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At least 487 records · Page 27Linked to original sources
PubMed1989

Reverse self-splicing of the tetrahymena group I intron: implication for the directionality of splicing and for intron transposition.

Using short oligoribonucleotides as ligated exon substrates, we show that splicing of the Tetrahymena rRNA group I intron is fully reversible in vitro. Incubation of ligated exon RNA with linear intron produces a molecule in which the splice site sequences of the precursor are reformed. Reversal of self-splicing is favored by high RNA concentration, high magnesium and temperature, and the absence of guanosine. 5' exon sequences that can pair with the internal guide sequence of the intron are required, whereas 3' exon sequences are not essential. Integration of the intron into ligated exon substrates that have the ability to form stem-loop structures is reduced at least one order of magnitude over short, unstructured substrates. We propose that the formation of these structures helps drive splicing in the forward direction. We also show that the Tetrahymena intron can integrate into a beta-globin transcript. This has implications for transposition of group I introns.

Animals↗
PubMed1995

Subnuclear localization of WT1 in splicing or transcription factor domains is regulated by alternative splicing.

WT1 is a tumor suppressor gene with a key role in urogenital development and the pathogenesis of Wilms' tumor. Two alternative splice sites in the WT1 transcript allow the gene to encode four proteins. These carry four Krüppel-type zinc fingers and to date have primarily been implicated in transcriptional control of genes involved in growth regulation. However, here we demonstrate colocalization of WT1 with splicing factors in the fetal kidney and testis and in expressing cell lines. Using immunoprecipitation, we show that two WT1 isoforms directly associate with one or a limited number of components in the spliceosomes and coiled bodies. Moreover, COS cell expression studies suggest that alternative splicing within the WT1 zinc finger region determines whether the protein localizes mainly with splicing factors or with DNA in transcription factor domains in the nucleus. We propose that WT1 plays roles in posttranscriptional processing of RNA as well as in transcription.

Alternative Splicing↗
PubMed1994

Mechanisms for selecting 5' splice sites in mammalian pre-mRNA splicing.

Identification of 5' splice sites requires that limited and dispersed sequence information be interpreted precisely. Both snRNAs and proteins are required for this process. The selection of 5' splice sites in alternative splicing is closely related to that in constitutive splicing, and uses the same components in somewhat different ways.

Animals↗
PubMed1998

Alternative splicing of mouse IL-15 is due to the use of an internal splice site in exon 5.

IL-15 is a pleiotropic cytokine modulating growth and differentiation of several hematopoietic cell types. Recently, we have demonstrated that mouse microglial cells, the brain macrophages, express both IL-15 and IL-15/IL-2 receptors. Based on single-cell RT-PCR data, we describe here an alternatively spliced IL-15 mRNA variant found in a small subpopulation of mouse microglia (5%, 3 out of 60 cells expressing IL-15 transcripts). PCR cycle sequencing of this larger transcript revealed the mouse homologue of the alternatively spliced exon A as it is known from the human IL-15 gene. Analysis of the corresponding mouse IL-15 gene region shows that the larger IL-15 transcript contains an yet unidentified 5' sequence of exon 5 while the shorter transcript uses an internal splice acceptor site. The mouse exon 5A segment has a length of 136 nt (17 nt longer than the human exon A). It contains five in-frame stop codons at its 5' end and a new translation initiation site at its 3' end. This new start site is surrounded by a favourable Kozak consensus sequence suggesting a more efficient translation rate. Further translational control by stem-loop binding factors is inferred by a predicted RNA stem-loop structure around the start site. Insertion of exon 5A would lead to an IL-15 polypeptide with a shortened leader sequence of 26 amino acids, as compared to the 48 amino acid leader sequence encoded by the transcript lacking exon 5A. Thus, the final IL-15 protein of the two splice variants is identical; different leader sequences could, however, lead to differences in the intracellular sorting, processing and/or secretion of IL-15.

Alternative Splicing↗
PubMed2000

Heart-specific splice-variant of a human mitochondrial ribosomal protein (mRNA processing; tissue specific splicing).

It has been proposed that splice-variants of proteins involved in mitochondrial RNA processing and translation may be involved in the tissue specificity of mitochondrial DNA disease mutations (Fischel-Ghodsian, 1998. Mol. Genet. Metab. 65, 97-104). To identify and characterize the structural components of mitochondrial RNA processing and translation, the Mammalian Mitochondrial Ribosomal Consortium has been formed. The 338 amino acid (aa) residues long MRP-L5 was identified (O'Brien et al., 1999. J. Biol. Chem. 274, 36043-36051), and its transcript was screened for tissue specific splice-variants. Screening of the EST databases revealed a single putative splice-variant, due to the insertion of an exon consisting of 89 nucleotides prior to the last exon. Screening of multiple cDNA libraries revealed this inserted exon to be present only in heart tissue, in addition to the predominant MRP-L5 transcript. Sequencing of this region confirmed the EST sequence, and showed in the splice-variant a termination triplet at the beginning of the last exon. Thus the inserted exon replaces the coding sequence of the regular last exon, and creates a new 353 aa long protein (MRP-L5V1). Sequence analysis and 3D modeling reveal similarity between MRP-L5 and threonyl-t-RNA synthetases, and a likely RNA binding site within MRP-L5, with the C-terminus in proximity to the RNA binding site. Sequence analysis of MRP-L5V1 also suggests a likely transmembrane domain at the C-terminus. Thus it is possible that the MRP-L5V1 C-terminus could interfere with RNA binding and may have gained a transmembrane domain. Further studies will be required to elucidate the functional significance of MRP-L5V1.

Amino Acid Sequence↗
PubMed1996

Initial splice-site recognition and pairing during pre-mRNA splicing.

Advances over the past year have provided new insights into the mechanisms involved in the initial recognition and pairing of the 5' and 3' splice sites in complex metazoan pre-mRNAs. Highlights include the demonstration that exonic enhancers can promote trans splicing and that an excess of the serine and arginine rich family of splicing proteins can obviate the requirement for U1 small nuclear ribonucleoprotein particle in splicing.

Amino Acid Sequence↗
PubMed2000

Identification of twenty alternatively spliced estrogen receptor alpha mRNAs in breast cancer cell lines and tumors using splice targeted primer approach.

Estrogen receptor (ER) alpha splice variant transcript profiles were analyzed by RT PCR in six ER positive breast cancer cell lines, MCF-7, T47D, ZR-75, LCC1, LCC2 and LCC9, three ER negative cell lines, MDA-MB-435, MDA-MB-235 and LCC6, and three ER positive malignant breast tumors using targeted primers which specifically anneal to the splice junctions of exon 2Delta, exon 3Delta, exons 2-3Delta, exon 4Delta, exon 5Delta, exon 6Delta and exon 7Delta. The partner primers were chosen such that largest possible transcripts were amplified between exons 1 and 8. The results described here show that each splice specific primer amplified not only the single exon deleted transcript but also a number of related transcripts that have deletions in various combinations of exons. The exon 2Delta specific primer amplified five transcripts that have deletions in exon 2, exons 2 and 7, exons 2, 5, and 7, exons 2 and 4-5, and exons 2 and 4-6. The exon 3Delta specific primer amplified two transcripts that have deletions in exon 3, and exons 3 and 7. The exon 2-3Delta specific primer amplified three products that have deletions in exons 2-3, exons 2-3 and 7 and exons 2-3, 5 and 7. The exon 4Delta specific primer amplified two products that have deletions in exon 4, and exons 4 and 7. The exon 5Delta specific primer amplified three transcripts, that have deletions in exon 5, exons 5 and 2, and exons 5, and 2-3. The 6Delta specific primer amplified only one transcript that has a deletion in exon 6. The 7Delta specific primer amplified four transcripts, that have deletions in exon 7, exons 7 and 4, exons 7 and 3-4, and exons 7 and 3-5. None of the above splice specific primers amplified the wild type ER sequences. The six ER positive cell lines differed in the patterns of the variant transcripts and among the three ER negative cell lines analyzed, only MDA-MB-435 showed the presence of exon 2Delta and exon 4Delta transcripts. Analyses in the tumor samples indicated that the above transcripts are extensively modified.

Alternative Splicing↗
PubMed1998

Silent nucleotide substitution in the sterol 27-hydroxylase gene (CYP 27) leads to alternative pre-mRNA splicing by activating a cryptic 5' splice site at the mutant codon in cerebrotendinous xanthomatosis patients.

A functionally silent nucleotide substitution of the sterol 27-hydroxylase gene (CYP 27), identified in two families with cerebrotendinous xanthomatosis (CTX), was confirmed to cause alternative pre-mRNA splicing of the gene. Full-length RT-PCR analysis of the CYP 27 gene in a patient from one of the CTX families revealed one major and an additional faint band. Sequence analysis of the cloned RT-PCR product showed three species of cDNA: 3' terminal 13 bp of exon 2 deleted cDNA, exon 2 skipped cDNA, and full-length cDNA with a functionally silent G to T mutation at codon 112 (GGG 112Gly to GGT 112Gly). Only a single base change was identified by genomic DNA sequence analysis of the CYP 27 gene in the patient: T replaced G at the third position of codon 112, 13 bp upstream from the 3' terminus of exon 2. Transfection of constructed minigenes, with or without the mutation, confirmed that this silent mutation resulted in alternative pre-mRNA splicing by activating a cryptic 5' splice site around the mutant codon. The mutation was also identified in two patients from another CTX family, with a compound heterozygous pattern of A for G substitution at codon 372, a mutation reported previously by our group. The results elucidate a novel molecular basis for the CTX and suggest the significance of a silent nucleotide substitution with regard to pre-RNA splicing.

Alternative Splicing↗
PubMed2001

The splice of life: alternative splicing and neurological disease.

Splicing of pre-messenger RNA is regulated differently in the brain compared with other tissues. Recognition of aberrations in splicing events that are associated with neurological disease has contributed to our understanding of disease pathogenesis in some cases. Neuron-specific proteins involved in RNA splicing and metabolism are also affected in several neurological disorders. These findings have begun to bridge what we know about the mechanisms regulating neuron-specific splicing and our understanding of neural function and disease.

Alternative Splicing↗
PubMed2006

Auxiliary splice factor U2AF26 and transcription factor Gfi1 cooperate directly in regulating CD45 alternative splicing.

By alternative splicing, different isoforms of the transmembrane tyrosine phosphatase CD45 are generated that either enhance or limit T cell receptor signaling. We report here that CD45 alternative splicing is regulated by cooperative action of the splice factor U2AF26 and the transcription factor Gfi1. U2AF26 promoted formation of the less-active CD45RO by facilitating exon exclusion. Gfi1 antagonized that process by directly interacting with U2AF26, identifying a previously unknown link between a transcription factor and alternative splicing. The presence of Gfi1 led to formation of the more-active CD45RB, whereas loss of Gfi1 favored CD45RO production. We propose that the relative abundance of U2AF26 and Gfi1 determines the ratio of CD45 isoforms, thereby regulating T cell activation.

3T3 Cells↗
PubMed2000

The mouse Nkx-1.2 homeobox gene: alternative RNA splicing at canonical and noncanonical splice sites.

A mouse homeobox gene, Nkx-1.2, (previously termed Sax-1) that is closely related to the Drosophila NK-1/S59 gene was cloned, and genomic DNA and cDNA were sequenced. Nine Nkx-1.2 cDNA clones were found that correspond to three species of Nkx-1.2 mRNA that are formed by alternative splicing at conventional 5' donor and 3' acceptor splice sites; however, seven cDNA clones were found that correspond to three species of Nkx-1.2 mRNA from testes that have novel TG/AC 5' and 3' splice sites. The consensus splice sequences are: 5' donor, CC downward arrowTGGAAG; 3' acceptor, ACTTAC downward arrow. Predicted amino acid sequences suggest that some transcripts may be translated into proteins that lack part or all of the homeodomain. At least three bands of Nkx-1.2 mRNA were found in RNA from the testes. Nkx-1.2 mRNA was shown to be present in postmeiotic germ cells of the testis and in mature spermatozoa. Nkx-1.2 mRNA also was found in regions of the adult cerebral cortex, hippocampus, diencephalon, pons/medulla, and cerebellum. Nkx-1.2 mRNA was found in embryos in highest abundance in 10-day embryos; the mRNA levels decrease during further development. Nkx-1.2 mRNA also was found in discrete zones of the embryonic mesencephalon and myelencephalon.

Alternative Splicing↗
PubMed1989

Alternative splicing of HLA-DQB transcripts and secretion of HLA-DQ beta-chain proteins: allelic polymorphism in splicing and polyadenylylation sites.

HLA class II antigens are highly polymorphic cell-surface proteins involved in initiation and regulation of the immune response. Allelic sequence variation primarily affects the structure of the first external domains of alpha and beta component chains. Here we provide evidence for other types of allelic polymorphism for the genes encoding these chains. Sequences of two cDNA clones corresponding to HLA-DQB mRNAs from an HLA-homozygous cell line exhibit both alternative splicing and read-through of polyadenylylation. Furthermore, alternative splicing that deletes the transmembrane exon is associated with only a subset of HLA-DQB alleles, while the polyadenylylation-site read-through is found in a larger subset. This suggest that polymorphic cis-acting elements within the HLA-DQB gene control both processing steps. Proteins, presumably encoded by alternatively spliced mRNAs lacking transmembrane exons, are immunoprecipitated with a monomorphic monoclonal antibody directed against HLA-DQ. These proteins are found in supernatants of cultured cell lines for which secretion is predicted, but not in those of cell lines that do not contain alternatively spliced mRNAs.

Alleles↗
PubMed1997

Cloning of human 2H9 heterogeneous nuclear ribonucleoproteins. Relation with splicing and early heat shock-induced splicing arrest.

Using antibody 2H9 from our heterogeneous nuclear ribonucleoproteins (anti-hnRNP) monoclonal antibody library, we previously showed in HeLa cells that a 35-37-kDa protein doublet switches from the hnRNP complexes to the nuclear matrix following a 10-min heat shock at 45 degrees C (1 Lutz, Y., Jacob, M., and Fuchs, J. P. (1988) Exp. Cell Res. 175, 109-124). cDNA cloning and sequencing revealed an hnRNP protein (2H9) which is a new member of the hnRNP F, H/H' family. Protein 2H9 displays two consensus sequence-type RNA binding domains (CS-RBD) showing 80-90% homology with two of the three CS-RBDs of hnRNP F and H/H'. Another common feature is the presence of two glycine/tyrosine-rich auxiliary domains located at the C terminus and between the two CS-RBDs. At the functional level we show that specific anti-2H9 peptide antibodies can directly inhibit an in vitro splicing system. Moreover, the 2H9 protein doublet is no more present in nuclear extracts from such briefly stressed cells, which interestingly correlates with the inability of these extracts to catalyze in vitro splicing reactions. Taken together, our data suggest that these proteins are involved in the splicing process and also participate in early heat shock-induced splicing arrest by transiently leaving the hnRNP complexes. These 2H9 proteins, which are encoded by a single gene located on human chromosome 10, were also found to be associated with nuclear bodies in situ.

Amino Acid Sequence↗
PubMed2003

Human RGS6 gene structure, complex alternative splicing, and role of N terminus and G protein gamma-subunit-like (GGL) domain in subcellular localization of RGS6 splice variants.

RGS proteins are defined by the presence of a semiconserved RGS domain that confers the GTPase-activating activity of these proteins toward certain G alpha subunits. RGS6 is a member of a subfamily of RGS proteins distinguished by the presence of DEP and GGL domains, the latter a G beta 5-interacting domain. Here we report identification of 36 distinct transcripts of human RGS6 that arise by unusually complex processing of the RGS6 gene, which spans 630 kilobase pairs of genomic DNA in human chromosome 14 and is interrupted by 19 introns. These transcripts arise by use of two alternative transcription sites and complex alternative splicing mechanisms and encode proteins with long or short N-terminal domains, complete or incomplete GGL domains, 7 distinct C-terminal domains and a common internal domain where the RGS domain is found. The role of structural diversity in the N-terminal and GGL domains of RGS6 splice variants in their interaction with G beta 5 and subcellular localization and of G beta 5 on RGS6 protein localization was examined in COS-7 cells expressing various RGS6 splice variant proteins. RGS6 splice variants with complete GGL domains interacted with G beta 5, irrespective of the type of N-terminal domain, while those lacking a complete GGL domain did not. RGS6 protein variants displayed subcellular distribution patterns ranging from an exclusive cytoplasmic to exclusive nuclear/nucleolar localization, and co-expression of G beta 5 promoted nuclear localization of RGS6 proteins. Analysis of our results show that the long N-terminal and GGL domain sequences of RGS6 proteins function as cytoplasmic retention sequences to prevent their nuclear/nucleolar accumulation. These findings provide the first evidence for G beta 5-independent functions of the GGL domain and for a role of G beta 5 in RGS protein localization. This study reveals extraordinary complexity in processing of the human RGS6 gene and provides new insights into how structural diversity in the RGS6 protein family is involved in their localization and likely function(s) in cells.

Alternative Splicing↗
PubMed2003

Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria.

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.

Cloning, Molecular↗
PubMed1998

Characterization of an intron splice enhancer that regulates alternative splicing of human GH pre-mRNA.

Splicing of pre-mRNA transcripts is regulated by consensus sequences at intron (intervening sequence, IVS) boundaries and the branch site. In vitro studies have shown that the small introns of some genes also require intron splice enhancers (ISE) to modulate splice site selection. An autosomal dominant form of isolated GH deficiency (IGHD-II) is caused by mutations in IVS3 of the GH-1 gene that cause exon 3 (E3) skipping, resulting in truncated hGH products that prevent secretion of normal hGH. Interestingly, some of these IGHD-II mutations perturb an ISE that is buried in IVS3. We localized this ISE by quantitating the effects of deletions within IVS3 on E3 skipping. The importance of individual nucleotides to ISE function was determined by analyzing the effects of point mutants and additional deletions. Our results show that (i) an ISE with a G2X1-4G3motif resides in IVS3 of GH-1; (ii) both runs of Gs are required for ISE function; (iii) a single copy of the ISE regulates E3 skipping and (iv) ISE function can be modified by an adjacent AC element. Our findings reveal a new mechanism by which mutations can cause inherited human endocrine disorders and suggest that (i) ISEs may regulate splicing of transcripts of other genes and (ii) mutations of these ISEs or of the trans -acting factors that bind them may cause other genetic disorders.

Alternative Splicing↗
PubMed1984

Elimination of mRNA splicing by a point mutation outside the conserved GU at 5' splice sites.

Nearly all mRNA introns begin with the dinucleotide GU. Mutations in either of these virtually invariant bases have been found to inactivate the corresponding 5' splice site. Until now single base changes in neighboring bases have not been found to completely inactivate a 5' splice site. Here we show that a single A----U transversion in the third position of the adenovirus 2 E1A 13S mRNA intron does prevent RNA splicing at the corresponding 5' splice site.

Adenoviruses, Human↗
PubMed1989

Structure-function relationships in a self-splicing group II intron: a large part of domain II of the mitochondrial intron aI5 is not essential for self-splicing.

An oligonucleotide-directed deletion of 156 nucleotides has been introduced into the yeast mitochondrial group II intron al5 (887 nt). The deletion comprises almost all of domain II, which is one of the six phylogenetically conserved structural elements of group II introns. This mutant displays reduced self-splicing activity, but results of chemical probing with dimethylsulphate suggest that sequences at the site of the deletion interfere with the normal folding of the intron. This is supported by computer analyses, which predict a number of alternative structures involving conserved intron sequences. Splicing activity could be restored by insertion of a 10-nucleotide palindromic sequence into the unique Smal site of the deletion mutant, resulting in the formation of a small stable stem-loop element at the position of domain II. These results provide a direct correlation between folding of the RNA and its activity. We conclude that at least a large part of domain II of the group II intron al5 is not required for self-splicing activity. This deletion mutant with a length of 731 nucleotides represents the smallest self-splicing group II intron so far known.

Base Sequence↗