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PubMed2005

Identification of a splicing enhancer in MLH1 using COMPARE, a new assay for determination of relative RNA splicing efficiencies.

Exonic splicing enhancers (ESEs) are sequences that facilitate recognition of splice sites and prevent exon-skipping. Because ESEs are often embedded within protein-coding sequences, alterations in them can also often be interpreted as nonsense, missense or silent mutations. To correctly interpret exonic mutations and their roles in diseases, it is important to develop strategies that identify ESE mutations. Potential ESEs can be found computationally in many exons but it has proven difficult to predict whether a given mutation will have effects on splicing based on sequence alone. Here, we describe a flexible in vitro method that can be used to functionally compare the effects of multiple sequence variants on ESE activity in a single in vitro splicing reaction. We have applied this method in parallel with conventional splicing assays to test for a splicing enhancer in exon 17 of the human MLH1 gene. Point mutations associated with hereditary non-polyposis colorectal cancer (HNPCC) have previously been found to correlate with exon-skipping in both lymphocytes and tumors from patients. We show that sequences from this exon can replace an ESE from the mouse IgM gene to support RNA splicing in HeLa nuclear extracts. ESE activity was reduced by HNPCC point mutations in codon 659, indicating that their primary effect is on splicing. Surprisingly, the strongest enhancer function mapped to a different region of the exon upstream of this codon. Together, our results indicate that HNPCC point mutations in codon 659 affect an auxillary element that augments the enhancer function to ensure exon inclusion.

Adaptor Proteins, Signal Transducing↗
PubMed1991

Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary.

Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Evidence that a nuclear matrix protein participates in premessenger RNA splicing.

The role of nuclear matrix proteins in premessenger RNA splicing has been investigated using antibodies raised against isolated rat liver nuclear matrix and cross-reactive with a 65-kDa HeLa cell nuclear matrix protein (IGA-65). IGA-65 is an internal nuclear matrix component which can be solubilized as a component of nuclear splicing extracts, by the action of endogenous ribonucleases, EDTA, and DTT during extract preparation. Preincubation of splicing extract with antibodies against IGA-65 (anti-IGA-65) inhibited in vitro splicing of exogenous adenovirus precursor RNA. Furthermore, assembly of precursor RNA into active spliceosome complexes was inhibited by pretreatment of extracts with anti-IGA-65, suggesting a role for IGA-65 during early spliceosome assembly. The IGA-65 present in splicing extracts was distinguishable from known U-snRNP and hnRNP proteins on protein gels. Furthermore, electrophoresis of splicing extract on native gels indicated that IGA-65 was present in protein complexes different from those containing U-snRNPs or hnRNP C protein. The data support identification of complexes containing IGA-65 as nuclear factors involved in pre-mRNA splicing and, by extension, suggest a role for the nuclear matrix during processing in vivo.

Antibodies↗
PubMed1983

RNA splice site selection: evidence for a 5' leads to 3' scanning model.

Human G gamma-globin genes containing tandem duplications of the donor (5') or acceptor (3') RNA splice sites of the second intervening sequence were constructed in order to ascertain the directionality of RNA splice site selection. These genes were introduced into cultured monkey cells, and their transcripts were analyzed. Transcripts of these duplication variants were spliced only at the proximal copy of the duplicated splice sites. These data are consistent with a 5' leads to 3' model of splice site selection.

Animals↗
PubMed1987

Nonmuscle and smooth muscle myosin light chain mRNAs are generated from a single gene by the tissue-specific alternative RNA splicing.

We have isolated two cDNA clones for myosin alkali light chain (MLC) mRNA from two respective cDNA libraries of chick gizzard and fibroblast cells by cross-hybridization to the previously isolated cDNA of skeletal muscle MLC. Sequence analysis of the two cloned cDNAs revealed that both of them are homologous to but distinct from the cDNA sequence used as the probe so that they may be classified into members of the MLC family, that they are identical with each other in the 3' and 5' untranslated sequence as well as in the coding sequence with a notable exception of a 39-nucleotide insertion in the fibroblast cDNA, 26 nucleotides of which are used for encoding the C-terminal amino acid sequence, and, therefore, that they encode the identical 142-amino acid sequence with different C-terminals of nine amino acids, each specific for fibroblast and gizzard smooth muscle MLC. The position of the inserted block corresponds exactly to one of the exon-intron junctions in the other MLC genes whose structures have so far been elucidated. DNA blot analysis suggested that the two MLC mRNAs of gizzard (smooth muscle) and fibroblast cells (nonmuscle) are generated from a single gene, probably through alternative RNA splicing mechanisms. RNA blot analysis and S1 nuclease mapping analysis using RNA preparations from fibroblast and gizzard tissues showed that the fibroblast MLC mRNA is expressed predominantly in fibroblast cells, but not, or very scantily if at all, in the gizzard, whereas the reverse is true for the gizzard smooth muscle MLC mRNA.

Amino Acid Sequence↗
PubMed2002

The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation.

Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis. The inhibition of relaxation correlated with the ability of various deletion mutants of the two proteins to interact directly, suggesting that an interaction between the RS-domain of ASF/SF2 and a region between amino acid residues 208-735 on topoisomerase I accounts for the observed effect. Consistently, phosphorylation of the RS-domain with either topoisomerase I or a human cell extract reduced the inhibition of relaxation activity. Taken together with the previously published studies of the topoisomerase I kinase activity, these observations suggest that topoisomerase I activity is shifted from relaxation to kinasing by specific interaction with SR-splicing factors.

Catalysis↗
PubMed1980

A mechanism for RNA splicing.

The most abundant of the stable small nuclear RNAs of eukaryotic cells, U-1 small nuclear RNA, is exactly complementary to the consensus sequences at RNA splice sites. We propose that this RNA is the recognition component of the nuclear RNA splicing enzyme and forms base pairs with both ends of an intron so as to align them for cutting and splicing.

Animals↗
PubMed2006

Identification of a family of DNA-binding proteins with homology to RNA splicing factors.

We describe a unique family of human proteins that are capable of binding to the cAMP regulatory element (CRE) and that are homologous to RNA splicing proteins. A human cDNA was isolated that encodes a protein with a distinctive combination of modular domain structures: 2 leucine-zipper-like domains, a DNA-binding zinc-finger-like domain, an RNA-binding zinc-finger-like domain, and 2 coiled-coil protein-protein interaction domains. It also has a serine-arginine-rich domain, commonly found in proteins involved in RNA splicing. The protein was discovered using the CRE as bait in a yeast 1-hybrid assay. It was then shown to bind specifically to the CRE in vitro using gel shift assays. We have named the protein CRE-associated protein (CREAP). We show that it is widely expressed in human tissues but is highly expressed in several fetal tissues and in several regions of the adult brain. CREAP is closely related to 2 human proteins of unknown function. CREAP shows significant homology with a small nuclear ribonucleoprotein of yeast, Luc7p, involved in 5' splice site recognition. The 3 human CREAP proteins form a unique family with the potential to act as transcription factors that link to RNA processing.

Amino Acid Sequence↗
PubMed2000

Aberrant RNA splicing in the hMSH2 gene: molecular identification of three aberrant RNA in Scottish patients with colorectal cancer in the West of Scotland.

Blood samples from 47 unselected patients with colorectal cancer were used as a source of hMSH2 mRNA. We identified three new hMSH2 aberrant mRNAs including: 1) IVS15 +5 G-->C resulting in exon 15 skipping from transcript; 2) an mRNA deletion of exons 2 to 6 inclusive; and 3) an mRNA deletion of exons 2 to 8 inclusive. In order to find out whether or not exon skipping is a natural consequence of alternative mRNA splicing, total RNA from 20 healthy individuals was converted to cDNA by reverse-transcriptase polymerase chain reaction, and our results show that none of the healthy individuals have the above aberrant mRNA. Our results also show that the presence of mutations in colorectal cancer cases, which do not fully meet the hereditary non-polyposis colon cancer criteria, would suggest that all familial cases should be investigated for germ line mutations in the mismatch repair genes.

Adult↗
PubMed1989

Characterization of the mode of binding of substrates to the active site of Tetrahymena self-splicing RNA using 5-fluorouracil-substituted mini-exons.

Splice-site selection specificity in Tetrahymena self-splicing RNA is thought to be mediated by a base-paired complex between a CUCUCU sequence on the end of the 5' exon and a GGGAGG guide sequence in the intron. The substitution of uracil (U) in oligonucleotide mini-exons with 5-fluorouracil (UF), an analogue bearing a much more acidic N-3 proton, allowed us to test the role of hydrogen bonding between complementary bases in the splice-site selection process. The affinities of (U) and (UF) mini-exons for the ribozyme active site were similar and several orders of magnitude greater than expected from base pairing alone. In contrast to CUCU, the CUFCUF mini-exon lost substrate activity with increasing pH, presumably due to ionization of the UF residues. However, the apparent pK values of these residues were several pK units above that of free UF, indicating that the mini-exon is shielded from the solvent by an active site of low polarity. Loss of the pyrimidine N-3 hydrogen bond by selective ionization of the UF residues decreased the binding of CUFCUF to the ribozyme only 3-fold but did prevent its ligation to the 3' exon. Temperature dependence of substrate activity was identical for both (U) and (UF) mini-exons, whereas the UF-substituted ribozyme lost activity at a considerably lower temperature than did the natural (U) ribozyme. These observations indicate that hydrogen-bonded base pairs involving the U residues contribute little to the total binding energy of the 5' splice site with the active site of the ribozyme, but probably help to align the splice sites properly for ligation.

Animals↗
PubMed2005

An intron-encoded protein assists RNA splicing of multiple similar introns of different bacterial genes.

Four group II introns were found in an unusually intron-rich dnaN gene (encoding the beta subunit of DNA polymerase III) of the cyanobacterium Trichodesmium erythraeum, and they have strong similarities to two introns of the RIR gene (encoding ribonucleotide reductase) of the same organism. Of these six introns, only the RIR-3 intron encodes a maturase protein and showed efficient RNA splicing when expressed in Escherichia coli cells. The other five introns do not encode a maturase protein and did not show RNA splicing in E. coli. But these maturase-less introns showed efficient RNA splicing when the RIR-3 intron-encoded maturase protein was co-expressed from a freestanding gene in the same cell. These findings demonstrated that an intron-encoded protein could function as a general maturase for multiple introns of different genes. Major implications may include an intron-mediated co-regulation of the different genes and a resemblance of the evolutionary origin of spliceosomal introns.

Alternative Splicing↗
PubMed1996

Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures.

We have used peripheral blood T-lymphocyte cultures to analyze the hprt mutation in two Lesch-Nyhan syndrome males who are cousins and to confirm the carrier status of female members of the family. Both cDNA and genomic DNA sequencing studies show that this patient carries a hitherto undescribed single base deletion in the exon 5 donor splice site sequence (I5: +1, delta G, base number 31635). The largest cDNA product contained all nine hprt exons plus an insertion of 66 bases of intron 5, consistent with the use of a cryptic splice site in intron 5 (aag67/gtaagc). This splicing error would result in a chain terminating codon immediately after exon 5 (I5:2-4, taa) and predicts a polypeptide of 133 amino acids. This loss of the normal splice donor site also results in multiple hprt mRNA species, combining the use of the cryptic splice site in intron 5 and splicing errors involving exons 2-6. In addition to defining a new Lesch-Nyhan mutation (hprtHenryville), these results provide insight into aberrant splicing of hprt mRNA in T-lymphocytes.

Base Sequence↗
PubMed1998

RNA splicing of bacterial genes in eukaryotes.

The presence of intervening sequences or introns in eukaryotic genes has been known for more than 20 years, and the mechanisms underlying RNA splicing have been studied in depth both genetically and biochemically. In recent years, however, an increasing number of bacterial genes have been introduced into higher eukaryotes as important tools for genetic studies. Their gene products are frequently used as an indirect measure for cell type-specific promoter activity, as, for example, in the case of chloramphenicol acetyl transferase (CAT assay) or beta-galactosidase. Here we show that RNA splicing of two prokaryotic genes encoding site-specific DNA recombinases occurs in eukaryotic cells. In one case, splicing is only observed after treatment of cells with the cytokine alpha interferon. We further demonstrate that mutating an intragenic donor splice site in a bacterial gene apparently activates a second, alternative splicing pathway. In conjunction with previous reports, our findings should also be regarded as a warning that splicing of bacterial genes in higher eukaryotes is a more common phenomenon than presently recognized, which may be difficult to overcome and may cause problems in the interpretation of experimental results.

Base Sequence↗
PubMed2004

A DNA enzyme that mimics the first step of RNA splicing.

We have discovered an artificial DNA enzyme that mimics the first step of RNA splicing. In vitro selection was used to identify DNA enzymes that ligate RNA. One of the new DNA enzymes carries out splicing-related catalysis by specifically recognizing an unpaired internal adenosine and facilitating attack of its 2'-hydroxyl onto a 5'-triphosphate. This reaction forms 2',5'-branched RNA and is analogous to the first step of in vivo RNA splicing, in which a ribozyme cleaves itself with formation of a branched intermediate. Unlike a natural ribozyme, the new DNA enzyme has no 2'-hydroxyl groups to aid in the catalytic mechanism. Our finding has two important implications. First, branch-site adenosine reactivity seems to be mechanistically favored by nucleic acid enzymes. Second, hydroxyl groups are not obligatory components of nucleic acid enzymes that carry out biologically related catalysis.

Adenosine↗
PubMed2003

SCNM1, a putative RNA splicing factor that modifies disease severity in mice.

The severity of many inherited disorders is influenced by genetic background. We describe a modifier interaction in C57BL/6Jmice that converts a chronic movement disorder into a lethal neurological disease. The primary mutation (medJ) changes a splice donor site of the sodium channel gene Scn8a (Nav1.6). The modifier mutation is characteristic of strain C57BL/6Jand introduces a nonsense codon into sodium channel modifier 1 (SCNM1), a zinc finger protein and a putative splice factor. An internally deleted SCNM1 protein is also predicted as a result of exon skipping associated with disruption of a consensus exonic splicing enhancer. The effect of the modifier mutation is to reduce the abundance of correctly spliced sodium channel transcripts below the threshold for survival. Our finding that genetic variation in a putative RNA splicing factor influences disease susceptibility in mice raises the possibility that a similar mechanism modifies the severity of human inherited disorders.

Alleles↗
PubMed1985

Differential RNA splicing and post-translational cleavages in the human salivary proline-rich protein gene system.

The nucleotide sequences of cDNAs coding for human salivary proline-rich proteins (PRPs) were determined. Clones cP1 and cP2 contain repetitive regions in which sites for the restriction enzyme HaeIII occur repeatedly; they code for the precursors of acidic PRPs. Clones cP3 to cP7 contain repetitive regions in which BstNI sites occur repeatedly; they code for precursors of basic and glycosylated PRPs. The clones cP3, cP4, and cP5 are identical except that cP4 and cP5 are missing 399 and 459 base pairs, respectively, from the repetitive region of cP3. The sequences at these deletion end points are homologous to the consensus sequences of RNA splicing donor and acceptor sites. This strongly suggests that all three cDNAs are derived from the transcript of a single gene via differential RNA splicing. All of the precursor proteins share a feature--the N-terminal region, following the signal peptide, is acidic, while the remainder of the molecule, made of proline-rich repeats of about 21 amino acids, is basic. Each precursor can generate multiple PRPs by various post-translational cleavages on the carboxylic side of specific arginine residues. The data show how differential RNA splicing and post-translational cleavages could generate a large number of proteins, such as those found in saliva, from a much smaller number of genes.

Animals↗