Incipient mitochondrial evolution in yeasts. I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae

We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii. Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S. douglasii mitochondrial genome. The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient. The mitochondrial genome of S. douglasii displays many interesting features in comparison with that of S. cerevisiae. The three mosaic genes present in both genomes are quite different with regard to their structure. The S. douglasii COXI gene has two new introns and is missing the five introns of the S. cerevisiae gene. The S. douglasii cytochrome b gene has one new intron and lacks two introns of the S. cerevisiae gene. Finally, the L-rRNA gene of S. douglasii, like that of S. cerevisiae, has one intron of which the structure is different. Another salient feature of the S. douglasii mitochondrial genome reported here is that the gene order is different in comparison with S. cerevisiae mitochondrial DNA. In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.     

The intron of tRNA-TrpCCA is dispensable for growth and translation of Saccharomyces cerevisiae

A part of eukaryotic tRNA genes harbor an intron at one nucleotide 3' to the anticodon, so that removal of the intron is an essential processing step for tRNA maturation. While some tRNA introns have important roles in modification of certain nucleotides, essentiality of the tRNA intron in eukaryotes has not been tested extensively. This is partly because most of the eukaryotic genomes have multiple genes encoding an isoacceptor tRNA. Here, we examined whether the intron of tRNA-Trp(CCA) genes, six copies of which are scattered on the genome of yeast, Saccharomyces cerevisiae, is essential for growth or translation of the yeast in vivo. We devised a procedure to remove all of the tRNA introns from the yeast genome iteratively with marker cassettes containing both positive and negative markers. Using this procedure, we removed all the introns from the six tRNA-Trp(CCA) genes, and found that the intronless strain grew normally and expressed tRNA-Trp(CCA) in an amount similar to that of the wild-type genes. Neither incorporation of (35)S-labeled amino acids into a TCA-insoluble fraction nor the major protein pattern on SDS-PAGE/2D gel were affected by complete removal of the intron, while expression levels of some proteins were marginally affected. Therefore, the tRNA-Trp(CCA) intron is dispensable for growth and bulk translation of the yeast. This raises the possibility that some mechanism other than selective pressure from translational efficiency maintains the tRNA intron on the yeast genome.     

Evolution of small nuclear RNAs in S. cerevisiae, C. albicans, and other hemiascomycetous yeasts

The spliceosome is a large, dynamic ribonuclear protein complex, required for the removal of intron sequences from newly synthesized eukaryotic RNAs. The spliceosome contains five essential small nuclear RNAs (snRNAs): U1, U2, U4, U5, and U6. Phylogenetic comparisons of snRNAs from protists to mammals have long demonstrated remarkable conservation in both primary sequence and secondary structure. In contrast, the snRNAs of the hemiascomycetous yeast Saccharomyces cerevisiae have highly unusual features that set them apart from the snRNAs of other eukaryotes. With an emphasis on the pathogenic yeast Candida albicans, we have now identified and compared snRNAs from newly sequenced yeast genomes, providing a perspective on spliceosome evolution within the hemiascomycetes. In addition to tracing the origins of previously identified snRNA variations present in Saccharomyces cerevisiae, we have found numerous unexpected changes occurring throughout the hemiascomycetous lineages. Our observations reveal interesting examples of RNA and protein coevolution, giving rise to altered interaction domains, losses of deeply conserved snRNA-binding proteins, and unique snRNA sequence changes within the catalytic center of the spliceosome. These same yeast lineages have experienced exceptionally high rates of intron loss, such that modern hemiascomycetous genomes contain introns in only approximately 5% of their genes. Also, the splice site sequences of those introns that remain adhere to an unusually strict consensus. Some of the snRNA variations we observe may thus reflect the altered intron landscape with which the hemiascomycetous spliceosome must contend.     

Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation.

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.     

Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns

As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5′ end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns’ features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts.     

Intron-loss evolution of hatching enzyme genes in Teleostei

Background  

Hatching enzyme, belonging to the astacin metallo-protease family, digests egg envelope at embryo hatching. Orthologous genes of the enzyme are found in all vertebrate genomes. Recently, we found that exon-intron structures of the genes were conserved among tetrapods, while the genes of teleosts frequently lost their introns. Occurrence of such intron losses in teleostean hatching enzyme genes is an uncommon evolutionary event, as most eukaryotic genes are generally known to be interrupted by introns and the intron insertion sites are conserved from species to species. Here, we report on extensive studies of the exon-intron structures of teleostean hatching enzyme genes for insight into how and why introns were lost during evolution.     

Ⅱ组内含子(Group Ⅱ Intron)的分布及多样性

Ⅱ组内含子(group Ⅱ intron)存在于原生生物、真菌、藻类、植物细胞器以及细菌和古细菌基因组中.在体内,Ⅱ组内含子可通过两步连续的转酯反应从前体RNA中自剪接,并连接两 侧外显子.许多Ⅱ组内含子的剪接反应是由蛋白质辅助完成的,这种蛋白质有的是由内含子编码,有的是由宿主基因编码.Ⅱ组内含子能够有效地归巢进入无内含子的等位基因,也能 够以低频率逆转座进入非等位基因.转座过程依赖内含子RNA和内含子编码的蛋白质（内切核酸酶活性和逆转录酶活性）.本论文在总结Ⅱ组内含子最新研究成果的基础上,分析Ⅱ组内含子可能的起源和进化途径     

Intron–Genome Size Relationship on a Large Evolutionary Scale

The intron–genome size relationship was studied across a wide evolutionary range (from slime mold and yeast to human and maize), as well as the relationship between genome size and the ratio of intervening/coding sequence size. The average intron size is scaled to genome size with a slope of about one-fourth for the log-transformed values; i.e., on the global scale its increase in evolution is lower than the increase in genome size by four orders of magnitude. There are exceptions to the general trend. In baker's yeast introns are extraordinarily long for its genome size. Tetrapods also have longer introns than expected for their genome sizes. In teleost fish the mean intron size does not differ significantly, notwithstanding the differences in genome size. In contrast to previous reports, avian introns were not found to be significantly shorter than introns of mammals, although avian genomes are smaller than genomes of mammals on average by about a factor of 2.5. The extra-/intragenic ratio of noncoding DNA can be higher in fungi than in animals, notwithstanding the smaller fungal genomes. In vertebrates and invertebrates taken separately, this ratio is increasing as the increase in genome size. Two hypotheses are proposed to explain the variation in the extra-/intragenic ratio of noncoding DNA in organisms with similar numbers of genes: transition (dynamic) and equilibrium (static). According to the transition model, this variation arises with the rapid shift of genome size because the bulk of extragenic DNA can be changed more rapidly than the finely interspersed intron sequences. The equilibrium model assumes that this variation is a result of selective adjustment of genome size with constraints imposed on the intron size due to its putative link to chromatin structure (and constraints of the splicing machinery). Received: 23 October 1997 / Accepted: 14 April 1999     

Remarkable interkingdom conservation of intron positions and massive,lineage-specific intron loss and gain in eukaryotic evolution

Sequencing of eukaryotic genomes allows one to address major evolutionary problems, such as the evolution of gene structure. We compared the intron positions in 684 orthologous gene sets from 8 complete genomes of animals, plants, fungi, and protists and constructed parsimonious scenarios of evolution of the exon-intron structure for the respective genes. Approximately one-third of the introns in the malaria parasite Plasmodium falciparum are shared with at least one crown group eukaryote; this number indicates that these introns have been conserved through >1.5 billion years of evolution that separate Plasmodium from the crown group. Paradoxically, humans share many more introns with the plant Arabidopsis thaliana than with the fly or nematode. The inferred evolutionary scenario holds that the common ancestor of Plasmodium and the crown group and, especially, the common ancestor of animals, plants, and fungi had numerous introns. Most of these ancestral introns, which are retained in the genomes of vertebrates and plants, have been lost in fungi, nematodes, arthropods, and probably Plasmodium. In addition, numerous introns have been inserted into vertebrate and plant genes, whereas, in other lineages, intron gain was much less prominent.     

Nonsense-Mediated Decay Enables Intron Gain in Drosophila

Intron number varies considerably among genomes, but despite their fundamental importance, the mutational mechanisms and evolutionary processes underlying the expansion of intron number remain unknown. Here we show that Drosophila, in contrast to most eukaryotic lineages, is still undergoing a dramatic rate of intron gain. These novel introns carry significantly weaker splice sites that may impede their identification by the spliceosome. Novel introns are more likely to encode a premature termination codon (PTC), indicating that nonsense-mediated decay (NMD) functions as a backup for weak splicing of new introns. Our data suggest that new introns originate when genomic insertions with weak splice sites are hidden from selection by NMD. This mechanism reduces the sequence requirement imposed on novel introns and implies that the capacity of the spliceosome to recognize weak splice sites was a prerequisite for intron gain during eukaryotic evolution.     

The intron of the mitochondrial 21S rRNA gene: distribution in different yeast species and sequence comparison between Kluyveromyces thermotolerans and Saccharomyces cerevisiae

We have screened numerous different yeast species for the presence of sequences homologous to the intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (intron r1) and found them in all Kluyveromyces species, some of the Saccharomyces species and none of the other yeasts tested. We have determined the nucleotide sequence of the r1-intron in K. thermotolerans and compared it with that of S. cerevisiae. The two introns are inserted at the same position within the 21S rRNA gene. They contain homologous internal open reading frames (ORFs) initiated at the same AUG codon which can be aligned over their entire length. Several silent multi-substitutions indicate that these intronic ORFs represent selectively conserved functional genes. Other intron segments, on the contrary, reveal short blocks of extensive homology separated by non-homologous stretches and/or additions-deletions. Comparison of our two yeast r1-introns with equivalent introns of N. crassa and A. nidulans mitochondria reveals that introns with very similar RNA secondary structures can accommodate different types of ORFs.     

Analysis of ribosomal protein gene structures: implications for intron evolution

Many spliceosomal introns exist in the eukaryotic nuclear genome. Despite much research, the evolution of spliceosomal introns remains poorly understood. In this paper, we tried to gain insights into intron evolution from a novel perspective by comparing the gene structures of cytoplasmic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs), which are held to be of archaeal and bacterial origin, respectively. We analyzed 25 homologous pairs of CRP and MRP genes that together had a total of 527 intron positions. We found that all 12 of the intron positions shared by CRP and MRP genes resulted from parallel intron gains and none could be considered to be “conserved,” i.e., descendants of the same ancestor. This was supported further by the high frequency of proto-splice sites at these shared positions; proto-splice sites are proposed to be sites for intron insertion. Although we could not definitively disprove that spliceosomal introns were already present in the last universal common ancestor, our results lend more support to the idea that introns were gained late. At least, our results show that MRP genes were intronless at the time of endosymbiosis. The parallel intron gains between CRP and MRP genes accounted for 2.3% of total intron positions, which should provide a reliable estimate for future inferences of intron evolution.     

Test of intron predictions reveals novel splice sites, alternatively spliced mRNAs and new introns in meiotically regulated genes of yeast

Correct identification of all introns is necessary to discern the protein-coding potential of a eukaryotic genome. The existence of most of the spliceosomal introns predicted in the genome of Saccharomyces cerevisiae remains unsupported by molecular evidence. We tested the intron predictions for 87 introns predicted to be present in non-ribosomal protein genes, more than a third of all known or suspected introns in the yeast genome. Evidence supporting 61 of these predictions was obtained, 20 predicted intron sequences were not spliced and six predictions identified an intron-containing region but failed to specify the correct splice sites, yielding a successful prediction rate of <80%. Alternative splicing has not been previously described for this organism, and we identified two genes (YKL186C/MTR2 and YML034W) which encode alternatively spliced mRNAs; YKL186C/MTR2 produces at least five different spliced mRNAs. One gene (YGR225W/SPO70) has an intron whose removal is activated during meiosis under control of the MER1 gene. We found eight new introns, suggesting that numerous introns still remain to be discovered. The results show that correct prediction of introns remains a significant barrier to understanding the structure, function and coding capacity of eukaryotic genomes, even in a supposedly simple system like yeast.     

Updated Progress on Group II Intron Splicing Factors in Plant Chloroplasts

Group II introns are large catalytic RNAs (ribozymes) in the bacteria and organelle genomes of several lower eukaryotes. Many critical photosynthesis-related genes in the plant chloroplast genome also contain group II introns, and their splicing is critical for chloroplast biogenesis and photosynthesis processes. The structure of chloroplast group II introns was altered during evolution, resulting in the loss of intron self-splicing. Therefore, the assistance of protein factors was required for their splicing processes. As an increasing number of studies focus on the mechanism of chloroplast intron splicing; many new nuclear-encoded splicing factors that are involved in the chloroplast intron splicing process have been reported. This report reviewed the research progress of the updated splicing factors found to be involved in the splicing of chloroplast group II introns. We discuss the main problems that remain in this research field and suggest future research directions.     

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.     

A very high fraction of unique intron positions in the intron-rich diatom Thalassiosira pseudonana indicates widespread intron gain

Although spliceosomal introns are present in all characterized eukaryotes, intron numbers vary dramatically, from only a handful in the entire genomes of some species to nearly 10 introns per gene on average in vertebrates. For all previously studied intron-rich species, significant fractions of intron positions are shared with other widely diverged eukaryotes, indicating that 1) large numbers of the introns date to much earlier stages of eukaryotic evolution and 2) these lineages have not passed through a very intron-poor stage since early eukaryotic evolution. By the same token, among species that have lost nearly all of their ancestral introns, no species is known to harbor large numbers of more recently gained introns. These observations are consistent with the notion that intron-dense genomes have arisen only once over the course of eukaryotic evolution. Here, we report an exception to this pattern, in the intron-rich diatom Thalassiosira pseudonana. Only 8.1% of studied T. pseudonana intron positions are conserved with any of a variety of divergent eukaryotic species. This implies that T. pseudonana has both 1) lost nearly all of the numerous introns present in the diatom-apicomplexan ancestor and 2) gained a large number of new introns since that time. In addition, that so few apparently inserted T. pseudonana introns match the positions of introns in other species implies that insertion of multiple introns into homologous genic sites in eukaryotic evolution is less common than previously estimated. These results suggest the possibility that intron-rich genomes may have arisen multiple times in evolution. These results also provide evidence that multiple intron insertion into the same site is rare, further supporting the notion that early eukaryotic ancestors were very intron rich.     

Mitochondrial-encoded endonucleases drive recombination of protein-coding genes in yeast

Mitochondrial recombination in yeast is well recognized, yet the underlying genetic mechanisms are not well understood. Recent progress has suggested that mobile introns in mitochondrial genomes (mitogenomes) can facilitate the recombination of their corresponding intron-containing genes through a mechanism known as intron homing. As many mitochondrial genes lack introns, there is a critical need to determine the extent of recombination and underlying mechanism of intron-lacking genes. This study leverages yeast mitogenomes to address these questions. In Saccharomyces cerevisiae, the 3′-end sequences of at least three intron-lacking mitochondrial genes exhibit elevated nucleotide diversity and recombination hotspots. Each of these 3′-end sequences is immediately adjacent to or even fused as overlapping genes with a stand-alone endonuclease. Our findings suggest that SAEs are responsible for recombination and elevated diversity of adjacent intron-lacking genes. SAEs were also evident to drive recombination of intron-lacking genes in Lachancea kluyveri, a yeast species that diverged from S. cerevisiae more than 100 million years ago. These results suggest SAEs as a common driver in recombination of intron-lacking genes during mitogenome evolution. We postulate that the linkage between intron-lacking gene and its adjacent endonuclease gene is the result of co-evolution.