit of the human asialoglycoprotein receptor had been cloned, differing only by the presence (H2a) or absence (H2b) of a segment of 15 base pairs (bp), encoding five amino acids (Glu-Gly-His-Arg-Gly) immediately carboxylterminal (exoplasmic) to the single membrane-spanning segment. We have cloned and sequenced this region of the H2 gene and showed that the two H2 forms are alternatively spliced variants differing in the presence of a 15-bp miniexon. Both H2 messenger RNAs were found in HepG2 cells, H2b accounting for about 92% of the H2 mRNAs. When expressed in NIH 3T3 cells without the H1 receptor subunit, the two-variant polypeptides exhibit different subcellular fates. H2a is completely retained in and degraded in the endoplasmic reticulum or a related pre-Golgi compartment. In contrast a substantial amount of H2b is processed by Golgi enzymes and reaches the cell surface. Thus, the sole difference determining the subcellular localization of the two forms if the five-amino acid insert in H2a. When a virion-packaged retroviral vector containing H2a cDNA infected 3T3 cells, 70% of the resulting clones expressed H2b and 30% H2a. Thus the 15-bp H2a miniexon can be spliced out, at least during the retrovirus life cycle.

Amino Acid Sequence↗

PubMed2004

Identification of an hnRNP A1-dependent splicing silencer in the human papillomavirus type 16 L1 coding region that prevents premature expression of the late L1 gene.

We have previously identified cis-acting RNA sequences in the human papillomavirus type 16 (HPV-16) L1 coding region which inhibit expression of L1 from eukaryotic expression plasmids. Here we have determined the function of one of these RNA elements, and we provide evidence that this RNA element is a splicing silencer which suppresses the use of the 3' splice site located immediately upstream of the L1 AUG. We also show that this splice site is inefficiently utilized as a result of a suboptimal polypyrimidine tract. Introduction of point mutations in the L1 coding region that altered the RNA sequence without affecting the L1 protein sequence resulted in the inactivation of the splicing silencer and induced splicing to the L1 3' splice site. These mutations also prevented the interaction of the RNA silencer with a 35-kDa cellular protein identified here as hnRNP A1. The splicing silencer in L1 inhibits splicing in vitro, and splicing can be restored by the addition of RNAs containing an hnRNP A1 binding site to the reaction, demonstrating that hnRNP A1 inhibits splicing of the late HPV-16 mRNAs through the splicing silencer sequence. While we show that one role of the splicing silencer is to determine the ratio between partially spliced L2/L1 mRNAs and spliced L1 mRNAs, we also demonstrate that it inhibits splicing from the major 5' splice site in the early region to the L1 3' splice site, thereby playing an essential role in preventing late gene expression at an early stage of the viral life cycle. We speculate that the activity of the splicing silencer and possibly the concentration of hnRNP A1 in the HPV-16-infected cell determines the ability of the virus to establish a persistent infection which remains undetected by the host immune surveillance.

3' Untranslated Regions↗

PubMed2003

The circadian clock regulated RNA-binding protein AtGRP7 autoregulates its expression by influencing alternative splicing of its own pre-mRNA.

The clock-regulated RNA-binding protein AtGRP7 is part of a negative feedback circuit through which the protein influences circadian oscillations of its own transcript. Constitutive overexpression of AtGRP7 in transgenic plants leads to the appearance of a low amount of an alternatively spliced Atgrp7 transcript with a premature stop codon. It is generated by the use of a 5' cryptic splice site in the middle of the intron at the expense of the fully spliced mRNA, indicating a role for AtGRP7 in splice site selection. Accelerated decay of this transcript accounts for its low steady state abundance. This implicates a mechanism for the AtGRP7 feedback loop: Atgrp7 expression is downregulated, as AtGRP7 protein accumulates over the circadian cycle, partly by the generation of an alternate transcript that due to its instability does not accumulate to high levels and does not produce a functional protein. Recombinant AtGRP7 protein specifically interacts with the 3' untranslated region and the intron of its transcript, suggesting that the shift in splice site selection and downregulation involves binding of AtGRP7 to its pre-mRNA. AtGRP7 also influences the choice of splice sites in the Atgrp8 transcript encoding a related RNA-binding protein, favoring the production of an alternatively spliced, unstable Atgrp8 transcript. This conservation points to the importance of this regulatory mechanism to control the level of the clock-regulated glycine-rich RNA-binding proteins and shows how AtGRP7 can control abundance of target transcripts.

Alternative Splicing↗

PubMed1995

Leaky splicing mutation in the acid maltase gene is associated with delayed onset of glycogenosis type II.

An autosomal recessive deficiency of acid alpha-glucosidase (GAA), type II glycogenosis, is genetically and clinically heterogeneous. The discovery of an enzyme-inactivating genomic deletion of exon 18 in three unrelated genetic compound patients--two infants and an adult--provided a rare opportunity to analyze the effect of the second mutation in patients who displayed dramatically different phenotypes. A deletion of Lys-903 in one patient and a substitution of Arg for Leu-299 in another resulted in the fatal infantile form. In the adult, a T-to-G base change at position -13 of intron 1 resulted in alternatively spliced transcripts with deletion of exon 2, the location of the start codon. The low level of active enzyme (12% of normal) generated from the leakage of normally spliced mRNA sustained the patient to adult life.

Adult↗

PubMed2004

Intron-containing beta-tubulin transcripts in Cryptosporidium parvum cultured in vitro.

The genome of Cryptosporidium parvum contains a relatively small number of introns, which includes the beta-tubulin gene with only a single intron. Recently, it was observed that the intron was not removed from some of the beta-tubulin transcripts in the late life cycle stages cultured in vitro. Although normally spliced beta-tubulin mRNA was detected in all parasite intracellular stages by RT-PCR (e.g. HCT-8 or Caco-2 cells infected with C. parvum for 12-72 h), at 48-72 h post-infection unprocessed beta-tubulin transcripts containing intact introns started to appear in parasite mRNA within infected host cells. The intron-containing transcripts could be detected by fluorescence in situ hybridization (FISH) using an intron-specific probe. The intron-containing beta-tubulin transcripts appeared unique to the in vitro-cultured C. parvum, since they were not detected in parasite-infected calves at 72 h. As yet, it is unclear whether the late life cycle stages of C. parvum are partially deficient in intron-splicing or the intron-splicing processes have merely slowed, both of which would allow the detection of intron-containing transcripts. Another possible explanation is that the decay in transcript processing might simply be due to the onset of parasite death. Nonetheless, the appearance of intron-containing transcripts coincides with the arrest of C. parvum development in vitro. This unusual observation prompts speculation that the abnormal intron-splicing of beta-tubulin transcripts may be one of the factors preventing complete development of this parasite in vitro. Furthermore, the presence of both processed and unprocessed introns in beta-tubulin transcripts in vitro may provide a venue for studying overall mechanisms for intron-splicing in this parasite.

Animals↗

PubMed1994

A temporal study of RNAs produced in maedi-visna virus infection of choroid plexus cells.

We have examined the types and cellular distribution of transcripts of maedi-visna virus during infection of choroid plexus fibroblasts. Early in infection (19 hours post infection) only small spliced transcripts are found in the cytoplasm. Little virus specific RNA is detected in the nucleus at this stage. Later in infection structural gene transcripts are detectable in the cytoplasm as well as the nucleus. We have measured the half life of each type of transcript in total cellular RNA late in infection by two different methods (northern blotting or S1 protection assays after alpha-amanitin treatment, or specific hybridisation after pulse chase). Both of these methods suggest half lives for unspliced viral RNAs of 8-12 hours. Although the gel based methods were unsuitable for the accurate determination of half life for the shorter transcripts, they did confirm a long half life for these transcripts: pulse chase measurements suggested that this was similar to that seen for the unspliced transcripts. These observations are consistent with the virus exerting temporal control on protein synthesis by a method analogous to that of the HIV Rev protein.

Animals↗

PubMed2004

A survey of SL1-spliced transcripts from the root-lesion nematode Pratylenchus penetrans.

Plant-parasitic nematodes are important and cosmopolitan pathogens of crops. Here, we describe the generation and analysis of 1928 expressed sequence tags (ESTs) of a splice-leader 1 (SL1) library from mixed life stages of the root-lesion nematode Pratylenchus penetrans. The ESTs were grouped into 420 clusters and classified by function using the Gene Ontology (GO) hierarchy and the Kyoto KEGG database. Approximately 80% of all translated clusters show homology to Caenorhabditis elegans proteins, and 37% of the C. elegans gene homologs had confirmed phenotypes as assessed by RNA interference tests. Use of an SL1-PCR approach, while ensuring the cloning of the 5' ends of mRNAs, has demonstrated bias toward short transcripts. Putative nematode-specific and Pratylenchus -specific genes were identified, and their implications for nematode control strategies are discussed.

Animals↗

PubMed2005

The HLA-G genotype is associated with IL-10 levels in activated PBMCs.

Human leukocyte antigen (HLA)-G is an MHC class Ib molecule that is expressed at the feto-maternal interface during pregnancy. However, recent results have also shown that it may have important functions as an immuno-modulatory factor in adult life. Differences in the pattern of alternative splicing and in the stability of HLA-G mRNA transcripts have been associated with HLA-G polymorphisms, especially a 14 bp deletion/insertion polymorphism in the 3' untranslated region of the HLA-G gene. We have investigated the secretion of HLA-G5/soluble HLA-G1 and interleukin-10 (IL-10) in lipopolysaccharide (LPS)-activated peripheral blood mononuclear lymphocytes (PBMCs) in relation to the HLA-G 14 bp genotype. No HLA-G5/sHLA-G1 could be detected in the non-activated control PBMC culture media, and there were no significant differences among the three HLA-G 14 bp genotypes regarding IL-10 concentrations. In LPS-activated PBMC cultures, no significant differences among the three HLA-G 14 bp genotypes regarding HLA-G5/sHLA-G1 concentrations were observed. However, this was in contrast to the IL-10 levels (P=0.0004, Kruskal-Wallis test). The +14/+14 bp PBMC samples expressed higher levels of IL-10 when compared to the -14/+14 bp genotype and the -14/-14 bp genotype. Interestingly, the IL-10 G/G polymorphism at position -1082 was more frequent in the +14/+14 bp genotype (P=0.024, chi2 test). These results support an autocrine loop between HLA-G5/sHLA-G1 and IL-10 expression in activated PBMCs, which may result in higher IL-10 levels in +14/+14 bp HLA-G genotypes.

3' Untranslated Regions↗

PubMed2005

Cyclin D1 polymorphism and the risk of endometrial cancer.

OBJECTIVES: The common G to A single nucleotide polymorphism (G870A) in the splice donor region of exon 4 enhances alternate splicing, and produces a longer half-life cyclin D1 (CCND1). This study was aimed at investigating the possible association between the G870A polymorphism in CCND1 and the risk of endometrial cancer. METHODS: We assessed the association between the CCND1 G870A polymorphism and the risk of endometrial cancer in a hospital-based case-control study among 231 Korean women (77 cases; 154 matched controls). Controls were matched to cases with respect to age, menopausal status, and hormone therapy status. RESULT: The allele frequencies of the case subjects (A, 0.45; G, 0.55) were significantly different from those of control subjects (A, 0.58; G, 0.42) (P = 0.008). All case and control subjects were in Hardy-Weinberg equilibrium. The AA genotype was associated with a significantly elevated odds ratio (OR) of 3.18 [95% confidence interval (CI) 1.38-7.37, P = 0.007], and the AG genotype was associated with an OR of 1.38 (95% CI 0.65-2.89). When we combined the GG and AG genotypes as a reference genotype, we found that the OR for the AA genotype was 2.53 (95% CI 1.34-4.80, P = 0.004), supporting a recessive model for the A allele. Conditional logistic regression adjusted for various risk factors of endometrial cancer revealed positive associations between the AA genotype and an increased risk of endometrial cancer (OR 3.16, 95% CI 1.18-8.43, P = 0.022). However, no significant difference in endometrial cancer stage or grade was observed between the CCND1 genotypes. CONCLUSION: Our data suggest that the CCND1 polymorphism is associated with an increased risk of endometrial cancer. To validate this association, a large-scale population-based study is needed.

Adult↗

PubMed1991

Molecular cloning and analysis of small optic lobes, a structural brain gene of Drosophila melanogaster.

Mutations in the small optic lobes (sol) gene of Drosophila melanogaster cause specific cells to degenerate in the developing optic lobes, resulting in the absence of certain classes of columnar neurons. These neuronal defects lead to specific alterations in behavioral characteristics, particularly during flight and walking maneuvers. We have isolated the wild-type sol locus by microcloning and chromosomal walking and have established its genetic and molecular limits. Two major transcripts of 5.8 and 5.2 kilobases are produced from this locus by alternative splicing and are present throughout the entire life cycle. Sequence analyses of cDNAs corresponding to these two classes of transcripts predict two proteins of 1597 and 395 amino acids. The first shows similarity in its carboxyl-terminal region to the catalytic domain of a vertebrate calcium-activated neutral protease (calpain), whereas its amino-terminal region contains several zinc-finger-like repeats of the form WXCX2CX10-11CX2C. The second predicted protein contains only the first two of the zinc-finger-like repeats and is missing the calpain domain. By constructing transgenic flies carrying a single wild-type copy of the sol gene in a homozygous sol mutant background, we have restored the normal neuroanatomical phenotype to individuals that would have developed mutant brains.

Amino Acid Sequence↗

PubMed2005

Protein splicing elements and plants: from transgene containment to protein purification.

Protein splicing elements, termed inteins, have been discovered in all the domains of life. Basic research on inteins has led to a greater understanding of how they mediate the protein splicing process. Because inteins are natural protein engineering elements they have been harnessed for use in a number of applications, including protein purification, protein semisynthesis, and in vivo and in vitro protein modifications. This review focuses on the use of inteins in plants. A split-gene technique utilizes inteins to reconstitute the activity of a transgene product with the goal of limiting the spread of transgenes from a genetically modified plant to a weedy relative. Furthermore, merging the intein tag for protein purification with the large protein yields possible with plants has the potential to produce pharmaceutically important proteins. Finally, relevant techniques that may be used in plants in the future are discussed.

Acetolactate Synthase↗

PubMed2006

Group I intron renders differential susceptibility of Candida albicans to Bleomycin.

The alarming increase in drug resistance gained by fungal pathogens has raised an urgent need to develop drugs against novel targets. Candida albicans, an opportunistic fungal pathogen, harbors in its 25S rRNA gene, a self-splicing Group I intron, which can act as a selective drug target. We report that Bleomycin selectively inhibits the self-splicing of Group I intron of C. albicans at IC(50) = 1.2 microM, leading to accumulation of precursor RNA as evinced by Reverse Transcriptase PCR. Drug susceptibility assays including MIC determination, growth curve analysis and disc diffusion assays indicate a strong susceptibility of the intron-containing strain (4-1) than the intronless strain (62-1). These results on the preferential targeting of Group I intron of C. albicans by Bleomycin might form a basis for design of small molecules that inhibit self-splicing of RNA as a antimicrobial tool against life-threatening microorganisms.

Antifungal Agents↗

PubMed1994

Protozyme: emerging evidence in nature.

The VMA1 locus in Saccharomyces cerevisiae contains a nested genetic element, the VDE gene, and expresses two functional proteins. A single VMA1 translational product seems to catalyze a self protein splicing in which an internal domain is excised out to produce a site-specific DNA endonuclease and the N- and C-domains are ligated by a transpeptidation reaction to yield a catalytic subunit of the vacuolar ATPase. Accumulating evidence in the past few years suggests that the VMA1 locus encodes a protozyme (for protos en zyme) which has dual roles in life as a protean catalyst for self protein splicing and for self gene homing. Four protozymes that share a common mechanism in protein splicing have been found in four organisms covering three major phylogenic trees.

Amino Acid Sequence↗

PubMed2001

Cloning of two novel P450 cDNAs from German cockroaches, Blattella germanica (L.): CYP6K1 and CYP6J1.

Two novel P450 cDNAs, CYP6K1 and CYP6J1, were isolated from German cockroaches, Blattella germanica (L). Both CYP6K1 and CYP6J1 are typical microsomal P450s and their deduced amino acid sequences share a number of common characteristics with other members of the P450 superfamily. Both CYP6K1 and CYP6J1 showed the highest per cent identity (based on the deduced amino acid sequence) to CYP6L1 from B. germanica and CYP6H1, a putative ecdysone 20-hydroxylase from Locusta migratoria. Using a CYP6K1 probe, two mRNA signals (~2.5 and ~2.1 kb) were detected in all life stages. Both signals were just detectable in the eggs and became stronger in later instars. The strongest signals were detected in the fifth and sixth instars as well as in adults. These two bands were also detected in the abdomens and in the remainder of bodies of both male and female adults. Southern blots suggest the two mRNA bands detected in the Northern blot might be a result of alternative splicing. No signal could be detected at any life stage using the CYP6J1 probe, suggesting that CYP6J1 was expressed at a low level.

Amino Acid Sequence↗

PubMed2000

The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions.

Eukaryotic mRNAs exist in vivo as ribonucleoprotein particles (mRNPs). The protein components of mRNPs have important functions in mRNA metabolism, including effects on subcellular localization, translational efficiency and mRNA half-life. There is accumulating evidence that pre-mRNA splicing can alter mRNP structure and thereby affect downstream mRNA metabolism. Here, we report that the spliceosome stably deposits several proteins on mRNAs, probably as a single complex of approximately 335 kDa. This complex protects 8 nucleotides of mRNA from complete RNase digestion at a conserved position 20-24 nucleotides upstream of exon-exon junctions. Splicing-dependent RNase protection of this region was observed in both HeLa cell nuclear extracts and Xenopus laevis oocyte nuclei. Immunoprecipitations revealed that five components of the complex are the splicing-associated factors SRm160, DEK and RNPS1, the mRNA-associated shuttling protein Y14 and the mRNA export factor REF. Possible functions for this complex in nucleocytoplasmic transport of spliced mRNA, as well as the nonsense-mediated mRNA decay pathway, are discussed.

Animals↗

PubMed1990

Identical G+1 to A mutations in three different introns of the type III procollagen gene (COL3A1) produce different patterns of RNA splicing in three variants of Ehlers-Danlos syndrome. IV. An explanation for exon skipping some mutations and not others.

Identical G+1 mutations in three different introns of the gene for type III procollagen (COL3A1) that cause aberrant splicing of RNA were found in three probands with life-threatening variants of Ehlers-Danlos syndrome. Because the three mutations were in a gene with multiple and homologous exons, they provided an interesting test for factors that influence aberrant splicing. The G+1 to A mutation in intron 16 caused extensive exon skipping, the G+1 to A mutation in intron 20 caused both use of a cryptic splice site and retention of all the intron sequences, and the G+1 to A mutation in intron 42 caused efficient use of a single cryptic splice site. The different patterns of RNA splicing were not explained by evaluation of potential cryptic splice sites in the introns by either their homology with 5'-splice sites from other genes or by their delta G(0)37 values for binding to U1 RNA. Instead, the results suggested that the patterns of aberrant RNA splicing were primarily determined by the relative rates at which adjacent introns were normally spliced.

Adenine↗

PubMed1995

Transposon insertions causing constitutive Sex-lethal activity in Drosophila melanogaster affect Sxl sex-specific transcript splicing.

Sex-lethal (Sxl) gene products induce female development in Drosophila melanogaster and suppress the transcriptional hyperactivation of X-linked genes responsible for male X-chromosome dosage compensation. Control of Sxl functioning by the dose of X-chromosomes normally ensures that the female-specific functions of this developmental switch gene are only expressed in diplo-X individuals. Although the immediate effect of X-chromosome dose is on Sxl transcription, during most of the life cycle "on" vs. "off" reflects alternative Sxl RNA splicing, with the female (productive) splicing mode maintained by a positive feedback activity of SXL protein on Sxl pre-mRNA splicing. "Male-lethal" (SxlM) gain-of-function alleles subvert Sxl control by X-chromosome dose, allowing female Sxl functions to be expressed independent of the positive regulators upstream of Sxl. As a consequence, SxlM haplo-X animals (chromosomal males) die because of improper dosage compensation, and SxlM chromosomal females survive the otherwise lethal effects of mutations in upstream positive regulators. Five independent spontaneous SxlM alleles were shown previously to be transposon insertions into what was subsequently found to be the region of regulated sex-specific Sxl RNA splicing. We show that these five alleles represent three different mutant types: SxlM1, SxlM3, and SxlM4. SxlM1 is an insertion of a roo element 674 bp downstream of the translation-terminating male-specific exon. SxlM3 is an insertion of a hobo transposon (not 297 as previously reported) into the 3' splice site of the male exon, and SxlM4 is an insertion of a novel transposon into the male-specific exon itself. We show that these three gain-of-function mutants differ considerably in their ability to bypass the sex determination signal, with SxlM4 being the strongest and SxlM1 the weakest. This difference is also reflected in effects of these mutations on sex-specific RNA splicing and on the rate of appearance of SXL protein in male embryos. Transcript analysis of double-mutant male-viable SxlM derivatives in which the SxlM insertion is cis to loss-of-function mutations, combined with other results reported here, indicates that the constitutive character of these SxlM alleles is a consequence of an alteration of the structure of the pre-mRNA that allows some level of female splicing to occur even in the absence of functional SXL protein. Surprisingly, however, most of the constitutive character of SxlM alleles appears to depend on the mutant alleles' responsiveness, perhaps greater than wild-type, to the autoregulatory splicing activity of the wild-type SXL proteins they produce.

Amino Acid Sequence↗

PubMed1985

Site-specific deletion in cauliflower mosaic virus DNA: possible involvement of RNA splicing and reverse transcription.

A frequent site-specific deletion was observed in the life cycle of cauliflower mosaic virus (S strain). Analysis of the sequence around the deletion site and the parental sequence implied that the deletion was promoted at sequences similar to the donor and acceptor consensus sequences of RNA splicing, designated as the deletion donor and acceptor sequences, respectively. To elucidate the mechanism of this site-specific deletion, point mutations were introduced into the deletion donor sequence (GT to GG or GA transversion). Deletion at the original deletion donor site did not occur in these mutants, instead, new (cryptic) donor sites were activated. All of these activated cryptic sites had sequences similar to the splicing consensus sequence. In all cases except one, the original deletion acceptor site was used. These results can be most readily explained by postulating that the site-specific deletion occurs by reverse transcription of spliced viral RNA. This frequent site-specific deletion was not observed in other strains. For a virus which replicates by reverse transcription, a mechanism to regulate the rate of splicing is required to ensure the intactness of the viral genome. We discuss the possibility that the S strain has a mutation in this regulatory mechanism.

Journal Article↗