Protein splicing: selfish genes invade cellular proteins

Protein splicing is a series of enzymatic events involving intramolecular protein breakage, rejoining and intron homing, in which introns are able to promote the recombinative transposition of their own coding sequences. Eukaryotic and prokaryotic spliced proteins have conserved similar gene structure, but little amino acid identity. The genes coding for these spliced proteins contain internal in-frame introns that encode polypeptides that apparently self-excise from the resulting host protein sequences. Excision of the ‘protein intron’ is coupled with joining of the two flanking protein regions encoded by exons of the host gene. Some introns of this type encode DNA endonucleases, related to Group I RNA intron gene products, that stimulate gene conversion and self-transmission.     

Discovery of a group I intron in the SSU rDNA of Ploeotia costata (Euglenozoa)

The gene coding for the small ribosomal subunit RNA of Ploeotia costata contains an actively splicing group I intron (Pco.S516) which is unique among euglenozoans. Secondary structure predictions indicate that paired segments P1-P10 as well as several conserved elements typical of group I introns and of subclass IC1 in particular are present. Phylogenetic analyses of SSU rDNA sequences demonstrate a well-supported placement of Ploeotia costata within the Euglenozoa; whereas, analyses of intron data sets uncover a close phylogenetic relation of Pco.S516 to S-516 introns from Acanthamoeba, Aureoumbra lagunensis (Stramenopila) and red algae of the order Bangiales. Discrepancies between SSU rDNA and intron phylogenies suggest horizontal spread of the group I intron. Monophyly of IC1 516 introns from Ploeotia costata, A. lagunensis and rhodophytes is supported by a unique secondary structure element: helix P5b possesses an insertion of 19 nt length with a highly conserved tetraloop which is supposed to take part in tertiary interactions. Neither functional nor degenerated ORFs coding for homing endonucleases can be identified in Pco.S516. Nevertheless, degenerated ORFs with His-Cys box motifs in closely related intron sequences indicate that homing may have occurred during evolution of the investigated intron group.     

The Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene. I. Complete DNA sequence and analysis of the nine intervening sequences

The nucleotide sequence of 6225 base pairs (bp) of Euglena gracilis chloroplast DNA including the complete DNA sequence of the chloroplast-encoded ribulose-1,5-bisphosphate carboxylase large subunit gene along with the flanking DNA sequences is presented. The gene is greater than 5.5 kilobase pairs in length and is organized as 10 exons coding for 475 amino acids, separated by 9 introns. The exons range in size from 45 to 438 bp, while the introns range in size from 382 to 568 bp. The introns have highly conserved boundary sequences with the consensus, 5'-N GTGTGGATTT...(intron)...TTAATTTTAT N-3'. The introns are 82-85 mol% AT, with a pronounced T greater than A greater than G greater than C base bias in the RNA-like strand. They do not appear to encode any polypeptides. In addition, the introns have a conserved sequence 30-50 bp from their 3'-ends with the consensus, 5'-TACAGTTTGAAAATGA-3'. The 5'-TACA sequence bears some homology to the 5'-end of the TACTAACA sequence found in a similar location in yeast nuclear mRNA introns. The conserved sequences of the Euglena rbcL introns may be indicative of a splicing mechanism similar to that of eucaryotic nuclear mRNA introns and group II mitochondrial introns.     

Evolutionary implications of intron–exon distribution and the properties and sequences of the RPL10A gene in eukaryotes

The RPL10A gene encodes the RPL10 protein, required for joining 40S and 60S subunits into a functional 80S ribosome. This highly conserved gene, ubiquitous across all eukaryotic super-groups, is characterized by a variable number of spliceosomal introns, present in most organisms. These properties facilitate the recognition of orthologs among distant taxa and thus comparative studies of sequences as well as the distribution and properties of introns in taxonomically distant groups of eukaryotes. The present study examined the multiple ways in which RPL10A conservation vs. sequence changes in the gene over the course of evolution, including in exons, introns, and the encoded proteins, can be exploited for evolutionary analysis at different taxonomic levels. At least 25 different positions harboring introns within the RPL10A gene were determined in different taxa, including animals, plants, fungi, and alveolates. Generally, intron positions were found to be well conserved even across different kingdoms. However, certain introns seemed to be restricted to specific groups of organisms. Analyses of several properties of introns, including insertion site, phase, and length, along with exon and intron GC content and exon–intron boundaries, suggested biases within different groups of organisms. The use of a standard primer pair to analyze a portion of the intron-containing RPL10A gene in 12 genera of green algae within Chlorophyta is presented as a case study for evolutionary analyses of introns at intermediate and low taxonomic levels. Our study shows that phylogenetic reconstructions at different depths can be achieved using RPL10A nucleotide sequences from both exons and introns as well as the amino acid sequences of the encoded protein.     

Exon junction sequences as cryptic splice sites: implications for intron origin

Introns are flanked by a partially conserved coding sequence that forms the immediate exon junction sequence following intron removal from pre-mRNA. Phylogenetic evidence indicates that these sequences have been targeted by numerous intron insertions during evolution, but little is known about this process. Here, we test the prediction that exon junction sequences were functional splice sites that existed in the coding sequence of genes prior to the insertion of introns. To do this, we experimentally identified nine cryptic splice sites within the coding sequence of actin genes from humans, Arabidopsis, and Physarum by inactivating their normal intron splice sites. We found that seven of these cryptic splice sites correspond exactly to the positions of exon junctions in actin genes from other species. Because actin genes are highly conserved, we could conclude that at least seven actin introns are flanked by cryptic splice sites, and from the phylogenetic evidence, we could also conclude that actin introns were inserted into these cryptic splice sites during evolution. Furthermore, our results indicate that these insertion events were dependent upon the splicing machinery. Because most introns are flanked by similar sequences, our results are likely to be of general relevance.     

Bioinformatics analysis of plant orthologous introns: identification of an intronic tRNA-like sequence

Orthologous introns have identical positions relative to the coding sequence in orthologous genes of different species. By analyzing the complete genomes of five plants we generated a database of 40,512 orthologous intron groups of dicotyledonous plants, 28,519 orthologous intron groups of angiosperms, and 15,726 of land plants (moss and angiosperms). Multiple sequence alignments of each orthologous intron group were obtained using the Mafft algorithm. The number of conserved regions in plant introns appeared to be hundreds of times fewer than that in mammals or vertebrates. Approximately three quarters of conserved intronic regions among angiosperms and dicots, in particular, correspond to alternatively-spliced exonic sequences. We registered only a handful of conserved intronic ncRNAs of flowering plants. However, the most evolutionarily conserved intronic region, which is ubiquitous for all plants examined in this study, including moss, possessed multiple structural features of tRNAs, which caused us to classify it as a putative tRNA-like ncRNA. Intronic sequences encoding tRNA-like structures are not unique to plants. Bioinformatics examination of the presence of tRNA inside introns revealed an unusually long-term association of four glycine tRNAs inside the Vac14 gene of fish, amniotes, and mammals.     

Protein Polymorphism Is Negatively Correlated with Conservation of Intronic Sequences and Complexity of Expression Patterns in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We report a significant negative correlation between nonsynonymous polymorphism and intron length in Drosophila melanogaster. This correlation is similar to that between protein divergence and intron length previously reported in Drosophila. We show that the relationship can be explained by the content of conserved noncoding sequences (CNS) within introns. In addition, genes with a high regulatory complexity and many genetic interactions also exhibit larger amounts of CNS within their introns and lower values of nonsynonymous polymorphism. The present study provides relevant evidence on the importance of intron content and expression patterns on the levels of coding polymorphism. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. Dmitri Petrov]     

A large multigene family codes for the polypeptides of the crystalline trichocyst matrix in Paramecium.

The secretory granules (trichocysts) of Paramecium are characterized by a highly constrained shape that reflects the crystalline organization of their protein contents. Yet the crystalline trichocyst content is composed not of a single protein but of a family of related polypeptides that derive from a family of precursors by protein processing. In this paper we show that a multigene family, of unusually large size for a unicellular organism, codes for these proteins. The family is organized in subfamilies; each subfamily codes for proteins with different primary structures, but within the subfamilies several genes code for nearly identical proteins. For one subfamily, we have obtained direct evidence that the different members are coexpressed. The three subfamilies we have characterized are located on different macronuclear chromosomes. Typical 23-29 nucleotide Paramecium introns are found in one of the regions studied and the intron sequences are more variable than the surrounding coding sequences, providing gene-specific markers. We suggest that this multigene family may have evolved to assure a microheterogeneity of structural proteins necessary for morphogenesis of a complex secretory granule core with a constrained shape and dynamic properties: genetic analysis has shown that correct assembly of the crystalline core is necessary for trichocyst function.     

A Group I Intron in the 18S Ribosomal DNA from the Parasitic Fungus Isaria japonica

The nucleotide sequence of the 18S rDNA coding gene in the ascomycetes parasitic fungus Isaria japonica contains a group I intron with a length of 379 nucleotides. The identification of the DNA sequence as a group I intron is based on its sequence homology to other fungal group I introns. Its group I intron contained the highly conserved sequence elements P, Q, R, and S found in other group I introns. Surprisingly, the intron sequence of I. japonica is more similar to that of Ustilago maydis than to the one found in Sclerotinia sclerotiorum. This is in contrast to the sequence identity found on the neighboring rDNA. This is an interesting finding and suggests a horizontal transfer of group I intron sequences. Received: 19 September 1997 / Accepted: 10 September 1998     

Phylogenetic and Exon–Intron Structure Analysis of Fungal Subtilisins: Support for a Mixed Model of Intron Evolution

Phylogenetic and exon–intron structure analyses of intra- and interspecific fungal subtilisins in this study provided support for a mixed model of intron evolution: a synthetic theory of introns-early and introns-late speculations. Intraspecifically, there were three phase zero introns in Pr1A and its introns 1 and 2 located at the highly conserved positions were phylogentically congruent with coding region, which is in favor of the view of introns-early speculation, while intron 3 had two different sizes and was evolutionarily incongruent with coding region, the evidence for introns-late speculation. Noticeably, the subtilisin Pr1J gene from different strains of M. ansiopliae contained different number of introns, the strong evidence in support of introns-late theory. Interspecifically, phylogenetic analysis of 60 retrievable fungal subtilisins provided a clear relationship between amino acid sequence and gene exon–intron structure that the homogeneous sequences usually have a similar exon–infron structure. There were 10 intron positions inserted by highly biased phase zero introns across examined fungal subtilisin genes, half of these positions were highly conserved, while the others were species-specific, appearing to be of recent origins due to intron insertion, in favor of the introns-late theory. High conservations of positions 1 and 2 inserted by the high percentage of phase zero introns as well as the evidence of phylogenetic congruence between the evolutionary histories of intron sequences and coding region suggested that the introns at these two positions were primordial.Reviewing Editor:Dr. Manyuan Long     

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. The cytochrome b gene has an intron closely related to the first two introns in the Saccharomyces cerevisiae cox1 gene

The DNA sequence of the cob region of the Schizosaccharomyces pombe mitochondrial DNA has been determined. The cytochrome b structural gene is interrupted by an intron of 2526 base-pairs, which has an open reading frame of 2421 base-pairs in phase with the upstream exon. The position of the intron differs from those found in the cob genes of Saccharomyces cerevisiae, Aspergillus nidulans or Neurospora crassa. The Sch. pombe cob intron has the potential of assuming an RNA secondary structure almost identical to that proposed for the first two cox1 introns (group II) in S. cerevisiae and the p1-cox1 intron in Podospora anserina. It has most of the consensus nucleotides in the central core structure described for this group of introns and its comparison with other group II introns allows the identification of an additional conserved nucleotide stretch. A comparison of the predicted protein sequences of group II intronic coding regions reveals three highly conserved blocks showing pairwise amino acid identities of 34 to 53%. These regions comprise over 50% of the coding length of the intron but do not include the 5' region, which has strong secondary structural features. In addition to the potential intron folding, long helical structures involving repetitive sequences can be formed in the flanking cob exon regions. A comparison of the Sch. pombe cytochrome b sequence with those available from other organisms indicates that Sch. pombe is evolutionarily distant from both budding yeasts and filamentous fungi. As was seen for the Sch. pombe cox1 gene (Lang, 1984), the cob exons are translated using the universal genetic code and this distinguishes Sch. pombe mitochondria from all other fungal and animal mitochondrial systems.     

Nucleotide sequence and intron structure of the apocytochrome b gene of Neurospora crassa mitochondria

The sequence of the apocytochrome b (cob) gene of Neurospora crassa has been determined. The structural gene is interrupted by two intervening sequences of approximately 1260 bp each. The polypeptide encoded by the exons shows extensive homology with the cob proteins of Aspergillus nidulans and Saccharomyces cerevisiae (79% and 60%, respectively). The two introns are, however, located at sites different from those of introns in the cob genes of A. nidulans and S. cerevisiae (which contain highly homologous introns at the same site within the gene). The introns share several short regions of sequence homology (10-12 bp long) with each other and with other fungal mitochondrial introns. Moreover, the second intron contains a 50 nucleotide long sequence that is highly homologous with sequences within every ribosomal intron of fungal mitochondria sequenced to date. The conserved sequences may allow the formation of a core secondary structure, which is nearly identical in many mitochondrial introns. The conserved secondary structure may be required for intron splicing. The second intron contains an open reading frame, continuous with the preceding exon, of approximately 290 codons. Two stretches of 10 amino acid residues, conserved in many introns, are present in the open reading frame.     

Architecture and anatomy of the chromosomal locus in human chromosome 21 encoding the Cu/Zn superoxide dismutase.

The SOD-1 gene on chromosome 21 and approximately 100 kb of chromosomal DNA from the 21q22 region have been isolated and characterized. The gene which is present as a single copy per haploid genome spans 11 kb of chromosomal DNA. Heteroduplex analysis and DNA sequencing reveals five rather small exons and four introns that interrupt the coding region. The donor sequence at the first intron contains an unusual variant dinucleotide 5'-G-C, rather than the highly conserved 5'-GT. The unusual splice junction is functional in vivo since it was detected in both alleles of the SOD-1 gene, which were defined by differences in the length of restriction endonuclease fragments (RFLPs) that hybridize to the cDNA probe. Genomic blots of human DNA isolated from cells trisomic for chromosome 21 (Down's syndrome patients) show the normal pattern of bands. At the 5' end of gene there are the 'TATA' and 'CAT' promoter sequences as well as four copies of the -GGCGGG- hexanucleotide. Two of these -GC- elements are contained within a 13 nucleotide inverted repeat that could form a stem-loop structure with stability of -33 kcal. The 3'-non coding region of the gene contains five short open reading-frames starting with ATG and terminating with stop codons.