Complete, precise, and innocuous loss of multiple introns in the currently intronless, active cathepsin L-like genes, and inference from this event

Retrotransposition typically generates pseudogenes. Here we demonstrate a different fate of the retro-processed genes through a novel mechanism in which the retro-processed genes still maintain their sequence intactness and the original functions. We show that the shrimp cathepsin L (CatL) gene MeCatL has lost all of its five introns. Also, ProEPB, the ancestor of the CatL-like barley EPBs and rice REP1, has lost all of its three introns. The multiple introns in a gene might have been eliminated simultaneously and precisely at the original locus for the CatL-like genes of shrimp, barley, rice, Drosophila, and Theileria. We reason that retrotransposition is not responsible for the generation of a processed active intronless (PAI) gene when the gene product retains its sequence intactness and its original function. We propose that double-strand-break repair (DSBR) machinery might play a role in cDNA-mediated homologous recombination (cDMHR) that causes the loss of introns. The cDMHR/DSBR pathway is probably a fundamental mechanism for intron loss in PAI genes and in some asymmetric-intron genes.     

Origin and Inheritance of Group I Introns in 26S rRNA Genes of Gaeumannomyces graminis

Studies of the distribution of the three group I introns (intron A, intron T, and intron AT) in the 26S rDNA of Gaeumannomyces graminis had suggested that they were transferred to a common ancestor of G. graminis var. avenae and var. tritici after it had branched off from var. graminis. Intron AT and intron A exhibited vertical inheritance and coevolved in concert with their hosts. Intron loss could occur after its acquisition. Loss of any one of the three introns could occur in var. tritici whereas only loss of intron T had been found in the majority of var. avenae isolates. The existence of isolates of var. tritici and var. avenae with three introns suggested that intron loss could be reversed by intron acquisition and that the whole process is a dynamic one. This process of intron acquisition and intron loss reached different equilibrium points for different varieties and subgroups, which explained the irregular distribution of these introns in G. graminis. Each of the three group I introns was more closely related to other intron sequences that share the same insertion point in the 26S rDNA than to each other. These introns in distantly related organisms appeared to have a common ancestry. This system had provided a good model for studies on both the lateral transfer and common ancestry of group I introns in the 26S rRNA genes. Received: 17 May 1996 / Accepted: 14 January 1997     

Group I introns as mobile genetic elements: facts and mechanistic speculations--a review

Group I introns form a structural and functional group of introns with widespread but irregular distribution among very diverse organisms and genetic systems. Evidence is now accumulating that several group I introns are mobile genetic elements with properties similar to those originally described for the omega system of Saccharomyces cerevisiae: mobile group I introns encode sequence-specific double-strand (ds) endoDNases, which recognize and cleave intronless genes to insert a copy of the intron by a ds-break repair mechanism. This mechanism results in: the efficient propagation of group I introns into their cognate sites; their maintenance at the site against spontaneous loss; and, perhaps, their transposition to different sites. The spontaneous loss of group I introns occurs with low frequency by an RNA-mediated mechanism. This mechanism eliminates introns defective for mobility and/or for RNA splicing. Mechanisms of intron acquisition and intron loss must create an equilibrium, which explains the irregular distribution of group I introns in various genetic systems. Furthermore, the observed distribution also predicts that horizontal transfer of intron sequences must occur between unrelated species, using vectors yet to be discovered.     

Multiple barriers to nonhomologous DNA end joining during meiosis in Drosophila

Repair of meiotic double-strand breaks (DSBs) uses the homolog and recombination to yield crossovers while alternative pathways such as nonhomologous end joining (NHEJ) are suppressed. Our results indicate that NHEJ is blocked at two steps of DSB repair during meiotic prophase: first by the activity of the MCM-like protein MEI-218, which is required for crossover formation, and, second, by Rad51-related proteins SPN-B (XRCC3) and SPN-D (RAD51C), which physically interact and promote homologous recombination (HR). We further show that the MCM-like proteins also promote the activity of the DSB repair checkpoint pathway, indicating an early requirement for these proteins in DSB processing. We propose that when a meiotic DSB is formed in the absence of both MEI-218 and SPN-B or SPN-D, a DSB substrate is generated that can enter the NHEJ repair pathway. Indeed, due to its high error rate, multiple barriers may have evolved to prevent NHEJ activity during meiosis.     

Rare genomic characters do not support Coelomata: intron loss/gain

Recently, a new phylogenetic method employing intron positionsharing across species was proposed and support for a Coelomateclade reported (Zheng et al. 2007. A rigorous analysis of thepattern of intron conservation supports the Coelomata cladeof animals. Mol Biol Evol. 24:2583–2592.). Here, we showthat the previous analysis depends on: 1) an idiosyncratic definitionof "conserved" introns, 2) exclusion of all phylogeneticallyinformative introns present in outgroups, 3) incorrect inferenceof change along the critical branch, and 4) lack of variationin rates across branches. The method thus seems unlikely togive accurate results. In addition, we address differences inrates of loss across intron sites, which Zheng et al. claimedinvalidates our previous analysis that supported Ecdysozoa (Royand Gilbert. 2005a. Resolution of a deep animal divergence bythe pattern of intron conservation. Proc Natl Acad Sci USA.102:4403–4408.). Instead, we show that our conclusionsare likely to be robust to such concerns.     

Higher intron loss rate in Arabidopsis thaliana than A. lyrata is consistent with stronger selection for a smaller genome

The number of introns varies considerably among different organisms. This can be explained by the differences in the rates of intron gain and loss. Two factors that are likely to influence these rates are selection for or against introns and the mutation rate that generates the novel intron or the intronless copy. Although it has been speculated that stronger selection for a compact genome might result in a higher rate of intron loss and a lower rate of intron gain, clear evidence is lacking, and the role of selection in determining these rates has not been established. Here, we studied the gain and loss of introns in the two closely related species Arabidopsis thaliana and A. lyrata as it was recently shown that A. thaliana has been undergoing a faster genome reduction driven by selection. We found that A. thaliana has lost six times more introns than A. lyrata since the divergence of the two species but gained very few introns. We suggest that stronger selection for genome reduction probably resulted in the much higher intron loss rate in A. thaliana, although further analysis is required as we could not find evidence that the loss rate increased in A. thaliana as opposed to having decreased in A. lyrata compared with the rate in the common ancestor. We also examined the pattern of the intron gains and losses to better understand the mechanisms by which they occur. Microsimilarity was detected between the splice sites of several gained and lost introns, suggesting that nonhomologous end joining repair of double-strand breaks might be a common pathway not only for intron gain but also for intron loss.     

Evolution of rbcL group IA introns and intron open reading frames within the colonial Volvocales (Chlorophyceae)

Mobile group I introns sometimes contain an open reading frame (ORF) possibly encoding a site-specific DNA endonuclease. However, previous phylogenetic studies have not clearly deduced the evolutionary roles of the group I intron ORFs. In this paper, we examined the phylogeny of group IA2 introns inserted in the position identical to that of the chloroplast-encoded rbcL coding region (rbcL-462 introns) and their ORFs from 13 strains of five genera (Volvox, Pleodorina, Volvulina, Astrephomene, and Gonium) of the colonial Volvocales (Chlorophyceae) and a related unicellular green alga, Vitreochlamys. The rbcL-462 introns contained an intact or degenerate ORF of various sizes except for the Gonium multicoccum rbcL-462 intron. Partial amino acid sequences of some rbcL-462 intron ORFs exhibited possible homology to the endo/excinuclease amino acid terminal domain. The distribution of the rbcL-462 introns is sporadic in the phylogenetic trees of the colonial Volvocales based on the five chloroplast exon sequences (6021 bp). Phylogenetic analyses of the conserved intron sequences resolved that the G. multicoccum rbcL-462 intron had a phylogenetic position separate from those of other colonial volvocalean rbcL-462 introns, indicating the recent horizontal transmission of the intron in the G. multicoccum lineage. However, the combined data set from conserved intron sequences and ORFs from most of the rbcL-462 introns resolved robust phylogenetic relationships of the introns that were consistent with those of the host organisms. Therefore, most of the extant rbcL-462 introns may have been vertically inherited from the common ancestor of their host organisms, whereas such introns may have been lost in other lineages during evolution of the colonial Volvocales. In addition, apparently higher synonymous substitutions than nonsynonymous substitutions in the rbcL-462 intron ORFs indicated that the ORFs might evolve under functional constraint, which could result in homing of the rbcL-462 intron in cases of spontaneous intron loss. On the other hand, the presence of intact to largely degenerate ORFs of the rbcL-462 introns within the three isolates of Gonium viridistellatum and the rare occurrence of the ORF-lacking rbcL-462 intron suggested that the ORFs might degenerate to result in the spontaneous intron loss during a very short evolutionary time following the loss of the ORF function. Thus, the sporadic distribution of the rbcL-462 introns within the colonial Volvocales can be largely explained by an equilibrium between maintenance of the introns by the intron ORF and spontaneous loss of introns when the introns do not have a functional ORF.     

Vestiges of lost introns in the thermal stability map of DNA

The absence of introns in prokaryotic genes has been explained by intron loss on various bases. Here we report another piece of evidence on intron loss, which was found in the thermal stability map of DNA. We calculated the local melting temperature of cDNA sequences and found that (i) gaps in thermal stability tend to occur near intron positions with a statistical significance, and (ii) one-third of the gaps far from intron positions can be assigned to lost introns. From these results we conclude that the gaps of thermal stability in protein coding regions are the vestiges of lost introns.     

The Agaricus bisporus cox1 gene: the longest mitochondrial gene and the largest reservoir of mitochondrial group i introns

In eukaryotes, introns are located in nuclear and organelle genes from several kingdoms. Large introns (up to 5 kbp) are frequent in mitochondrial genomes of plant and fungi but scarce in Metazoa, even if these organisms are grouped with fungi among the Opisthokonts. Mitochondrial introns are classified in two groups (I and II) according to their RNA secondary structure involved in the intron self-splicing mechanism. Most of these mitochondrial group I introns carry a "Homing Endonuclease Gene" (heg) encoding a DNA endonuclease acting in transfer and site-specific integration ("homing") and allowing intron spreading and gain after lateral transfer even between species from different kingdoms. Opposed to this gain mechanism, is another which implies that introns, which would have been abundant in the ancestral genes, would mainly evolve by loss. The importance of both mechanisms (loss and gain) is matter of debate. Here we report the sequence of the cox1 gene of the button mushroom Agaricus bisporus, the most widely cultivated mushroom in the world. This gene is both the longest mitochondrial gene (29,902 nt) and the largest group I intron reservoir reported to date with 18 group I and 1 group II. An exhaustive analysis of the group I introns available in cox1 genes shows that they are mobile genetic elements whose numerous events of loss and gain by lateral transfer combine to explain their wide and patchy distribution extending over several kingdoms. An overview of intron distribution, together with the high frequency of eroded heg, suggests that they are evolving towards loss. In this landscape of eroded and lost intron sequences, the A. bisporus cox1 gene exhibits a peculiar dynamics of intron keeping and catching, leading to the largest collection of mitochondrial group I introns reported to date in a Eukaryote.     

Evidence for extensive recent intron transposition in closely related fungi

Though spliceosomal introns are a major structural component of most eukaryotic genes and intron density varies by more than three orders of magnitude among eukaryotes [1-3], the origins of introns are poorly understood, and only a few cases of unambiguous intron gain are known [4-8]. We utilized population genomic comparisons of three closely related fungi to identify crucial transitory phases of intron gain and loss. We found 74 intron positions showing intraspecific presence-absence polymorphisms (PAPs) for the entire intron. Population genetic analyses identified intron PAPs at different stages of fixation and showed that intron gain or loss was very recent. We found direct support for extensive intron transposition among unrelated genes. A substantial proportion of highly similar introns in the genome either were recently gained or showed a transient phase of intron PAP. We also identified an intron transfer among paralogous genes that created a new intron. Intron loss was due mainly to homologous recombination involving reverse-transcribed mRNA. The large number of intron positions in transient phases of either intron gain or loss shows that intron evolution is much faster than previously thought and provides an excellent model to study molecular mechanisms of intron gain.     

Factors determining DNA double-strand break repair pathway choice in G2 phase

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.     

双翅目昆虫（黑腹果蝇和冈比亚按蚊）内含子丢失的比较分析

内含子插入和丢失的进化动力及机制尚存在许多疑问。通过对真核生物的105个同源基因的蛋白质高度保守区域内含子-外显子结构的研究,对人Homo sapiens、小鼠Mus musculus、大鼠Rattus norvegicus、黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae和秀丽隐杆线虫Caenorhabditis elegans的3 574个内含子、1 001个的内含子保守位点进行分析,推断出不同系统中内含子的变化途径。发现在进化早期,脊椎动物、双翅目昆虫和线虫的共同祖先中含有大量内含子,在进化过程中,双翅目昆虫和线虫发生了大量的内含子丢失,甚至在双翅目昆虫中内含子丢失较线虫更严重。线虫获得的内含子略多于丢失的内含子, 而在双翅目昆虫中则显示出内含子的丢失明显多于内含子的获得。该结果合理地解释了内含子在脊椎动物、线虫及昆虫中数量的分布呈下降趋势。     

The linear form of a group II intron catalyzes efficient autocatalytic reverse splicing, establishing a potential for mobility

Self-splicing group II introns catalyze their own excision from pre-RNAs, thereby joining the flanking exons. The introns can be released in a lariat or linear form. Lariat introns have been shown to reverse the splicing reaction; in contrast, linear introns are generally believed to perform no or only poor reverse splicing. Here, we show that a linear group II intron derived from ai5γ can reverse the second step of splicing with unexpectedly high efficiency and precision. Moreover, the linear intron generates dramatically more reverse-splicing product than its lariat equivalent. The finding that linear group II introns can readily undergo the critical first step of mobility by catalyzing efficient reverse splicing into complementary target molecules demonstrates their innate potential for mobility and transposition and raises the possibility that reverse splicing by linear group II introns may have played a significant role in certain forms of intron mobility and lateral gene transfer during evolution.     

Intron evolution: a statistical comparison of two models.

The two most frequently occurring explanations for the existence and distribution of introns in the genes of different species are: (1) introns are remnants of the original genetic material. (2) Introns were introduced during evolution. We construct mathematical models corresponding to these two explanations, and calculate the probabilities that the intron distribution in genes from different species coding for actin, alpha-tubulin, triosephosphate isomerase and superoxide dismutase are described by these models. In both models, the branch lengths as well as the structure of the corresponding evolutionary tree is taken into account. Every branch in the evolutionary tree is assumed to have its own individual rate of loss of introns for the first model and rate of gain of introns for the second model. These rate constants are estimated from the actual number of introns. Using the rate constants we stimulate the intron evolution and calculate the probabilities that the actual intron arrangements are produced. The results for actin and alpha-tubulin, which are the two genes we have the most data for, favor the model corresponding conjecture (1), i.e. the idea that introns are old. This contradicts the results from an earlier attempt to model intron evolution where almost the same data was used (Dibb & Newman, 1989, EMBO J. 8, 2015-2021).