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Developmentally regulated alternative splicing in the Xenopus laevis c-Myc gene creates an intron-1 containing c-Myc RNA present only in post-midblastula embryos.

Two distinct c-Myc RNA classes have been identified in Xenopus laevis, presumably expressed from the duplicated c-Myc locus (1, 6). The major Xenopus c-Myc transcripts arise from sites termed P1 and P2 similarly to those of the mammalian c-Myc genes. I have used a cloned Xenopus c-Myc gene to examine the regulated pattern of expression from this gene during early Xenopus embryogenesis. Analysis of the pattern of transcript processing indicates that not only are P1 and P2 differentially active during early development but alternatively spliced c-Myc RNAs are generated which contain sequences of the first intron. These intron-1 containing c-Myc RNAs are generated by alternative splicing of transcripts initiated from the major transcription start site, P2, and are observed only in RNA samples from post-midblastula embryos or Xenopus tissue culture cells. Xenopus tissue culture cells synthesize two major c-Myc proteins (p61 and p64). Xenopus RNAs that do not contain intron-1 sequences synthesize only the p61 species. Two closely spaced ATG codons at the 5' end of exon-2 are utilized equivalently to generate a p61 doublet. Intron-1 containing RNAs utilize an ATG codon in the intron sequences to synthesize the p64 species as well as the exon-2 ATG codons to synthesize the p61 doublet.

Animals↗

Plant nonsense suppressor tRNA(Tyr) genes are expressed at very low levels in vitro due to inefficient splicing of the intron-containing pre-tRNAs.

Oligonucleotide-directed mutagenesis was used to generate amber, ochre and opal suppressors from cloned Arabidopsis and Nicotiana tRNA(Tyr) genes. The nonsense suppressor tRNA(Tyr) genes were efficiently transcribed in HeLa and yeast nuclear extracts, however, intron excision from all mutant pre-tRNAs(Tyr) was severely impaired in the homologous wheat germ extract as well as in the yeast in vitro splicing system. The change of one nucleotide in the anticodon of suppressor pre-tRNAs leads to a distortion of the potential intron-anticodon interaction. In order to demonstrate that this caused the reduced splicing efficiency, we created a point mutation in the intron of Arabidopsis tRNA(Tyr) which affected the interaction with the wild-type anticodon. As expected, the resulting pre-tRNA was also inefficiently spliced. Another mutation in the intron, which restored the base-pairing between the amber anticodon and the intron of pre-tRNA(Tyr), resulted in an excellent substrate for wheat germ splicing endonuclease. This type of amber suppressor tRNA(Tyr) gene which yields high levels of mature tRNA(Tyr) should be useful for studying suppression in higher plants.

Base Sequence↗

Splice site selection by intron aI3 of the COX1 gene from Saccharomyces cerevisiae.

Interactions of the 5' and 3' splice sites with intron internal sequences of the yeast mitochondrial group I intron aI3 were studied using mutation analysis. The results can be fully explained by the splice guide model in which the splice sites are defined by the Internal Guide Sequence. No evidence was found for an alternative interaction between intron nucleotides preceding the 3' splice site and nucleotides in the vicinity of the core region as was found for the Tetrahymena intron. Our results also suggest that binding of the 5' and 3' splice site nucleotides to the IGS can not take place simultaneously. The intron must therefore undergo conformational changes as the reaction proceeds from the first step of self splicing, GTP attack at the 5' splice site, to exon ligation, the second step.

Base Sequence↗

The yeast actin intron contains a cryptic promoter that can be switched on by preventing transcriptional interference.

We show that the single intron of the actin gene of the yeast Saccharomyces cerevisiae contains a cryptic promoter for transcription of the second exon. This promoter is inactive in the normal actin gene, but can be activated when the actin gene promoter is deleted. An identical activation was induced by placing efficient transcriptional terminators at position 61 of the 309 bp intron. In all cases transcripts with identical 5' ends close to the boundary of the intron and the second exon were produced. These results indicate that the cryptic promoter in the actin intron is occluded in the normal actin gene by transcriptional interference with the actin gene promoter. Transcription initiation near the intron/exon 2 boundary is enabled by protection from traversing polymerases, that initiated transcription at the upstream located actin gene promoter. A partial promoter protection using leaky terminators resulted in small amounts of transcripts initiated from the cryptic promoter. Although we do not know any function of the cryptic promoter in actin gene expression, it is tentative to speculate that the cryptic intron promoter might be a relict of a promoter that was functional earlier in evolution.

Actins↗

Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons.

Circularly permuted group I intron precursor RNAs, containing end-to-end fused exons which interrupt half-intron sequences, were generated and tested for self-splicing activity. An autocatalytic RNA can form when the primary order of essential intron sequence elements, splice sites, and exons are permuted in this manner. Covalent attachment of guanosine to the 5' half-intron product, and accurate exon ligation indicated that the mechanism and specificity of splicing were not altered. However, because the exons were fused and the order of the splice sites reversed, splicing released the fused-exon as a circle. With this arrangement of splice sites, circular exon production was a prediction of the group I splicing mechanism. Circular RNAs have properties that would make them attractive for certain studies of RNA structure and function. Reversal of splice site sequences in a context that allows splicing, such as those generated by circularly permuted group I introns, could be used to generate short defined sequences of circular RNA in vitro and perhaps in vivo.

Anabaena↗

The protein sequence and some intron positions are conserved between the switching gene swi10 of Schizosaccharomyces pombe and the human excision repair gene ERCC1.

The switching gene swi10+ has a function in mating-type switching as well as in the repair of radiation damages. We have cloned the genomic swi10+ gene by functional complementation of the switching defect of the swi10-154 mutant. The swi10+ gene is not essential for viability. The DNA sequence revealed an open reading frame of 759 nucleotides interrupted by three introns of 127, 52 and 60 bp, respectively. The positions of intron I as well as of intron III of swi10 are evolutionary conserved in comparison to the introns III and IV of the human ERCC1 gene. The analysis of cDNA clones isolated by PCR amplification confirmed the structure of the swi10 gene. The putative Swi10 protein has homologies to the human and mouse ERCC1 protein, to Rad10 of Saccharomyces cerevisiae and to parts of UvrA and UvrC of E. coli. All these proteins are essential components for excision repair of damaged DNA. The Swi10 protein contains a putative DNA binding domain previously found in other proteins. Northern blot experiments and the analyses of cDNA clones indicate that intron I of the swi10 gene is not efficiently spliced.

Amino Acid Sequence↗

Variation and in vitro splicing of group I introns in rRNA genes of Pneumocystis carinii.

The sequences of the rRNA genes of Pneumocystis carinii from rat and human sources demonstrate three distinct genotypes based on the group I introns present in these genes. One rat isolate (Pc1) contains such introns in its 16S and 26S rRNA genes, while another rat isolate (Pc2) and a human isolate (Pc3) only contain an intron in the 26S rRNA gene. The four introns all catalyze their own excision from RNA transcripts, and this reaction is inhibited by the anti-pneumocystis drug pentamidine and its analogues. Although they differ in sequence, they are more similar to one another than to group I introns found in other eukaryotic microbes.

Animals↗

Kinetic analysis of the 5' splice junction hydrolysis of a group II intron promoted by domain 5.

The 5' splice junction (5'SJ) of Group II intron transcripts is subject to a specific hydrolysis reaction (SJH). This reaction occurs either within a single transcript containing intron sequences through domain 5 (D5) or by cooperation of two separate transcripts, one bearing the 5'SJ and another contributing D5 (1). In this report we describe the latter reaction in terms of its kinetic parameters. A minimal D5 RNA of 36 nts (GGD5) was sufficient to promote SJH of a second transcript containing the 5' exon plus intron domains 1, 2, and 3 (E1:123). Equimolar production of two RNAs, the 5' exon (E1) and an intron fragment containing domains 1, 2, and 3 (123) was observed. The kinetic coefficients were evaluated by an excess GGD5 approach. The apparent Km was complex, varying with GGD5 concentration. This behavior indicates heterogeneity in E1:123 with respect to GGD5 binding. The binding heterogeneity may result from formation of E1:123 dimers or from nicks in some molecules of each E1:123 preparation. The heterogeneity was always evident, but to a variable degree, regardless of the procedure by which E1:123 was isolated. The system may be described in terms of parameters analogous to kcat and Km. At infinite dilution of GGD5, the characterizing values were: k2 degrees (the analog of kcat) = 0.0055 min-1 and Km degrees = 0.22 microM. In the limit of GGD5 saturation, the values were: k2 infinity = 0.012 min-1 and Km infinity = 4.5 microM. A natural variant D5, representing the sequence from intron 1 of the yeast cytochrome-b gene, was also functional in SJH. This GGD5b1 was governed by similar Km degrees and Km infinity values, but was only one-third as active over the entire D5 concentration range. A different D5 isomer was entirely ineffective for SJH.

Base Sequence↗

U21, a novel small nucleolar RNA with a 13 nt. complementarity to 28S rRNA, is encoded in an intron of ribosomal protein L5 gene in chicken and mammals.

Following a search of sequence data bases for intronic sequences exhibiting structural features typical of snoRNAs, we have positively identified by Northern assays and sequence analysis another intron-encoded snoRNA, termed U21. U21 RNA is a 93 nt. long, metabolically stable RNA, present at about 10(4) molecules per HeLa cell. It is encoded in intron 5 of the ribosomal protein L5 gene, both in chicken and in the two mammals studied so far, human and mouse. U21 RNA is devoid of a 5'-trimethyl-cap and is likely to result from processing of intronic RNA. The nucleolar localization of U21 has been established by fluorescence microscopy after in situ hybridization with digoxigenin-labeled oligonucleotide probes. Like most other snoRNAs U21 contains the box C and box D motifs and is precipitated by anti-fibrillarin antibodies. By the presence of a typical 5'-3' terminal stem, U21 appears more particularly related to U14, U15, U16 and U20 intron-encoded snoRNAs. Remarkably, U21 contains a long stretch (13 nt.) of complementarity to a highly conserved sequence in 28S rRNA. Sequence comparisons between chicken and mammals, together with Northern hybridizations with antisense oligonucleotides on cellular RNAs from more distant vertebrates, point to the preferential preservation of this segment of U21 sequence during evolution. Accordingly, this complementarity, which overlaps the complementarity of 28S rRNA to another snoRNA, U18, could reflect an important role of U21 snoRNA in the biogenesis of large ribosomal subunit.

Animals↗

Evidence for a group II intron in Escherichia coli inserted into a highly conserved reading frame associated with mobile DNA sequences.

The distribution of group II introns in the living world is an important aspect of the hypothesis which postulates their evolutionary relation to the nuclear spliceosome. As an alternative to the restricted experimental approaches towards their identification we devised a strategy to recognize group II introns in sequence data. By this approach we identified a locus on a plasmid in the bacterium Escherichia coli. Modelling of the derived RNA secondary structure reveals the presence of perfectly conserved domains V and VI as typical features of group II introns. An intron internal reading frame upstream of domain V is homologous to group II intron encoded maturases. A reading frame downstream of the predicted 3'-splice site is highly similar to a small polypeptide encoded in the central part of the Agrobacterium tumefaciens T-DNA. With the TBLASTN algorithm a set of plasmid-borne insertion sequences in Agrobacteria and Rhizobia and surprisingly also in a Yersinia pseudotuberculosis strain was identified which contain this highly conserved reading frame.

Amino Acid Sequence↗

Two group I introns with a C.G basepair at the 5' splice-site instead of the very highly conserved U.G basepair: is selection post-translational?

In virtually all of the 200 group I introns sequenced thus far, the specificity of 5' splice-site cleavage is determined by a basepair between a uracil base at the end of the 5' exon and a guanine in an intron guide sequence which pairs with the nucleotides flanking the splice-site. It has been reported that two introns in the cytochrome oxidase subunit I gene of Aspergillus nidulans and Podospora anserina are exceptions to this rule and have a C.G basepair in this position. We have confirmed the initial reports and shown for one of them that RNA editing does not convert the C to a U. Both introns autocatalytically cleave the 5' splice-site. Mutation of the C to U in one intron reduces the requirement for Mg2+ and leads to an increase in the rate of cleavage. As the C base encodes a highly conserved amino acid, we propose that it is selected post-translationally at the level of protein function, despite its inferior splicing activity.

Ascomycota↗

Comparative analysis of splicing of the complete set of chloroplast group II introns in three higher plant mutants.

The barley mutant albostrians and the maize mutants crs1 and crs2 are defective in the splicing of various plastid group II introns. By analysing tRNA precursors and several mRNAs not previously examined, the investigation of in vivo splicing defects in these mutants has been completed. The albostrians mutation causes the loss of plastid ribosomes resulting secondarily in a disruption of splicing of all subgroup IIA introns in the chloroplast. Thus MatK, the only putative chloroplast intron-specific maturase of higher plants, might have evolved to function in splicing of multiple introns. We show that in the case of tRNA-Ala(UGC)the first step of splicing is affected, as suggested by the absence of lariat molecules. Thus the plastid-encoded splicing factor lacking in albostrians must participate in the formation of the catalytically active structure. In contrast, a mutation in the nuclear gene crs1 prevents splicing of only one intron but causes specific additional effects as precursor transcripts for tRNA-Ile(GAU), tRNA-Ala(UGC), tRNA-Lys(UUU)and tRNA-Val(UAC), but not tRNA-Gly(UCC), have significantly enhanced steady-state levels in this mutant. Our data provide evidence for a variety of splicing factors and pathways in the chloroplast, some encoded by nuclear and some by chloroplast genes, and possibly for a dual function of some of these factors.

Base Sequence↗

RecA-independent ectopic transposition in vivo of a bacterial group II intron.

RmInt1 is a group II intron of Sinorhizobium meliloti which was initially found within the insertion sequence ISRm2011-2. Although the RmInt1 intron-encoded protein lacks a recognizable endonuclease domain, it is able to mediate insertion of RmInt1 at an intron-specific location in intronless ISRm2011-2 recipient DNA, a phenomenon termed homing. Here we have characterized three additional insertion sites of RmInt1 in the genome of S.meliloti. Two of these sites are within IS elements closely related to ISRm2011-2, which appear to form a characteristic group within the IS630-Tc1 family. The third site is in the oxi1 gene, which encodes a putative oxide reductase. The newly identified integration sites contain conserved intron-binding site (IBS1 and IBS2) and delta' sequences (14 bp). The RNA of the intron-containing oxi1 gene is able to splice and the oxi1 site is a DNA target for RmInt1 transposition in vivo. Ectopic transposition of RmInt1 into the oxi1 gene occurs at 20-fold lower efficiency than into the homing site (ISRm2011-2) and is independent of the major RecA recombination pathway. The possibility that transposition of RmInt1 to the oxi1 site occurs by reverse splicing into DNA is discussed.

Base Sequence↗

Purification and characterization of the DNA cleavage and recognition site of I-ScaI mitochondrial group I intron encoded endonuclease produced in Escherichia coli.

The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGLIDADG motifs. Genetic and molecular studies have previously shown that this protein has a dual function in the wild-type strain. It acts as a specific homing endonuclease I- Sca I promoting intron mobility and as a maturase promoting intron splicing. Here we describe the synthesis of a universal code equivalent to the mitochondrial sequence coding for this protein and the in vitro characterization of I- Sca I endonuclease activity, using a truncated mutant form of the protein p28bi2 produced in Escherichia coli. We have also determined the cleavage pattern as well as the recognition site of p28bi2. It was found that p28bi2 generates a double-strand cleavage downstream from the intron insertion site with 4 nt long 3'-overhangs. Mutational analysis of the DNA target site shows that p28bi2 recognizes a 16-19 bp sequence from positions -11 to +8 with respect to the intron insertion site.

Amino Acid Sequence↗

Folding of the group I intron ribozyme from the 26S rRNA gene of Candida albicans.

Preincubation of the group I intron Ca.LSU from Candida albicans at 37 degrees C in the absence of divalent cations results in partial folding of this intron. This is indicated by increased resistance to T1 ribonuclease cleavage of many G residues in most local helices, including P4-P6, as well as the non-local helix P7, where the G binding site is located. These changes correlate with increased gel mobility and activation of catalysis by precursor RNA containing this intron after preincubation. The presence of divalent cations or spermidine during preincubation results in formation of the predicted helices, as indicated by protection of additional G residues. However, addition of these cations during preincubation of the precursor RNA alters its gel mobility and eliminates the preincubation activation of precursor RNA seen in the absence of cations. These results suggest that, in the presence of divalent cations or spermidine, Ca.LSU folds into a more ordered, stable but misfolded conformation that is less able to convert into the catalytically active form than the ribozyme preincubated without cations. These results indicate that, like the group I intron of Tetrahymena, multiple folding pathways exist for Ca.LSU. However, it appears that the role cations play in the multiple folding pathways leading to the catalytically active form may differ between folding of these two group I introns.

Base Sequence↗

Characterization of group I introns in generating circular RNAs as vaccines.

Circular RNAs are an increasingly important class of RNA molecules that can be engineered as RNA vaccines and therapeutics. Here, we screened eight different group I introns for their ability to circularize and delineated different features that are important for their function. First, we identified the Scytalidium dimidiatum group I intron as causing minimal innate immune activation inside cells, underscoring its potential to serve as an effective RNA vaccine without triggering unwanted reactogenicity. Additionally, mechanistic RNA structure analysis was used to identify the P9 domain as important for circularization, showing that swapping sequences can restore pairing to improve the circularization of poor circularizers. We also determined the diversity of sequence requirements for the exon 1 and exon 2 (E1 and E2) domains of different group I introns and engineered a S1 tag within the domains for positive purification of circular RNAs. In addition, this flexibility in E1 and E2 enables substitution with less immunostimulatory sequences to enhance protein production. Our work deepens the understanding of the properties of group I introns, expands the panel of introns that can be used, and improves the manufacturing process to generate circular RNAs for vaccines and therapeutics.

RNA, Circular↗

Activation of cryptic 3' splice sites within introns of cellular genes following gene entrapment.

Gene trap vectors developed for genome-wide mutagenesis can be used to study factors governing the expression of exons inserted throughout the genome. For example, entrapment vectors consisting of a partial 3'-terminal exon [i.e. a neomycin resistance gene (Neo), a poly(A) site, but no 3' splice site] were typically expressed following insertion into introns, from cellular transcripts that spliced to cryptic 3' splice sites present either within the targeting vector or in the adjacent intron. A vector (U3NeoSV1) containing the wild-type Neo sequence preferentially disrupted genes that spliced in-frame to a cryptic 3' splice site in the Neo coding sequence and expressed functional neomycin phosphotransferase fusion proteins. Removal of the cryptic Neo 3' splice site did not reduce the proportion of clones with inserts in introns; rather, the fusion transcripts utilized cryptic 3' splice sites present in the adjacent intron or generated by virus integration. However, gene entrapment with U3NeoSV2 was considerably more random than with U3NeoSV1, consistent with the widespread occurrence of potential 3' splice site sequences in the introns of cellular genes. These results clarify the mechanisms of gene entrapment by U3 gene trap vectors and illustrate features of exon definition required for 3' processing and polyadenylation of cellular transcripts.

Animals↗

Functional studies on the ATM intronic splicing processing element.

In disease-associated genes, the understanding of the functional significance of deep intronic nucleotide variants may represent a difficult challenge. We have previously reported a new disease-causing mechanism that involves an intronic splicing processing element (ISPE) in ATM, composed of adjacent consensus 5' and 3' splice sites. A GTAA deletion within ISPE maintains potential adjacent splice sites, disrupts a non-canonical U1 snRNP interaction and activates an aberrant exon. In this paper, we demonstrate that binding of U1 snRNA through complementarity within a approximately 40 nt window downstream of the ISPE prevents aberrant splicing. By selective mutagenesis at the adjacent consensus ISPE splice sites, we show that this effect is not due to a resplicing process occurring at the ISPE. Functional comparison of the ATM mouse counterpart and evaluation of the pre-mRNA splicing intermediates derived from affected cell lines and hybrid minigene assays indicate that U1 snRNP binding at the ISPE interferes with the cryptic acceptor site. Activation of this site results in a stringent 5'-3' order of intron sequence removal around the cryptic exon. Artificial U1 snRNA loading by complementarity to heterologous exonic sequences represents a potential therapeutic method to prevent the usage of an aberrant CFTR cryptic exon. Our results suggest that ISPE-like intronic elements binding U1 snRNPs may regulate correct intron processing.

Animals↗