Organization of the Euplotes crassus micronuclear genome

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Fungal Smn and Spf30 homologues are mainly present in filamentous fungi and genomes with many introns: implications for spinal muscular atrophy

Spinal muscular atrophy is an important rare genetic disease characterized by the loss of motor neurons, where the main gene responsible is smn1. Orthologous genes have only been characterized in a single fungal genome: Schizosaccharomyces pombe. We have searched for putative SMN orthologues in publically available fungal genomes, finding that they are predominately present in filamentous fungi. SMN binding partners and the SPF30 SMN paralogue, which are all involved in mRNA splicing, were found to be present in a similar but non-identical subset of fungal genomes. The Saccharomycces cerevisiae yeast genome contains neither smn1 orthologues nor paralogues and it has been suggested that this might be related to the low number of introns in this yeast. Here we have tested this hypothesis by looking at other fungal genomes. Significantly, we find that fungal genomes with high numbers of introns also possess an SMN orthologue or at least its paralogue, SPF30.     

Genome-wide survey of alternative splicing in the grass Brachypodium distachyon: a emerging model biosystem for plant functional genomics

A draft sequence of the genome of Brachypodium distachyon, the emerging grass model, was recently released. This represents a unique opportunity to determine its functional diversity compared to the genomes of other model species. Using homology mapping of assembled expressed sequence tags with chromosome scale pseudomolecules, we identified 128 alternative splicing events in B. distachyon. Our study identified that retention of introns is the major type of alternative splicing events (53%) in this plant and highlights the prevalence of splicing site recognition for definition of introns in plants. We have analyzed the compositional profiles of exon-intron junctions by base-pairing nucleotides with U1 snRNA which serves as a model for describing the possibility of sequence conservation. The alternative splicing isoforms identified in this study are novel and represent one of the potentially biologically significant means by which B. distachyon controls the function of its genes. Our observations serve as a basis to understand alternative splicing events of cereal crops with more complex genomes, like wheat or barley.     

Genome architecture drives protein evolution in ciliates

Studies of microbial eukaryotes have been pivotal in the discovery of biological phenomena, including RNA editing, self-splicing RNA, and telomere addition. Here we extend this list by demonstrating that genome architecture, namely the extensive processing of somatic (macronuclear) genomes in some ciliate lineages, is associated with elevated rates of protein evolution. Using newly developed likelihood-based procedures for studying molecular evolution, we investigate 6 genes to compare 1) ciliate protein evolution to that of 3 other clades of eukaryotes (plants, animals, and fungi) and 2) protein evolution in ciliates with extensively processed macronuclear genomes to that of other ciliate lineages. In 5 of the 6 genes, ciliates are estimated to have a higher ratio of nonsynonymous/synonymous substitution rates, consistent with an increase in the rate of protein diversification in ciliates relative to other eukaryotes. Even more striking, there is a significant effect of genome architecture within ciliates as the most divergent proteins are consistently found in those lineages with the most highly processed macronuclear genomes. We propose a model whereby genome architecture-specifically chromosomal processing, amitosis within macronuclei, and epigenetics-allows ciliates to explore protein space in a novel manner. Further, we predict that examination of diverse eukaryotes will reveal additional evidence of the impact of genome architecture on molecular evolution.     

nMAT1, a nuclear-encoded maturase involved in the trans-splicing of nad1 intron 1, is essential for mitochondrial complex I assembly and function

Mitochondrial genomes (mtDNAs) in angiosperms contain numerous group II-type introns that reside mainly within protein-coding genes that are required for organellar genome expression and respiration. While splicing of group II introns in non-plant systems is facilitated by proteins encoded within the introns themselves (maturases), the mitochondrial introns in plants have diverged and have lost the vast majority of their intron-encoded ORFs. Only a single maturase gene (matR) is retained in plant mtDNAs, but its role(s) in the splicing of mitochondrial introns is currently unknown. In addition to matR, plants also harbor four nuclear maturase genes (nMat 1 to 4) encoding mitochondrial proteins that are expected to act in the splicing of group II introns. Recently, we established the role of one of these proteins, nMAT2, in the splicing of several mitochondrial introns in Arabidopsis. Here, we show that nMAT1 is required for trans-splicing of nad1 intron 1 and also functions in cis-splicing of nad2 intron 1 and nad4 intron 2. Homozygous nMat1 plants show retarded growth and developmental phenotypes, modified respiration activities and altered stress responses that are tightly correlated with mitochondrial complex I defects.     

Origin and evolution of spliceosomal introns

ABSTRACT: Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded 'introns first' held that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns. Thus, the introns-first scenario is not supported by any evidence but exon-intron structure of protein-coding genes appears to have evolved concomitantly with the eukaryotic cell, and introns were a major factor of evolution throughout the history of eukaryotes. This article was reviewed by I. King Jordan, Manuel Irimia (nominated by Anthony Poole), Tobias Mourier (nominated by Anthony Poole), and Fyodor Kondrashov. For the complete reports, see the Reviewers' Reports section.     

AtnMat2, a nuclear-encoded maturase required for splicing of group-II introns in Arabidopsis mitochondria

Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as “maturases”), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established. Clues to other proteins are scarce, but these are likely encoded within the nucleus as there are no obvious candidates among the remaining ORFs within the mtDNA. Intriguingly, higher plants genomes contain four maturase-related genes, which exist in the nucleus as self-standing ORFs, out of the context of their evolutionary-related group-II introns “hosts.” These are all predicted to reside within mitochondria and may therefore act “in-trans” in the splicing of organellar-encoded introns. Here, we analyzed the intracellular locations of the four nuclear-encoded maturases in Arabidopsis and established the roles of one of these genes, At5g46920 (AtnMat2), in the splicing of several mitochondrial introns, including the single intron within cox2, nad1 intron2, and nad7 intron2.     

RNA在RNA剪接中的功能：从催化到调控

对非编码RNA功能的认识是后基因组时代的一个研究焦点,本文主要介绍非编码RNA在RNA剪接中的催化和调控功能。在RNA加工过程中,三大类内含子的剪接都是由RNA成员主导。其中Ⅰ型和Ⅱ型内含子能催化自身的切除和外显子连接反应;而核mRNA内含子的剪接则由剪接体里的小核RNA主导。Ⅰ型和Ⅱ型内含子存在于细菌、低等真核细胞和植物的细胞器内;而真核细胞的核编码蛋白质基因内全部是核mRNA内含子,并且其数目随生物体的复杂性而显著升高。一个多内含子前体mRNA通过选择性剪接产生多种,甚至上万种不同的mRNA和蛋白质,对蛋白质组的复杂度和时空表达调控至关重要。选择性剪接调控由剪接调控蛋白特异识别和结合前体mRNA里所富含的顺式RNA调控元件完成的;系统认识这两者之间的对应关系是揭示基因组表达调控网络的一把钥匙。     

Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III

We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36. The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E. coli. The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene. The operon contains at least 7 introns. We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns. Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene. Three introns resemble the classical group II introns of organelle genomes. The remaining 4 introns appear to be unique to the Euglena chloroplast DNA. They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns. We designate this new intron category as 'group III'.     

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     

Quantitative Analyses of Interbreeding in Populations of vannus-Morphotype Euplotes, with Special Attention to the Nominal Species E. vannus and E. crassus

ABSTRACT. Classical genetic techniques were applied to clonal cultures of the Euplotes vannus-crassus-minuta sibling species complex in an effort to provide some resolution to the species problem among these hypotrichs. Complex mating interactions were observed among clonal stock cultures derived from samples collected from sympatric and allopatric populations in a wide geographic survey. These results suggested that the classical model for the mating type inheritance and determination in these Euplotes is necessary but not sufficient to describe the mating mating interactions among populations of these ciliates. Successful conjugation between the nominal species E. crassus and E. vannus was observed routinely, and crosses between these two nominal species did not differ significantly from those among the other clonal stock cultures with respect to mating intensity and exconjugant survival. Data from backcrosses suggests that E. vannus and E. crassus can and do exchange genes. Based upon these data, we conclude that E. vannus and E. crassus comprise a single, highly polymorphic species with countless small populations, among which incomplete genetic exchange takes place.     

蛋白质基因组学：运用蛋白质组技术注释基因组

随着高通量DNA测序技术的飞速发展,越来越多的物种完成了基因组测序．定位编码基因、确定编码基因结构是基因组注释的基本任务,然而以往的基因组注释方法主要依赖于DNA及RNA序列信息．为了更加精确地解读完成测序的基因组,我们需要整合多种类型的组学数据进行基因组注释．近年来,基于串联质谱技术的蛋白质组学已经发展成熟,实现了对蛋白质组的高覆盖,使得利用串联质谱数据进行基因组注释成为可能．串联质谱数据一方面可以对已注释的基因进行表达验证,另一方面还可以校正原注释基因,进而发现新基因,实现对基因组序列的重新注释．这正是当前进展较快的蛋白质基因组学的研究内容．利用该方法系统地注释已完成测序的基因组已成为解读基因组的一个重要补充．本文综述了蛋白质基因组学的主要研究内容和研究方法,并展望了该研究方向未来的发展．