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Results for “RNA Splicing”

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PubMed1991

An RNA splice site mutation in the C1-inhibitor gene causes type I hereditary angio-oedema.

Restriction fragment length polymorphism analysis, the polymerase chain reaction and nucleotide sequencing have been used to characterise a single base substitution (G----T) at nucleotide 8863 in the C1-inhibitor gene. This destroys the 5' donor splice site recognition motif of the sixth intron. Family studies suggest that the mutation is responsible for type I hereditary angio-oedema in a studied kindred.

Angioedema↗

PubMed1987

Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing.

Adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, we synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNAs showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These changes do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency.

Adenosine Deaminase↗

PubMed1994

Molecular characterization of a ferrochelatase gene defect causing anomalous RNA splicing in erythropoietic protoporphyria.

Erythropoietic protoporphyria is an inherited disorder caused by deficient activity of the enzyme ferrochelatase. We have examined the ferrochelatase gene in an 11-year-old female with protoporphyria and have found that she is heterozygous for a mutation at a conserved residue in the exon 3 donor splice site consensus sequence (T(+2)-->G). This is inherited from her father, who also has deficient ferrochelatase activity. As a consequence of the mutation, ferrochelatase transcripts are aberrantly spliced and give rise to mRNA molecules in which sequences corresponding to exon 3 are absent. This leads to the expression of a ferrochelatase protein lacking a central region of 40 amino acids.

Base Sequence↗

PubMed1999

Expression of the gene for mitoribosomal protein S12 is controlled in human cells at the levels of transcription, RNA splicing, and translation.

The human gene RPMS12 encodes a protein similar to bacterial ribosomal protein S12 and is proposed to represent the human mitochondrial orthologue. RPMS12 reporter gene expression in cultured human cells supports the idea that the gene product is mitochondrial and is localized to the inner membrane. Human cells contain at least four structurally distinct RPMS12 mRNAs that differ in their 5'-untranslated region (5'-UTR) as a result of alternate splicing and of 5' end heterogeneity. All of them encode the same polypeptide. The full 5'-UTR contains two types of sequence element implicated elsewhere in translational regulation as follows: a short upstream open reading frame and an oligopyrimidine tract similar to that found at the 5' end of mRNAs encoding other growth-regulated proteins, including those of cytosolic ribosomes. The fully spliced (short) mRNA is the predominant form in all cell types studied and is translationally down-regulated in cultured cells in response to serum starvation, even though it lacks both of the putative translational regulatory elements. By contrast, other splice variants containing one or both of these elements are not translationally regulated by growth status but are translated poorly in both growing and non-growing cells. Reporter analysis identified a 26-nucleotide tract of the 5'-UTR of the short mRNA that is essential for translational down-regulation in growth-inhibited cells. Such experiments also confirmed that the 5'-UTR of the longer mRNA variants contains negative regulatory elements for translation. Tissue representation of RPMS12 mRNA is highly variable, following a typical mitochondrial pattern, but the relative levels of the different splice variants are similar in different tissues. These findings indicate a complex, multilevel regulation of RPMS12 gene expression in response to signals mediating growth, tissue specialization, and probably metabolic needs.

Animals↗

PubMed2003

SRp55 is a regulator of calcitonin/CGRP alternative RNA splicing.

Alternative splicing is an important mechanism for the regulation of gene expression. The mammalian calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA is alternatively spliced in a tissue-specific manner, leading to the production of calcitonin mRNA containing exons 1-4 in thyroid C cells and CGRP mRNA containing exons 1-3, 5, and 6 in neurons. The calcitonin-specific fourth exon contains an exonic splice enhancer (ESE) that binds SRp55. We define the RNA binding site of SRp55 in the ESE and demonstrate that base changes that decrease the level of SRp55 binding decrease the level of calcitonin splicing in vitro and calcitonin mRNA production in vivo. Base changes that increase the affinity of SRp55 for the ESE increase the level of calcitonin splicing in vitro and calcitonin mRNA levels in 293 cells. We also observe that SRp55 levels in different cell types correlate with the levels of calcitonin mRNA produced in these cells. Finally, we show that increasing the level of cellular expression of SRp55 stimulates calcitonin mRNA production in vivo. These observations suggest that SRp55 binding to a suboptimal RNA binding site in the calcitonin/CGRP pre-mRNA ESE is required for calcitonin mRNA production. Differential amounts of SRp55 present in different cell types would then control calcitonin/CGRP alternative splicing.

Alternative Splicing↗

PubMed1990

Evidence for alternative RNA splicing and possible alternative promoters in the human muscle phosphofructokinase gene at the 5' untranslated region.

Three mRNA species for human muscle phophofructokinase containing heterogeneous 5' untranslated sequences were identified through cDNA cloning. Type A mRNA was essentially the same as that reported previously (Nakajima, H., et al. (1987) FEBS Lett. 223, 113). Type B mRNA was considered to be the major gene product, which contained an extra non-coding sequence within the 5' untranslated region of type A mRNA. Amplification of mRNA by polymerase chain reaction revealed that types A and B mRNAs shared a common precursor RNA, and were alternatively spliced. Type C mRNA, homologous to the cDNA sequence from a placenta library (Sharma, P. M. et al., (1989) Gene 77, 177), was considered to be under the control of an alternative promotor.

Base Sequence↗

PubMed1988

Alternative RNA splicing that is spatially regulated: generation of transcripts from the Antennapedia gene of Drosophila melanogaster with different protein-coding regions.

We have shown previously that transcription of the Drosophila homeotic gene Antennapedia results in four major RNA species which differ in long 5'- and 3'-untranslated sequences. The protein-coding portion of these transcripts, however, is located in exons common to all. Using RNase protection assays and further cDNA clone isolation, we have now detected two alternative splicing events between exons of this region. These result in four RNA variations which, if translated, would encode a family of Antennapedia proteins. By analyzing transcripts from various developmental stages and isolated tissues, we show that alternative splicing is under strict temporal and spatial regulation. For example, while similar patterns of splicing were found for all wild-type thoracic imaginal disks examined, these differed distinctly from the patterns observed in neural tissues. Our results suggest that individual RNAs may be associated with different biological roles, and provide molecular evidence that the Antennapedia gene is involved in multiple functions.

Age Factors↗

PubMed1992

Functional isoforms of Pit-1 generated by alternative messenger RNA splicing.

Pit-1 is a pituitary-specific transcription factor that activates expression of the GH and PRL genes. We have isolated a cDNA encoding a variant isoform of Pit-1 that we term Pit-1 beta. Pit-1 beta contains an insertion of 26 amino acids in the transactivation domain as a consequence of the utilization of an alternative 3' splice acceptor at the end of the first intron. Like the previously described Pit-1 alpha, Pit-1 beta transactivates both the GH and PRL promoters. However, the larger isoform acts as a more potent inducer of GH.

Amino Acid Sequence↗

PubMed2006

Drosophila dSAP18 is a nuclear protein that associates with chromosomes and the nuclear matrix, and interacts with pinin, a protein factor involved in RNA splicing.

SAP18 is a highly conserved protein that was proposed to be involved in multiple cellular processes from autophagy to gene regulation and mRNA processing. In this paper we show that, in Drosophila, dSAP18 is a predominantly nuclear protein that associates to both chromosomes and the nuclear matrix. dSAP18 becomes nuclear early during development, at the onset of cellularization, and remains so all through embryo development. dSAP18 is also nuclear in salivary glands, ovaries and cultured S2 cells. Here we also show that dSAP18 forms a complex with the Drosophila homolog of pinin (dPnn), a protein factor involved in mRNA splicing. dSAP18-dPnn interaction was confirmed in vivo, through co-immunoprecipitation experiments, as well as in vitro, through GST pull-down assays. These results are discussed in the context of the possible functions played by SAP18.

Amino Acid Sequence↗

PubMed1992

The yeast nuclear gene MRF1 encodes a mitochondrial peptide chain release factor and cures several mitochondrial RNA splicing defects.

We report the molecular cloning, sequencing and genetic characterization of the first gene encoding an organellar polypeptide chain release factor, the MRF1 gene of the yeast Saccharomyces cerevisiae. The MRF1 gene was cloned by genetic complementation of a respiratory deficient mutant disturbed in the expression of the mitochondrial genes encoding cytochrome c oxidase subunit 1 and 2, COX1 and COX2. For COX1 this defect has been attributed to an impaired processing of several introns. Sequence analysis of the MRF1 gene revealed that it encodes a protein highly similar to prokaryotic peptide chain release factors, especially RF-1. Disruption of the gene results in a high instability of the mitochondrial genome, a hallmark for a strict lesion in mitochondrial protein synthesis. The respiratory negative phenotype of mrf1 mutants lacking all known mitochondrial introns and the reduced synthesis of mitochondrial translation products encoded by unsplit genes confirm a primary defect in mitochondrial protein synthesis. Over-expression of the MRF1 gene in a mitochondrial nonsense suppressor strain reduces suppression in a dosage-dependent manner, shedding new light on the role of the '530 region' of 16S-like ribosomal RNA in translational fidelity.

Amino Acid Sequence↗

PubMed1992

Human erythroid 5-aminolevulinate synthase. Gene structure and species-specific differences in alternative RNA splicing.

Erythroid 5-aminolevulinate synthase (ALAS) is expressed exclusively in differentiating erythroid cells as the principal isoform of the enzyme to catalyze the first step of the heme biosynthetic pathway. The human gene encoding this isozyme was isolated from a cosmid library, and its structure was characterized with restriction mapping followed by sequencing of fragments. The gene is 22 kilobases long and has 11 exons. Exon 2 encodes the N-terminal signal sequence required for mitochondrial import, exons 3 and 4 encode a variable portion of the N-terminal end, and exons 5-11 the highly conserved C-terminal portion of the mature protein, respectively. Enzymatic amplification of human reticulocyte RNA using PCR techniques revealed two erythroid ALAS mRNA transcripts predicted to encode both the prototypical 64-kDa isoform as well as a novel smaller isoform with a deletion of 37 amino acids near the N terminus. The two mRNA isoforms are generated by alternative splicing of exon 4 and are expressed in fetal erythroid cells as well as at all stages of erythroid development tested, so that there is no evidence of differentiation-specific regulation of exon 4 splicing. However, striking species-specific differences were observed in that alternative splicing of exon 4 was found in man but not dog or mouse; also, the previously described alternative splicing within exon 3 in mouse was not observed in man. This transcript heterogeneity suggests the existence of erythroid ALAS protein isoforms with potentially distinct functional or regulatory roles. The occurrence of species-specific splicing in the least conserved region of the enzyme may reflect another mechanism of gene evolution in eukaryotes.

5-Aminolevulinate Synthetase↗

PubMed1993

Molecular genetic study of a Japanese family with Lesch-Nyhan syndrome: a point mutation at the consensus region of RNA splicing (HPRTKeio).

Complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT) causes Lesch-Nyhan syndrome. A single nucleotide substitution of G to T at the 3'-end of intron 3 in the splicing consensus region has been identified in one allele of the HPRT gene from a mother predicted to be a heterozygous Lesch-Nyhan carrier. Utilizing a BfaI restriction site which was lost in the mutation as an indicator, family study showed that the mother and her only daughter were heterozygotes but the mother's sister did not have the mutant allele. The mutation generated splicing error and resulted in two types of abnormal mRNA. The major altered mRNA, named Type I, skipped the exon 4 and is predicted to produce a protein deleted of 22 amino acid residues. The other, Type II, having a 9-bp deletion at the 5'-end of exon 4, can result in a protein lacking 3 amino acids, from codon 107 to 109.

Base Sequence↗

PubMed2002

RNA splicing in higher plant mitochondria: determination of functional elements in group II intron from a chimeric cox II gene in electroporated wheat mitochondria.

Higher plant mitochondria mainly contain group II introns presenting a secondary structure with six helical domains linked to a central hub. Experimental evidence of functional elements in higher plant mitochondria introns is limited since they are unable to undergo self-splicing and the definition of functional domains is based on data obtained from yeast autocatalytic introns. Here we study the role of putative functional elements required for the splicing reaction. The exon-binding and intron-binding sites (EBS and IBS, respectively), and the domain 6, which is involved in lariat formation, were analysed by site-directed mutagenesis and transient expression in electroporated mitochondria. The data presented here demonstrate the role of EBS1-IBS1 and EBS2-IBS2 interactions and reveal a new secondary-structure interaction. The role of the C to U editing conversion in the IBS1 motif is discussed.

Base Sequence↗

PubMed1985

Generation of troponin T isoforms by alternative RNA splicing in avian skeletal muscle. Conserved and divergent features in birds and mammals.

We describe the isolation and sequence analysis of quail muscle cDNA clones encoding two closely related isoforms of the striated muscle contractile protein, troponin T. The cDNAs represent two troponin T mRNAs that exhibit an unusual sequence relationship. The two mRNAs have identical sequences over hundreds of nucleotides including 3' untranslated regions, but they differ dramatically in a discrete, internally located block of 38 nucleotides. The two alternative sequences of this 38-nucleotide block encode two different but related versions of amino acid residues 230-242, near the C terminus of the protein. These results are consistent with a novel mechanism of troponin T isoform generation by alternative mRNA splicing pathways from a single gene containing two different exons corresponding to amino acids 229-242, as recently proposed by Medford et al. (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). This proposal was based on analysis of a rat troponin T genomic DNA clone and a cDNA clone corresponding to one of the two alternatively spliced mRNAs. Our analysis of quail troponin T cDNA clones, apparently corresponding to two alternatively spliced mRNA species, provides important new evidence for this novel mechanism of troponin T isoform generation and reveals the differential splicing mechanism to be of great antiquity, antedating the bird-mammal divergence. One of the quail alternative isoform sequences clearly corresponds to one of the rat sequences, but the other quail alternative sequence does not correspond to either of the rat sequences. This result suggests a greater complexity of troponin T gene structure or a greater diversity of troponin T isoform genes than is currently known, and also has implications for the functional significance of the troponin T protein isoform heterogeneity. Comparison of quail and mammal alternative isoform sequences also reveals strongly conserved features which suggest that all the isoform alternative amino acid sequences are variations on a common structural theme.

Amino Acid Sequence↗

PubMed2026

A single-cell study of transcription and RNA splicing in MDD and ALC.

Major depressive disorder (MDD) and problematic alcohol use (ALC) commonly co-occur, yet the extent, genomic distribution, and biological context of their shared genetic architecture remain incompletely understood. Here, we integrated genome-wide and local genetic architecture analyses with tissue, spatial, single-cell, and multi-omics analyses to characterize the shared genetic basis of MDD and ALC. Across methods, the two phenotypes showed a consistent positive genetic correlation (rg = 0.380-0.582). MiXeR estimated that they shared approximately 5479 variants with non-zero additive genetic effects, with the shared component accounting for a larger proportion of the polygenic architecture of ALC than of MDD. Local analyses further indicated that shared genetic covariance was concentrated in a limited number of genomic segments. At the tissue and cellular levels, genetic signals were primarily associated with central nervous system tissues and neuronal lineages, with additional support for oligodendrocyte-related populations; the two phenotypes also differed in the distribution and within-cell-type heterogeneity of disease-relevance scores. Multi-omics integration prioritized MED19 and ACO2 as candidate genes and highlighted processes related to mitochondrial energy metabolism and synaptic function. These findings refine the genomic, tissue, and cellular context of the shared genetic architecture of MDD and ALC and provide prioritized genomic regions, cell types, and candidate genes for validation in independent populations and functional studies.

Major Depressive Disorder↗

PubMed1988

Human macrophage colony stimulating factor (M-CSF): alternate RNA splicing generates three different proteins that are expressed on the cell surface and secreted.

Human macrophage colony stimulating factor (M-CSF) cDNA clones were isolated from a pancreatic carcinoma cell line. Three different classes of M-CSF precursor protein (256, 554 and 438 amino acids in length) were predicted to be encoded by these cDNAs. Two of these, that we designate M-CSF alpha and M-CSF beta have already been described. The third, M-CSF gamma represents a novel class of M-CSF cDNA. All three precursors share a 32 amino acid signal sequence and the first 149 amino acids of the mature protein. At this position, M-CSF beta and gamma have insertions of 298 and 182 amino acids relative to M-CSF alpha. The first 182 amino acids of these insertions are shared between M-CSF beta and gamma. All three precursors share the C-terminal 75 amino acids that encode the transmembrane and cytoplasmic domains. Expression of all three cDNAs in COS-7 monkey kidney cells gave rise to soluble M-CSF activity, associated with proteins of subunit molecular weight 44 Kda (beta and gamma) or 28 Kda (alpha). In addition, M-CSF proteins could be detected on the surface of the transfected cells by indirect immunofluorescence. When the transmembrane and cytoplasmic domains of M-CSF alpha were removed by introducing a stop codon after amino acid 190, no membrane-bound M-CSF could be detected, but the truncated protein was secreted efficiently and was biologically active. This suggests that all three forms of M-CSF can exist as cell surface proteins, anchored by their hydrophobic transmembrane domains, and can be processed to soluble forms by proteolytic digestion. Although all soluble forms of M-CSF were biologically active in murine bone marrow colony and proliferation assays, they showed greatly reduced or no activity in similar assays using human bone marrow.

Amino Acid Sequence↗

PubMed2000

TLS-ERG leukemia fusion protein inhibits RNA splicing mediated by serine-arginine proteins.

The translocation liposarcoma (TLS) gene is fused to the ETS-related gene (ERG) in human myeloid leukemia, resulting in the generation of a TLS-ERG protein. We demonstrate that both TLS and the TLS-ERG leukemia fusion protein bind to RNA polymerase II through the TLS N-terminal domain, which is retained in the fusion protein; however, TLS recruits members of the serine-arginine (SR) family of splicing factors through its C-terminal domain, whereas the TLS-ERG fusion protein lacks the ability to recruit SR proteins due to replacement of the C-terminal domain by the fusion partner ERG. In transient-transfection assays, the TLS-ERG fusion protein inhibits E1A pre-mRNA splicing mediated by these TLS-associated SR proteins (TASR), and stable expression of the TLS-ERG fusion protein in K562 cells alters the splicing profile of CD44 mRNA. These results suggest that TLS fusion proteins may lead to cellular abnormalities by interfering with the splicing of important cellular regulators.

Adenovirus E1A Proteins↗

PubMed1988

Translation of globin RNA spliced in vitro.

Pre-mRNA (precursor mRNA) enriched for globin-gene transcription products was prepared from murine erythroleukaemia cells induced to differentiate with dimethyl sulphoxide. The pre-mRNA was prepared from nuclear RNA by oligo(dT)-cellulose chromatography followed by sucrose-gradient centrifugation. The pre-mRNA thus obtained was utilized as the substrate for a splicing assay based on sequential splicing and translation reactions. Small nuclear ribonucleoprotein particles, which were shown to inhibit translation, were removed by an intermediate high-speed centrifugation step. The assay developed is based on a three-step procedure: (1) incubation of pre-mRNA with HeLa-cell nuclear extract in the presence of ATP; (2) incorporation of 3H-labelled amino acid into proteins by wheat-germ extract by using RNA from step 1; and (3) immunoprecipitation of globin chains with rabbit anti-(mouse globin) antibody. No incorporation of label into immune complexes was observed in the absence of HeLa-cell extract or ATP. Preincubation with these components was unnecessary for the incorporation of label into immune complexes when mature cytoplasmic RNA was used as a template for protein synthesis. The assay was further validated by fluorography after polyacrylamide-gel electrophoresis, when the only band detectable corresponded to globin chains. These results demonstrate, for the first time, translational activity of transcripts spliced in vitro. The current assay can be utilized with a few micrograms of pre-mRNA and may have potential for further characterization of the splicing system.

Cell Line↗