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PubMed1998

The association of nonsense codons with exon skipping.

Some genes that contain premature nonsense codons express alternatively-spliced mRNA that has skipped the exon containing the nonsense codon. This paradoxical association of translation signals (nonsense codons) and RNA splicing has inspired numerous explanations. The first is based on the fact that premature nonsense codons often reduce mRNA abundance. The reduction in abundance of full-length mRNA then allows more efficient amplification during PCR of normal, minor, exon-deleted products. This mechanism has been demonstrated to explain an extensive correlation between nonsense codons and exon-skipping for the hamster Hprt gene. The second explanation is that the mutation producing an in-frame nonsense codon has an effect on exon definition. This has been demonstrated for the Mup and hamster Hprt gene by virtue of the fact that missense mutations at the same sites also are associated with the same exon-deleted mRNA. The third general explanation is that a hypothetical process takes place in the nucleus that recognizes nonsense codons, termed 'nuclear scanning', which then has an effect on mRNA splicing. Definitive evidence for nuclear scanning is lacking. My analysis of both nonsense and missense mutations associated with exon skipping in a large number of genes revealed that both types of mutations frequently introduce a T into a purine-rich DNA sequence and are often within 30 base pairs of the nearest exon boundary. This is intriguing given that purine-rich splicing enhancers are known to be inhibited by the introduction of a T. Almost all mutations associated with exon skipping occur in purine-rich or A/C-rich sequences, also characteristics of splicing enhancers. I conclude that most cases of exon skipping associated with premature termination codons may be adequately explained either by a structural effect on exon definition or by nonquantitative methods to measure mRNA, rather than an effect on a putative nuclear scanning mechanism.

Animals↗

PubMed1985

A multicomponent complex is involved in the splicing of messenger RNA precursors.

A multicomponent complex termed spliceosome (splicing body) is unique to the splicing of messenger RNA precursors in vitro. This 60S RNA-protein complex contains RNAs from the previously characterized bipartite splicing intermediate, the 5' exon RNA, and the lariat intervening sequence-3' exon RNA, as well as some intact 455 nucleotide precursor RNA. This complex contains snRNPs, particularly U1 RNP, as shown by immunoprecipitation with specific antisera. Formation of the 60S complex appears to be an early and essential step in splicing, because the 60S complex forms during the early stage, or lag time, of the reaction before the first covalent modification, cleavage at the 5' splice site of precursor RNA. The 60S complex forms only under conditions that permit splicing; both ATP and a precursor RNA containing authentic 5' and 3' splice sites are required for formation, while antiserum specific for U1 RNP inhibits its formation. RNA within the 60S complex, predominantly precursor RNA, was chased into products with accelerated kinetics and more complete conversion than purified precursor RNA.

Adenosine Triphosphate↗

PubMed1987

Splicing of messenger RNA precursors.

A general mechanism for the splicing of nuclear messenger RNA precursors in eukaryotic cells has been widely accepted. This mechanism, which generates lariat RNAs possessing a branch site, seems related to the RNA-catalyzed reactions of self-splicing introns. The splicing of nuclear messenger RNA precursors involves the formation of a multicomponent complex, the spliceosome. This splicing body contains at least three different small nuclear ribonucleoprotein particles (snRNPs), U2, U5, and U4 + U6. A complex containing precursor RNA and the U2 snRNP particle is a likely intermediate in the formation of the spliceosome.

Animals↗

PubMed1998

A cluster of latently expressed genes in Kaposi's sarcoma-associated herpesvirus.

Infection with Kaposi's sarcoma-associated herpesvirus (KSHV) is closely associated with Kaposi's sarcoma (KS) and primary effusion lymphoma, with viral genomes present in a latent state in the majority of tumor cells. Here we describe a cluster of latently expressed viral genes whose mRNAs are generated from a common promoter. Two mRNAs in this region encode the latency-associated nuclear antigen, the product of open reading frame 73 (ORF73). The larger RNA, of 5.8 kb, is an unspliced transcript that includes ORF72 and -71 at its 3' end; it initiates at nucleotides (nt) 127880 to 127886 from a promoter lacking recognizable TATA elements. A less abundant mRNA, of 5.4 kb, is a variant of this transcript, in which 336 nt of 5' noncoding information has been removed by RNA splicing. A third, more abundant RNA is generated from the same promoter region via splicing from the common splice donor at nt 127813 to an acceptor 5' to ORF72; this transcript is the presumed mRNA for ORF72, which encodes the viral cyclin D homolog. All three RNAs are 3' coterminal. In situ hybridization analysis with probes that can detect all three transcripts shows that the RNAs are detectable in a large fraction of BCBL-1 cells prior to lytic induction and in >70% of KS spindle cells in primary KS tumors. This confirms that these transcripts are indeed latent RNAs and suggests a role for their products in viral persistence and/or KSHV-associated proliferation.

Alternative Splicing↗

PubMed2001

Mitochondrial Mg(2+) homeostasis is critical for group II intron splicing in vivo.

The product of the nuclear MRS2 gene, Mrs2p, is the only candidate splicing factor essential for all group II introns in mitochondria of the yeast Saccharomyces cerevisiae. It has been shown to be an integral protein of the inner mitochondrial membrane, structurally and functionally related to the bacterial CorA Mg(2+) transporter. Here we show that mutant alleles of the MRS2 gene as well as overexpression of this gene both increase intramitochondrial Mg(2+) concentrations and compensate for splicing defects of group II introns in mit(-) mutants M1301 and B-loop. Yet, covariation of Mg(2+) concentrations and splicing is similarly seen when some other genes affecting mitochondrial Mg(2+) concentrations are overexpressed in an mrs2Delta mutant, indicating that not the Mrs2 protein per se but certain Mg(2+) concentrations are essential for group II intron splicing. This critical role of Mg(2+) concentrations for splicing is further documented by our observation that pre-mRNAs, accumulated in mitochondria isolated from mutants, efficiently undergo splicing in organello when these mitochondria are incubated in the presence of 10 mM external Mg(2+) (mit(-) M1301) and an ionophore (mrs2Delta). This finding of an exceptional sensitivity of group II intron splicing toward Mg(2+) concentrations in vivo is unprecedented and raises the question of the role of Mg(2+) in other RNA-catalyzed reactions in vivo. It explains finally why protein factors modulating Mg(2+) homeostasis had been identified in genetic screens for bona fide RNA splicing factors.

Alleles↗

PubMed1999

PUF60: a novel U2AF65-related splicing activity.

We have identified a new pyrimidine-tract binding factor, PUF, that is required, together with U2AF, for efficient reconstitution of RNA splicing in vitro. The activity has been purified and consists of two proteins, PUF60 and the previously described splicing factor p54. p54 and PUF60 form a stable complex in vitro when cotranslated in a reaction mixture. PUF activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA. This reaction is dependent upon the presence of the large subunit of U2AF, U2AF65, but not the small subunit U2AF35. PUF60 is homologous to both U2AF65 and the yeast splicing factor Mud2p. The C-terminal domain of PUF60, the PUMP domain, is distantly related to the RNA-recognition motif domain, and is probably important in protein-protein interactions.

Amino Acid Sequence↗

PubMed1991

The chicken tropomyosin 1 gene generates nine mRNAs by alternative splicing.

Skeletal muscle beta-tropomyosin, smooth muscle alpha-tropomyosin, and a low molecular weight fibroblast tropomyosin are generated by alternatively splicing RNA transcripts of the chicken tropomyosin 1 (TM 1) gene (Forry-Schaudies, S., Maihle, N. J., and Hughes, S. H. (1990) J. Mol. Biol. 211; 321-330). Two novel tropomyosin cDNAs that derive from mRNAs of the TM 1 gene have been isolated from a chicken embryo brain cDNA library. Brain cDNA BRT-1 is 2.2 kilobases in length and encodes 283 amino acids. It is identical to skeletal muscle beta-tropomyosin from amino acids 1 to 258. The sequence 3' of this point is unique to BRT-1; a comparison to genomic sequence indicates that a new carboxyl-terminal exon is used to generate this sequence. 1.4-kilobase brain cDNA BRT-2 contains sequences found in both fibroblast cDNA FT-beta (5'-end) and skeletal muscle cDNA SKT-beta (3'-end). RNase and S1 nuclease assays using RNA samples from leg muscle, gizzard, fibroblasts, and brain indicate that the TM 1 gene expresses four additional tropomyosin RNAs by alternately splicing previously characterized exons. These results demonstrate that the chicken TM 1 gene encodes nine tropomyosin RNAs through the use of two promoters, two internal exons that are mutually exclusive, and three 3'-exons. Implications for the regulation of alternative splicing are discussed.

Amino Acid Sequence↗

PubMed1989

Alterations in the polypyrimidine sequence affect the in vitro splicing reactions catalyzed by HeLa cell-free preparations.

The polypyrimidine tract, located at the 3' end of intron 1 of the adenovirus major late transcript, was studied for its role in splicing using cell-free preparations isolated from HeLa cells. A plasmid (pIz) was constructed in which seven purine bases were substituted for pyrimidine bases within the 14-nucleotide polypyrimidine sequence. Runoff transcripts extending to the middle of intron 2 were tested for their ability to support in vitro splicing. The efficiency of these reactions was compared with pre-mRNA transcripts made from the wild-type nonmutated plasmid (p1-2). Neither spliced products nor splicing intermediates were detected in reactions with the pIz pre-mRNA. The formation of the nucleoprotein complexes involved in splicing was examined with this altered pre-mRNA. No 55 S splicing complex was detected and only low levels of the 30 S presplicing complex formed (30-fold less than with wild-type pre-mRNA). However, when a longer runoff transcript was prepared from the polypyrimidine mutated plasmid pIz, spliced RNA was formed. This activity required specific downstream sequences, since transcripts produced from pIz which contained substituted downstream sequences were not spliced. Although intron 2 of the adenovirus major late transcript does not contain a discernible 3' polypyrimidine sequence, pre-mRNA (p2-3) containing this intron was efficiently spliced. However, when the 3' region of intron 2 was substituted for the polypyrimidine sequence of intron 1, the resulting pre-mRNA did not support efficient splicing in vitro. However, when the polypyrimidine sequence of intron 1 was substituted for the sequence at the 3' end of intron 2, efficient splicing occurred, and the rate of formation of splicing intermediates and the accumulation of nucleoprotein complexes was greater than with the wild-type pre-mRNA (p2-3).

Adenoviruses, Human↗

PubMed1999

The role of evolutionarily conserved sequences in alternative splicing at the 3' end of Drosophila melanogaster myosin heavy chain RNA.

Exon 18 of the muscle myosin heavy chain gene (Mhc) of Drosophila melanogaster is excluded from larval transcripts but included in most adult transcripts. To identify cis-acting elements regulating this alternative RNA splicing, we sequenced the 3' end of Mhc from the distantly related species D. virilis. Three noncoding regions are conserved: (1) the nonconsensus splice junctions at either end of exon 18; (2) exon 18 itself; and (3) a 30-nucleotide, pyrimidine-rich sequence located about 40 nt upstream of the 3' splice site of exon 18. We generated transgenic flies expressing Mhc mini-genes designed to test the function of these regions. Improvement of both splice sites of adult-specific exon 18 toward the consensus sequence switches the splicing pattern to include exon 18 in all larval transcripts. Thus nonconsensus splice junctions are critical to stage-specific exclusion of this exon. Deletion of nearly all of exon 18 does not affect stage-specific utilization. However, splicing of transcripts lacking the conserved pyrimidine sequence is severely disrupted in adults. Disruption is not rescued by insertion of a different polypyrimidine tract, suggesting that the conserved pyrimidine-rich sequence interacts with tissue-specific splicing factors to activate utilization of the poor splice sites of exon 18 in adult muscle.

Alternative Splicing↗

PubMed1994

Regulation of splicing at an intermediate step in the formation of the spliceosome.

In vivo experiments have demonstrated that the ribosomal protein L32 of Saccharomyces cerevisiae brings about the inhibition of splicing of the transcript of its own gene through an RNA structure comprised largely of the first exon. We now show that L32, itself, binds specifically to this RNA. Splicing of the RPL32 transcript in vitro is blocked by the presence of L32. Furthermore, addition of the 75-nucleotide RNA representing the 5' end of the RPL32 transcript stimulates specifically the splicing of the RPL32 substrate, presumably by competing for L32 present in the extract. Use of RNAs carrying mutations shown to abolish the regulation of splicing, either as substrates or as competitors, confirmed that the in vitro reaction is a faithful representation of the situation in vivo. We conclude that the regulation of splicing occurs through the specific binding of L32 to an RNA structure within the first 75 nucleotides of the RPL32 transcript. The RPL32 substrate, bound to L32, forms a complex with U1 snRNP, the first step in spliceosome assembly. The presence of L32 prevents the ATP-dependent association of the U2 snRNP necessary to form a complete spliceosome.

Base Sequence↗

PubMed1990

Trans-splicing of pre-mRNA is predicted to occur in a wide range of organisms including vertebrates.

Several known trans-splicing RNA structures were used to define a canonical trans-splicing structure which was then used to perform a computer search of the EMBL nucleotide database. In addition to most known trans-splicing structures, many putative new trans-splicing sites were detected. These were found in a broad range of organisms including the vertebrates. Control experiments indicate that the search predicts known false positives at a rate of only 20%. Trans-splicing may therefore be a very wide-spread phenomenon.

Animals↗

PubMed1998

A common human beta globin splicing mutation modeled in mice.

The betaIVS-2-654 C-->T mutation accounts for approximately 20% of beta thalassemia mutations in southern China; it causes aberrant RNA splicing and leads to beta0 thalassemia. To provide an animal model for testing therapies for correcting splicing defects, we have used the "plug and socket" method of gene targeting in murine embryonic stem cells to replace the two (cis) murine adult beta globin genes with a single copy of the human betaIVS-2-654 gene. No homozygous mice survive postnatally. Heterozygous mice carrying this mutant gene produce reduced amounts of the mouse beta globin chains and no human beta globin, and have a moderate form of beta thalassemia. The heterozygotes show the same aberrant splicing as their human counterparts and provide an animal model for testing therapies to correct splicing defects at either the RNA or DNA level.

Animals↗

PubMed1996

Circularizing ribozymes and decoy-competitors by autocatalytic splicing in vitro and in vivo.

An Anabaena group I intron was circularly permuted at loop 5, loop 6 and loop 8, and tested for self-splicing activity. Precursor RNAs from these constructs spliced efficiently and produced circular exons in vitro. Using group I permuted-intron-exon sequences, circular forms of the HDV ribozyme, the RNA component of RNaseP from B. subtilis, the HIV TAR and a short HIV Rev-binding element were generated and tested for activity and stability. The activity of circular ribozymes is comparable to the linear counterparts with similar core sequences. Circular forms of the TAR and Rev-binding element showed specific binding to Tfr-38 and Rev(35-50) peptide, respectively. To explore the potential for using this methodology to express circular RNA in vivo, circular forms of the HDV ribozyme and RNaseP RNA were produced in E. coli. Analysis of total RNA indicated that the precursor RNA spliced efficiently and accurately to produce circular ribozymes. The activity of in vivo expressed circular ribozymes could be demonstrated indicating that they fold into active conformation. These results suggest that self-splicing group I PIE sequences could prove useful for expressing small stable circular ribozyme/decoy-competitor or antisense RNAs in cells.

Anabaena↗

PubMedDate not supplied

Inhibition of in vitro pre-mRNA splicing in S. cerevisiae by branched oligonucleotides.

A series of V- and Y-shaped nucleic acids, related to the splicing intermediates derived from S. cerevisiae actin pre-mRNA, were prepared. The effects of such branched nucleic acids (bNAs) on the efficiency of in vitro pre-mRNA splicing in yeast were studied. The exogenous bNAs each effect the efficiency of splicing, yet to different degrees, depending on the sugar composition and topology of the molecules. Y-shaped RNAs inhibited the formation of mRNA (i.e. RNA splicing) to the greatest extent.

Oligonucleotides↗

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↗

PubMed1985

Splice-site signals of mRNA precursors as revealed by computer search. Site-specific mutagenesis and thalassemia.

Many eukaryotic genes are interrupted by introns, which are removed from mRNA precursors by the RNA splicing mechanism. Although nucleotide sequences around splice sites have considerable homology among various genes, exact splice-site signals are unknown. Previously, applying the computer searching method to primary nucleotide sequences of various genes, we studied what kinds of patterns are the necessary and sufficient sequences for recognition of the 5'-slice site (donor site). We proposed that four common patterns, AG/GTA, /GTAAGT, RG/GTGAG, and AG/GTXXGT, where R = A or G and X = A, T, G, or C, are often used as such signals. In the present paper, we examined a number of experimental results on site-specific mutagenesis around donor sites and on alpha- and beta-thalassemia in globin genes, and found that the above four common patterns could explain almost all of those results.

Adenoviridae↗

PubMed1983

A method for mapping RNA initiation, termination, splice, and protein binding sites. Ribosome binding sites on beta-globin messenger RNA.

A new method for mapping RNA initiation, termination, and splice sites was developed. The method involves: 1) hybridization of RNA to end-labeled single-stranded DNA; 2) mild digestion of the hybrid with a single strand specific nuclease; 3) high resolution gel electrophoresis and autoradiography. The regions of the labeled probe which are resistant to nuclease digestion are mapped by measuring the distance from the labeled end. The sequence can be determined by running the end-labeled probe sequenced according to the protocol of Maxam and Gilbert (Maxam, A.M., and Gilbert, W. (1980) Methods Enzymol. 65, 499-560) at the same time. The feasibility of this method was tested with adult chicken beta-globin mRNA, and we found that the transcription initiation, termination, and RNA splice sites can be determined to within a few bases of the known sites. Using this method we also found that ribosomes bind to beta-globin mRNA from 22 bases upstream from the translation start codon (AUG) to 15 bases past the stop codon (UAA).

Animals↗

PubMed1987

Three nuclear genes suppress a yeast mitochondrial splice defect when present in high copy number.

A gene bank of a yeast wild type DNA in the high copy number vector YEp13 was screened for recombinant plasmids which suppress the mitochondrial RNA splice defect exerted by mutant M1301, a -1 bp deletion in the first intron of the mitochondrial COB gene (bI1). A total of 17 recombinant plasmids with similar suppressor activity were found. Restriction mapping and cross-hybridization of the inserts revealed that these 17 plasmids contain three different inserts, all lacking any extended sequence homology. Each of the inserts, when present in high copy number, has a similar suppressor activity: high in the presence of mutation M1301 in bI1, a group II intron, and low but significant with the presence of few mutants in bI2 and bI3 of the COB gene, both of which are group I introns.

Alleles↗