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PubMed2001

Comparison of intron-containing and intron-lacking human genes elucidates putative exonic splicing enhancers.

Of the rules used by the splicing machinery to precisely determine intron-exon boundaries only a fraction is known. Recent evidence suggests that specific short sequences within exons help in defining these boundaries. Such sequences are known as exonic splicing enhancers (ESE). A possible bioinformatical approach to studying ESE sequences is to compare genes that harbor introns with genes that do not. For this purpose two non-redundant samples of 719 intron-containing and 63 intron-lacking human genes were created. We performed a statistical analysis on these datasets of intron-containing and intron-lacking human coding sequences and found a statistically significant difference (P = 0.01) between these samples in terms of 5-6mer oligonucleotide distributions. The difference is not created by a few strong signals present in the majority of exons, but rather by the accumulation of multiple weak signals through small variations in codon frequencies, codon biases and context-dependent codon biases between the samples. A list of putative novel human splicing regulation sequences has been elucidated by our analysis.

Alternative Splicing↗
PubMed1996

Fugu intron oversize reveals the presence of U15 snoRNA coding sequences in some introns of the ribosomal protein S3 gene.

We present here the analysis of the genomic organization of the Fugu gene coding for ribosomal protein S3 and its intron encoded U15 RNA, and compare it with the homologous human and Xenopus genes. Only two of the six Fugu S3 gene introns do not contain the U15 sequence and are in fact shorter than 100 nucleotides, as most Fugu introns. The other four introns are somewhat longer and contain sequences homologous to U15 RNA; two of these represent functional copies, as shown by microinjections of Fugu transcripts into Xenopus oocytes, whereas the other two appear to be nonfunctional pseudocopies. Thus Fugu turns out to be ideal for the study of intron encoded snoRNAs, partly because of the reduced cloning and sequencing workload, and partly because the intron length per se can be an indication of the presence of a snoRNA coding sequence.

Animals↗
PubMed1987

Variable occurrence of the nrdB intron in the T-even phages suggests intron mobility.

The bacteriophage T4 nrdB gene, encoding nucleoside diphosphate reductase subunit B, contains a self-splicing group I intervening sequence. The nrdB intron was shown to be absent from the genomes of the closely related T-even phages T2 and T6. Evidence for variable intron distribution was provided by autocatalytic 32P-guanosine 5'-triphosphate labeling of T-even RNAs, DNA and RNA hybridization analyses, and DNA sequencing studies. The results indicate the nonessential nature of the intron in nrdB expression and phage viability. Furthermore, they suggest that either precise intron loss from T2 and T6 or lateral intron acquisition by T4 occurred since the evolution of these phages from a common ancestor. Intron movement in the course of T-even phage divergence raises provocative questions about the origin of these self-splicing elements in prokaryotes.

DNA Transposable Elements↗
PubMed1998

A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point.

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3' splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3' splice site. Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5' splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.

Amino Acid Sequence↗
PubMed2001

Polymorphism of PCR-based markers targeting exons, introns, promoter regions, and SSRs in maize and introns and repeat sequences in oat.

Sequence databases could be efficiently exploited for development of DNA markers if it were known which gene regions reveal the most polymorphism when amplified by PCR. We developed PCR primer pairs that target specific regions of previously sequenced genes from Avena and Zea species. Primers were targeted to amplify 40 introns, 24 exons, and 23 promoter regions within 54 maize genes. We surveyed 48 maize inbred lines (previously assayed for simple-sequence repeat (SSR) polymorphism) for amplification-product polymorphism. We also developed primers to target 14 SSRs and 12 introns within 18 Avena genes, and surveyed 22 hexaploid oat cultivars and 2 diploid Avena species for amplification-product polymorphism. In maize, 67% of promoter markers, 58% of intron markers, and 13% of exon markers exhibited amplification-product polymorphisms. Among polymorphic primer pairs in maize, genotype diversity was highest for SSR markers (0.60) followed by intron markers (0.46), exon markers (0.42), and promoter markers (0.28). Among all Avena genotypes, 64% of SSR markers and 58% of intron markers revealed polymorphisms, but among the cultivars only, 21% of SSR markers and 50% of intron markers were polymorphic. Polymorphic-sequence-tagged sites for plant-breeding applications can be created easily by targeting noncoding gene regions.

Avena↗
PubMed1990

Recognition of exon-intron boundaries by the Halobacterium volcanii tRNA intron endonuclease.

The intron-containing tRNA(Trp) precursor from Halobacterium volcanii, like many intron-containing archaebacterial precursor tRNAs, can assume a structure in which the two intron endonuclease cleavage sites are localized in two three-nucleotide loops separated by four base pairs. To investigate the role of this structure in cleavage by the halophilic endonuclease, a series of mutant tRNA(Trp) RNAs were prepared and evaluated as substrates. We find that alterations in this structure result in the loss of cleavage at both 5' and 3' sites. Cleavage of a 35-nucleotide model RNA substrate, containing only these features, demonstrates that sequences and structures present at the exon-intron boundaries are sufficient for recognition and cleavage. We have also examined the mechanism used by the halophilic endonuclease to identify the cleavage sites. Addition of a single base, or a base pair in the anticodon stem above the cleavage sites, does not affect the cleavage site selection. The addition of nucleotides between the two cleavage sites significantly decreases cleavage efficiency and has an effect on the cleavage site selection. These results demonstrate that the halophilic endonuclease requires a defined structure at the exon-intron boundaries and does not identify its cleavage sites by a measurement mechanism like that employed by eukaryotic tRNA intron endonucleases.

Bacterial Proteins↗
PubMed1984

Two intervening sequences in the ATPase subunit 6 gene of Neurospora crassa. A short intron (93 base-pairs) and a long intron that is stable after excision.

A 3590 base-pair region of the mitochondrial genome of Neurospora crassa, including the gene for ATPase subunit 6 (oli2), has been sequenced. The oli2 gene is interrupted by two intervening sequences. The first intron, situated after the third codon of the gene, is 93 base-pairs long; two-thirds of this intron consist of a palindromic sequence. The second intron is 1370 base-pairs long and contains an extended open reading frame that is continuous and in frame with the upstream exon sequence. This intron has structural homology with most other fungal mitochondrial introns. Transcript analysis has yielded a complex pattern of RNA species and demonstrated that the second intron is quite stable after excision. An unknown reading frame (homologous to reading frames of other mitochondrial genomes) is located 1000 base-pairs upstream from the oli2 coding sequence.

Adenosine Triphosphatases↗
PubMed1985

Transposition of an intron in yeast mitochondria requires a protein encoded by that intron.

The optional 1143 bp intron in the yeast mitochondrial 21S rRNA gene (omega +) is nearly quantitatively inserted in genetic crosses into 21S rRNA alleles that lack it (omega -). The intron contains an open reading frame that can encode a protein of 235 amino acids, but no function has been ascribed to this sequence. We previously found an in vivo double-strand break in omega - DNA at or close to the intron insertion site only in zygotes of omega + X omega - crosses that appears with the same kinetics as intron insertion. We now show that mutations in the intron open reading frame that would alter the translation product simultaneously inhibit nonreciprocal omega recombination and the in vivo double-strand break in omega - DNA. These results provide evidence that the open reading frame encodes a protein required for intron transposition and support the role of the double-strand break in the process.

Base Sequence↗
PubMed1984

Intron splicing: a conserved internal signal in introns of animal pre-mRNAs.

Splicing of introns of yeast pre-mRNAs requires an internal conserved sequence T-A-C-T-A-A-C that is located 20-55 nucleotides from the 3' intron boundary. Sequences differing only in certain positions from this yeast signal have now been identified in the corresponding internal region of pre-mRNA introns of a variety of animal genes. A computer program that searches for homologues to a consensus structure and calculates the accuracy of match of each homologue is used to locate these sequences. We list here the signals found by this search in introns of sea urchin, mouse, rat, and human genes and give the consensus for each species. We also give the consensus found for Drosophila and chicken and duck signals. We then discuss the accumulating evidence that these internal signals are required for splicing in animals. It is also noted that a single-stranded region of small nuclear RNA U2 contains sequences complementary both to the proposed mammalian internal signal and to the neighboring CT-A-G at the 3' intron boundary. A role for U2 ribonucleoprotein in intron splicing is thus suggested.

Animals↗
PubMed1985

Splicing of the adenovirus-2 E1A 13S mRNA requires a minimal intron length and specific intron signals.

The adenovirus E1A region encodes three overlapping mRNAs, designated 9S, 12S and 13S. They differ from each other with regard to the length of the intron which is removed by RNA splicing. We have constructed E1A genes with deletions and insertions in the intervening sequence that is common to all three E1A mRNAs, in a search for signals which influence splicing of the 13S mRNA. Mutant plasmids were transfected into HeLa cells and the transiently expressed E1A mRNAs characterized by the S1 protection assay. The results show that five upstream and 20 downstream nucleotides are sufficient to allow for a correct utilization of the 5'-splice junction for the E1A 13S mRNA. Moreover, we show that a minimal intron length of 78 nucleotides is required for efficient 13S mRNA splicing. The ability of mutants with large intron deletions to maturate a 13S mRNA could partially be restored by expanding the intron length with phage lambda sequences. However, in no case was the normal splicing efficiency obtained with these mutants. In contrast, one mutant in which sequences from the authentic 13S mRNA intron were used to expand the intron expressed almost normal levels of 13S mRNA, thus suggesting that signals which specifically promote 13S mRNA splicing exist.

Adenoviruses, Human↗
PubMed1983

Functional domains in introns. RNA processing intermediates in cis- and trans-acting mutants in the penultimate intron of the mitochondrial gene for cytochrome b.

The penultimate intron of the split mitochondrial gene (cob) for apocytochrome b of Saccharomyces cerevisiae is of particular interest; it contains a long unassigned reading frame, is present in both long form (six exons) and short form (three exons) of the gene, and a product expressed from it is required for the removal of its transcript and that of an intron in the transcript of the oxi3 gene. Complementation analysis shows mutants in this intron to be either cis-dominant or transrecessive. Cis-dominant mutants are located in the first third (approximately 350 base pairs) of the open and near the 3'-end of the closed reading frame, while trans-recessive mutants are scattered throughout the remaining two-thirds (approximately 750 base pairs) of the open frame. Mutants in both classes exhibit the same pattern of splicing defects in their transcripts, but for different reasons. Those in the trans-recessive class lack a functional maturase (probably a protein of Mr = 27,000) encoded wholly within the 3'-terminal segment of the intron, and for this reason also fail to express oxi3. In contrast, cis-dominant mutants are incapable of providing the splicing complex with a substrate of appropriate 2 degrees structure. They also accumulate a novel transcript, 1900 nucleotides long, which contains the intron fused to the downstream (3') exons. This may reflect an inability of the splicing complex to complete the normal sequence of cleavage of the intron at its downstream junction and the ligation of the two exonic moieties.

Base Composition↗
PubMed1993

Interaction of intronic boundaries is required for the second splicing step efficiency of a group II intron.

Group II and nuclear pre-mRNAs introns share a common splicing pathway involving a lariat intermediate, as well as some primary sequence similarities at the splice junctions. In this work, we analyze the role of the conserved nucleotides at the first and penultimate positions (G1 and A886) of a group II self-splicing intron. We show that the G1 nucleotide is essential for the efficiency of both the first and the second splicing steps, while substitutions at the penultimate nucleotide affect mostly the efficiency of the second step. A reciprocal suppression of the second splicing step defect can be observed in some double mutants. This result is best explained by a non-Watson-Crick interaction between the first and the penultimate nucleotides of the intron, which occurs after lariat formation. The finding that an interaction between intron boundaries is required for the second splicing step in both group II and nuclear pre-mRNA introns strengthens the idea that both systems employ similar mechanisms, albeit with differences in the details of the nucleotide interactions.

Base Sequence↗
PubMed2004

Splicing of a cap-proximal human Papillomavirus 16 E6E7 intron promotes E7 expression, but can be restrained by distance of the intron from its RNA 5' cap.

Human papillomavirus 16 (HPV16) E6E7 pre-mRNA is bicistronic and has an intron in the E6 coding region with one 5' splice site and two alternative 3' splice sites, which produce E6(*)I and E6(*)II, respectively. If this intron remains unspliced, the resulting E6E7 mRNA expresses oncogenic E6. We found for the first time that the E6E7 pre-mRNA was efficiently spliced in vitro only when capped and that cellular cap-binding factors were involved in the splicing. The cap-dependent splicing of the E6E7 pre-mRNA was extremely efficient in cervical cancer-derived cells, producing mostly E6(*)I, but inefficient in cells transfected with a common retrovirus expression vector, pLXSN16E6E7, due to the large size of this vector's exon 1. Further studies showed that efficient splicing of the E6E7 pre-mRNA depends on the distance of the cap-proximal intron from the RNA 5' cap, with an optimal distance of less than 307nt in order to facilitate better association of U1 small nuclear RNA with the intron 5' splice site. The same was true for splicing of human beta-globin RNA. Splicing of the E6E7 RNA provided more E7 RNA templates and promoted E7 translation, whereas a lack of RNA splicing produced a low level of E7 translation. Together, our data indicate that the distance between the RNA 5' cap and cap-proximal intron is rate limiting for RNA splicing. HPV16 E6E7 pre-mRNA takes advantage of its small cap-proximal exon to confer efficient splicing for better E7 expression.

3' Flanking Region↗
PubMed1997

Properties of H. volcanii tRNA intron endonuclease reveal a relationship between the archaeal and eucaryal tRNA intron processing systems.

To better understand the relationship between archaeal and eucaryal tRNA introns and their processing systems, we have cloned the gene encoding the tRNA intron endonucleases from the archaeon H. volcanii. The gene encodes a 37 kDa protein that appears to be present as a homodimer under native conditions. Recombinant forms of this protein were expressed in E. coli and found to cleave precursor tRNAs lacking full mature tRNA structure, a property observed for the native endonuclease. Comparative sequence analysis revealed that similar proteins existed in other Archaea and that these proteins have significant similarity with two subunits of the yeast tRNA intron endonuclease. These results provide evidence that the archaeal and eucaryal tRNA intron processing systems are related and suggest a common origin for tRNA introns in these organisms.

Archaea↗
PubMed1998

U-richness is a defining feature of plant introns and may function as an intron recognition signal in maize.

Using a large set of plant gene sequences we compared individual introns to their flanking exons. Both Zea mays and Arabidopsis thaliana introns are U-rich but display no apparent bias for A. We identified fifteen 11-mer U-rich motifs as frequent elements of maize introns, and these are virtually absent from exons. By mutagenesis, we show that the single U-rich motif in the Bronze2 intron of maize plays a key role in intron processing in vivo.

Base Composition↗
PubMed2004

Alternative polyadenylation of adeno-associated virus type 5 RNA within an internal intron is governed by the distance between the promoter and the intron and is inhibited by U1 small nuclear RNP binding to the intervening donor.

Adeno-associated virus type 5 is unique among adeno-associated virus serotypes in that it uses a polyadenylation site in the center of the genome. The great majority of transcripts generated from the upstream P7 and P19 promoters are polyadenylated at a site in the central intron ((pA)p); however, most of the viral transcripts generated by the proximal P41 promoter are polyadenylated at the distal polyadenylation site at the 3' end of the genome (pA)d and subsequently spliced. Polyadenylation at (pA)p increases as the distance between the RNA initiation site and the intron and (pA)p site is increased. The steady-state level of RNAs polyadenylated at (pA)p is independent of the promoter used or of the intervening sequence but is dependent upon competition with splicing, inhibition by U1 snRNP binding to the intron donor, and the intrinsic efficiency of the cleavage/polyadenylation reaction. Each of these determinants shows a marked dependence on the distance between the RNA initiation site and the intron and (pA)p. Finally, unlike other reported systems, inhibition of (pA)p by U1 snRNP binding to the intron donor is decreased as the distance between the donor and (pA)p is increased.

Alternative Splicing↗
PubMed1999

In vivo expression of the nucleolar group I intron-encoded I-dirI homing endonuclease involves the removal of a spliceosomal intron.

The Didymium iridis DiSSU1 intron is located in the nuclear SSU rDNA and has an unusual twin-ribozyme organization. One of the ribozymes (DiGIR2) catalyses intron excision and exon ligation. The other ribozyme (DiGIR1), which along with the endonuclease-encoding I-DirI open reading frame (ORF) is inserted in DiGIR2, carries out hydrolysis at internal processing sites (IPS1 and IPS2) located at its 3' end. Examination of the in vivo expression of DiSSU1 shows that after excision, DiSSU1 is matured further into the I-DirI mRNA by internal DiGIR1-catalysed cleavage upstream of the ORF 5' end, as well as truncation and polyadenylation downstream of the ORF 3' end. A spliceosomal intron, the first to be reported within a group I intron and the rDNA, is removed before the I-DirI mRNA associates with the polysomes. Taken together, our results imply that DiSSU1 uses a unique combination of intron-supplied ribozyme activity and adaptation to the general RNA polymerase II pathway of mRNA expression to allow a protein to be produced from the RNA polymerase I-transcribed rDNA.

Amoeba↗