Molecular biology. RNA enzymes for RNA splicing.
Explore the source record for details and available documents.
Binding Sites↗
SEARCH · PubMed Health
Results for “RNA Splicing”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Another mechanism for creating diversity in gamma-aminobutyrate type A receptors: RNA splicing directs expression of two forms of gamma 2 phosphorylation site.
Diversity of gamma-aminobutyrate type A (GABAA) receptors has recently been proposed to be achieved by assembly of receptor subtypes from a multitude of subunits (alpha 1-6, beta 1-3, gamma 1-2, and delta) encoded by different genes. Here we report a further mechanism for creating GABAA receptor diversity: alternative RNA splicing. Two forms of bovine gamma 2 subunit cDNA were isolated (gamma 2S and gamma 2L) that differed by the presence or absence of a 24-base-pair (8-amino acid) insertion in the cytoplasmic domain between the third and fourth putative membrane-spanning regions. Polymerase chain reaction from RNA demonstrated that the two forms of gamma 2 subunit are expressed in bovine, human, and rat brain. Sequencing of genomic DNA clones encoding the gamma 2 subunit demonstrated that the 24-base-pair insert is organized as a separate exon. Analysis of the sequence of the 8-amino acid insert revealed that it contains a protein kinase C consensus phosphorylation site. Expression of the large cytoplasmic loop domains of gamma 2S and gamma 2L in Escherichia coli, followed by phosphorylation of the recombinant proteins by protein kinase C, demonstrated that gamma 2L, but not gamma 2S, could be phosphorylated. Thus the two forms of gamma 2 subunit differ by the presence or absence of a protein kinase C phosphorylation site. This mechanism for creating GABAA receptor diversity may allow differential regulation of the function of receptor subtypes.
Amino Acid Sequence↗
Mutations in U1 snRNA bypass the requirement for a cell type-specific RNA splicing factor.
Previous studies have demonstrated that efficient splicing of the primary transcript of the yeast MER2 gene requires the MER1 protein, which is produced only in meiotic cells. A genetic selection was devised to recover second-site mutations that bypass the requirement for MER1 in MER2 RNA-splicing. This selection identified a mutation in SNR19, the gene for U1 snRNA. The suppressor mutation affects the first residue in U1 snRNA, allowing this nucleotide to base pair with the eighth nucleotide in the MER2 intron. This base in MER2 lies outside the conserved hexanucleotide that defines the 5' splice site in yeast. The MER2 5' splice site (GUUCGU) differs from the consensus in yeast (GUAYGU) at the third position. When this nucleotide is mutated to restore the consensus, base pairing with U1 snRNA is increased and the requirement for MER1 is alleviated.
Base Sequence↗
Fixing human factor IX (fIX): correction of a cryptic RNA splice enables the production of biologically active fIX in the mammary gland of transgenic mice.
Transgenic mice and sheep secrete only low levels of human factor IX in their milk because of an aberrant splicing of the transgene RNA in the mammary gland. Removal of the cryptic 3' splice site prevents this splicing and leads to the production of relatively high levels of factor IX. The purified protein is fully active showing that the mammary gland is capable of the efficient post-translational modification of this protein and that transgenic animals are a suitable means of its production.
Animals↗
The bI4 group I intron binds directly to both its protein splicing partners, a tRNA synthetase and maturase, to facilitate RNA splicing activity.
The imported mitochondrial leucyl-tRNA synthetase (NAM2p) and a mitochondrial-expressed intron-encoded maturase protein are required for splicing the fourth intron (bI4) of the yeast cob gene, which expresses an electron transfer protein that is essential to respiration. However, the role of the tRNA synthetase, as well as the function of the bI4 maturase, remain unclear. As a first step towards elucidating the mechanistic role of these protein splicing factors in this group I intron splicing reaction, we tested the hypothesis that both leucyl-tRNA synthetase and bI4 maturase interact directly with the bI4 intron. We developed a yeast three-hybrid system and determined that both the tRNA synthetase and bI4 maturase can bind directly and independently via RNA-protein interactions to the large bI4 group I intron. We also showed, using modified two-hybrid and three-hybrid assays, that the bI4 intron bridges interactions between the two protein splicing partners. In the presence of either the bI4 maturase or the Leu-tRNA synthetase, bI4 intron transcribed recombinantly with flanking exons in the yeast nucleus exhibited splicing activity. These data combined with previous genetic results are consistent with a novel model for a ternary splicing complex (two protein: one RNA) in which both protein splicing partners bind directly to the bI4 intron and facilitate its self-splicing activity.
Base Sequence↗
Differential RNA splicing predicts two distinct nerve growth factor precursors.
Nerve growth factor (NGF) has a crucial role in the development of sensory and sympathetic neurones. However, although it can affect other neural cell types under certain experimental conditions, no biological role has been convincingly demonstrated elsewhere in the nervous system. The 5' end of the mouse NGF gene contains several relatively short exons. The NGF messenger RNA contains two in-frame initiator methionine codons; the second precedes the signal peptide sequence. Studies of the translation of other eukaryotic mRNAs indicate that the first AUG is preferred, suggesting that the signal for secretion might be ambiguous. We have analysed the NGF mRNA species from various cell types, some of which (clonal myoblast and fibroblast cell lines) are known to secrete NGF, to search for different NGF transcripts. One pathway of RNA splicing generates the transcript already described from a submaxillary gland complementary DNA clone. We demonstrate here that there is another splicing pathway, leading to a shorter transcript that lacks the second exon. This short transcript is the major form in most other mouse tissues and in the tissues of several other species, but both transcripts are usually present. In the short transcript, the initiator methionine is immediately upstream from a signal peptide-like sequence whereas in the long transcript the first methionine is 62 amino acids upstream from the signal peptide-like sequences. This may result in a different cellular localization of the NGF or alter the biological activity of the NGF precursor.
Animals↗
Viral spliced RNA are produced, encapsidated and reverse transcribed during in vivo woodchuck hepatitis virus infection.
By the use of reverse transcription followed by polymerase chain reaction (RT-PCR), we have identified one shorter than full-length, pregenomic viral RNA species in liver samples of woodchucks chronically infected with the woodchuck hepatitis virus (WHV). The spliced WHV RNA of about 2.4 kb in length was cloned and partially sequenced. The splicing donor and acceptor sites of this novel RNA are located, respectively, 130 nucleotides downstream of the ATG initiation codon of the core gene and 21 nucleotides upstream of the initiation codon of the pre-S2 surface gene. The splicing event generates a new core-polymerase fusion protein and removes the terminal protein domain and the spacer region of the polymerase gene. A nucleotide probe specific for the splice junction was used following RT-PCR, to further confirm the existence of this spliced RNA in the liver of seven WHV-infected woodchucks. Deleted viral DNA molecules corresponding to the 2.4 kb spliced RNA were also detected in the liver and, to a lesser extent, in the serum of infected woodchucks, suggesting that this spliced RNA can be encapsidated and reverse-transcribed during the course of natural WHV infection.
Amino Acid Sequence↗
Adenosine is inherently favored as the branch-site RNA nucleotide in a structural context that resembles natural RNA splicing.
We previously used in vitro selection to identify the 7S11 deoxyribozyme, which catalyzes formation of 2',5'-branched RNA using a branch-site adenosine nucleophile and a 5'-triphosphate electrophile. An unanswered question is whether the use of branch-site adenosine is inherently preferred or a chance event during the particular selection experiment. Here we have found that deoxyribozymes newly selected to use uridine as the branch-site RNA nucleotide in a structural context that resembles natural RNA splicing instead prefer a branch-site adenosine, although adenosine was never available during the selection itself. Our results support a chemical basis for nature's choice of the branch-site nucleotide, which is almost always adenosine in group II introns and the spliceosome.
Adenosine↗
Tumor cells of hairy cell leukemia express multiple clonally related immunoglobulin isotypes via RNA splicing.
Hairy cell leukemia (HCL) derives from a mature B cell and expresses markers associated with activation. Analysis of immunoglobulin variable region genes has revealed somatic mutation in most cases, consistent with an origin from a cell that has encountered the germinal center. One unusual feature of hairy cells (HCs) is the frequent expression of multiple immunoglobulin heavy chain isotypes, with dominance of immunoglobulin (Ig)--G3, but only a single light chain type. The origin and clonal relationship of these isotype variants have been unclear. In order to probe the isotype switching status of HCL, RNA transcripts of V(H)DJ(H)--constant region sequences from 5 cases of typical HCL, all expressing multiple surface immunoglobulin isotypes, were analyzed. Tumor V(H)DJ(H)--C(mu) sequences were identified and found to be somatically mutated (range, 1.4% to 6.5%), with a low level of intraclonal heterogeneity. Additional immunoglobulin isotypes of identical V(H)DJ(H) sequence were also identified, including IgD (5 of 5), IgG3 (5 of 5), IgG1 (3 of 5), IgG2 (2 of 5), IgA1 (4 of 5), and IgA2 (1 of 5). Derivation of multiple isotypes from individual cells was demonstrated by analyzing transcripts in single sorted cells from one patient, with evidence for coexistence of isotype variants in 10 of 10 cells. These findings indicate that clonally related multiple isotypes coexist in single HCs, with individual isotypes presumably generated via RNA splicing. Production of IgG3 appears common, but IgG1, IgG2, IgA1, and IgA2 also arise, indicating a continuing influence of a directed process on the tumor clone. These HCs appear to be arrested at the point of isotype switch, where RNA processing may precede deletional recombination. (Blood. 2001;98:1174-1181)
Adult↗
Combinatorial RNA splicing alters the surface charge on the NMDA receptor.
Transcripts encoding four NMDA receptor subunits, generated from the NMDAR1 gene by alternative RNA splicing, have been demonstrated in adult rat brain. RNA transcripts derived from cDNAs encoding each form direct the formation of functional NMDA receptors in Xenopus oocytes. The two amino acid cassettes of 21 and 37 amino acids found in the splice variants increase the positive extracellular surface charge on the subunits and may thereby modulate the functional properties of the receptor.
Amino Acid Sequence↗
Positive and negative modulation of human immunodeficiency virus type 1 Rev function by cis and trans regulators of viral RNA splicing.
Expression of the entire complement of human immunodeficiency virus type 1 (HIV-1) viral proteins depends on the competing activities of viral RNA splicing and export into the cytoplasm by Rev. To investigate the possibility that modulation of viral RNA metabolism may alter Rev function, we analyzed the impact of multiple SR proteins on both processes. While overexpression of several of the SR factors altered splicing of HIV-1 env mRNA, they had disparate effects on Rev function that varied with the cell line used. Subsequent examination of exon splicing enhancer (ESE) and/or silencer (ESS) deletions suggests that the effects of the SR proteins on Rev function are not mediated through interaction with these elements. However, analysis of the deletions did indicate that the ESE and/or ESS does have significant effects on Rev function, with deletion of the ESS augmenting the magnitude of the response to Rev and deletion of the ESE significantly reducing it. In situ hybridization and reverse transcription-PCR indicated that the loss of Rev response upon deletion of the ESE was due to a failure of Rev to induce transport of the unspliced RNA into the cytoplasm. Together, the data indicate that cellular splicing factors and viral regulatory elements can have significant stimulatory and inhibitory effects on Rev function, raising the possibility that cells can be rendered permissive or nonpermissive for virus replication by modulation of splicing activities.
Animals↗
A muscle-type tropomyosin in human fibroblasts: evidence for expression by an alternative RNA splicing mechanism.
We have isolated a cDNA clone from a human fibroblast cDNA library that contains the entire protein-coding region of a 1.1-kilobase mRNA. This mRNA encodes a 284-amino acid tropomyosin, the primary structure of which most closely resembles smooth muscle tropomyosin. Thus, the expression of both 284-amino acid muscle-type and 247-amino acid non-muscle-type tropomyosins appears to be a normal feature of human non-muscle cells. We also present evidence to suggest that this cytoskeletal tropomyosin and a human skeletal muscle beta-tropomyosin are derived from a common structural gene by an alternative RNA splicing mechanism.
Amino Acid Sequence↗
Regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gene transcription and alternative RNA splicing in a model of developing intestinal epithelium.
Transcriptional and post-transcriptional regulation of CFTR (cystic fibrosis transmembrane conductance regulator) gene expression was studied in HT29 cells. It is known that the abundance of CFTR mRNA increases during differentiation of pluripotent HT29-18 cells and is maintained at high levels in the stably differentiated HT29-18-C1 subclone. Nuclear run-on assays suggest that increased transcription of the CFTR gene explains the increased abundance of total CFTR mRNA in differentiated HT29 cells. The increased transcription cannot be ascribed to cell cycle-dependent expression of the CFTR gene or to changes in CFTR gene copy number between subcloned cells. Similar to native tissue cells, differentiated HT29 cells contain low copy numbers of CFTR transcripts (1-5/cell), and a portion of the CFTR transcripts are alternatively spliced to remove exon 9 (and make 9-mRNA). During differentiation of HT29-18 cells, the absolute amount of full-length CFTR mRNA increases 8-fold, whereas the amount of 9- mRNA increases 18-fold. The fraction of 9- mRNA in the CFTR mRNA pool is increased in differentiated HT29 cells. The results show that gene transcription regulates the abundance of CFTR transcripts and that regulatory control of alternative RNA splicing may also be a cellular mechanism to modulate CFTR function.
Base Sequence↗
Identification of exon sequences and an exon binding protein involved in alternative RNA splicing of calcitonin/CGRP.
Transcripts derived from the 6 exon CALC I gene are differentially processed in a tissue-specific fashion to include or exclude a calcitonin-specific exon 4. All cell types which transcribe a second calcitonin/CGRP gene, CALC II, exclude exon 4. Substitution of the first 30 nucleotides of CALC I exon 4 with analogous CALC II sequence was sufficient to prevent recognition of exon 4 in in vitro or in vivo RNA splicing systems. UV crosslinking detected a approximately 66 kDa RNA-binding protein in HeLa nuclear extract which interacted with CALC I proximal exon sequence, but not CALC II or mutant sequences. UV crosslinking of this protein was inhibited by addition of nuclear extract from a cell type which normally causes exclusion of exon 4. These results identify an important regulatory element within exon 4 and support a model in which calcitonin production requires protein interaction with this sequence to facilitate exon recognition.
Amino Acid Sequence↗
A single-base change at a splice acceptor site in the ornithine aminotransferase gene causes abnormal RNA splicing in gyrate atrophy.
Gyrate atrophy (GA) is an autosomal recessive eye disease involving a progressive loss of vision due to chorioretinal degeneration in which the mitochondrial matrix enzyme ornithine aminotransferase (OAT) is defective. Two sisters with GA are described in this study in whom an A-to-G substitution at the 3' splice acceptor site of intron 4 in one allele of the OAT gene results in a truncated OAT mRNA devoid of exon 5 sequence. The mutation in the other allele was identified to be a mis-sense mutation at codon 318 by denaturing gradient gel electrophoresis and direct sequencing of the polymerase chain reaction (PCR)-amplified DNA. Thus, these GA patients are compound heterozygotes with respect to mutations in the OAT gene that result in inactivation of OAT.
Adenine↗
CHD1 associates with NCoR and histone deacetylase as well as with RNA splicing proteins.
CHD1 is one of a family of nuclear proteins containing two chromodomains, a SWI/SNF-like helicase/ATPase domain and a DNA binding domain. We found that CHD1 co-immunoprecipitates with histone deacetylase (HDAC) activity and that CHD1 also associates with NCoR, a transcriptional corepressor, in yeast two-hybrid and in vitro pull-down assays. NCoR is known to associate with HDACs to effect its repressive activity, suggesting that the predicted chromatin remodeling activity of CHD1 plays a role in this repression. Yeast two-hybrid assays also showed that CHD1 interacts with splicing proteins mKIAA0164, Srp20, and SAF-B. Splicing assays show that CHD1 overexpression can affect alternative splicing. These results suggest that CHD1 may function in both chromatin mediated transcriptional repression and RNA splicing.
Base Sequence↗
Developmentally regulated alternative RNA splicing of rat brain sodium channel mRNAs.
Two rat brain Na channel alpha-subunit cDNAs, named RII and RIIA, have almost identical coding regions, with a divergence of only 36 nucleotides (0.6%) over a total length of 6015 residues. A cluster of 20 divergent residues occurs within a 90 nucleotide segment of cDNA sequence. We now demonstrate that this 90 nucleotide segment is encoded twice in the RII/RIIA genomic sequence. Furthermore, the mutually exclusive selection of these two exons is developmentally regulated. RII mRNAs are relatively abundant at birth but are gradually replaced by RIIA mRNAs as development proceeds. The two mRNAs also appear to have different regional distributions in the developing rat brain. Strikingly, although 30 amino acids are encoded by each alternative exon, only amino acid position 209 is altered between the two, specifying asparagine in RII and aspartate in RIIA. Alternative RNA splicing may modulate the RII/RIIA sodium channel properties during neuronal development.
Amino Acid Sequence↗
Isolation of a novel cDNA clone for human tyrosine hydroxylase: alternative RNA splicing produces four kinds of mRNA from a single gene.
Human tyrosine hydroxylase (TH) cDNA was isolated by molecular cloning. Lambda gt 11 cDNA library constructed from human pheochromocytoma was screened with a synthetic 23-mer oligonucleotide complementary to rat TH mRNA. We found a novel type of cDNA clone whose N-terminal sequence is similar to but clearly distinct from each of the three types (type 1, 2 and 3) of TH cDNA reported by Grima et al. [Nature (1987) 326, 707-711]. It contains both the 12-bp insert characteristic of type 2 cDNA and the 81-bp sequence of type 3. This novel cDNA clone was designated as type 4. Southern blot analysis of human genomic DNA indicated that TH is encoded by a single gene. This suggests that the four different forms of TH mRNA are produced by alternative RNA splicing from a single primary transcript.
Adrenal Gland Neoplasms↗