Box C/D Small Nucleolar RNA (snoRNA) U60 Regulates Intracellular Cholesterol Trafficking

Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Because mutant cells with defects in PM to endoplasmic reticulum cholesterol trafficking can be isolated on the basis of resistance to amphotericin B, we conducted an amphotericin B loss-of-function screen in Chinese hamster ovary (CHO) cells using insertional mutagenesis to identify genes that regulate this trafficking mechanism. Mutant line A1 displayed reduced cholesteryl ester formation from PM-derived cholesterol and increased de novo cholesterol synthesis, indicating a deficiency in retrograde cholesterol transport. Genotypic analysis revealed that the A1 cell line contained one disrupted allele of the U60 small nucleolar RNA (snoRNA) host gene, resulting in haploinsufficiency of the box C/D snoRNA U60. Complementation and mutational studies revealed the U60 snoRNA to be the essential feature from this locus that affects cholesterol trafficking. Lack of alteration in predicted U60-mediated site-directed methylation of 28 S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Our study adds to a growing body of evidence for participation of small noncoding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function.     

植物一个新box H/ACA snoRNA的鉴定及功能分析

通过生物信息学方法对拟南芥基因组序列进行搜索,发现两个新的非编码小分子RNA基因,分别命名为AthsnoR206 a和AthsnoR206b。它们相距约170nt,位于蛋白质基因间隔区。MFOLD二级结构预测这两个RNA均具有典型的box H/ACAsnoRNA"发夹-铰链-发夹-尾巴"结构,符合box H/ACA snoRNA的判定标准;两个RNA分子的反义序列一致,可以判定它们为同一基因的两个拷贝。分析预测snoR206的两段反义序列分别指导拟南芥rRNA小亚基U1717位点和大亚基U2181位点的假尿嘧啶化修饰。在其它13种包括单子叶植物和双子叶植物在内的植物搜索到14个snoR206同源分子,其中12个发现于表达序列标签中,表明该snoRNA在植物中表达且广泛存在。具有双功能的snoR206在人和酵母中的部分功能同源分子分别为U70和snR32,表明其祖先分子在进化过程中存在分子重组。     

18S rRNA processing requires base pairings of snR30 H/ACA snoRNA to eukaryote‐specific 18S sequences

The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non‐coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of ‘orphan’ H/ACA RNAs participate in pre‐rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 ‘orphan’ H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre‐rRNA processing, two evolutionarily conserved sequence elements in the 3′‐hairpin of snR30 base‐pair with short pre‐rRNA sequences located in the eukaryote‐specific internal region of 18S rRNA. The newly discovered snR30‐18S base‐pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3′‐hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein‐binding site.     

The Origin and Evolution of tRNA Inferred from Phylogenetic Analysis of Structure

The evolutionary history of the two structural and functional domains of tRNA is controversial but harbors the secrets of early translation and the genetic code. To explore the origin and evolution of tRNA, we reconstructed phylogenetic trees directly from molecular structure. Forty-two structural characters describing the geometry of 571 tRNAs and three statistical parameters describing thermodynamic and mechanical features of molecules quantitatively were used to derive phylogenetic trees of molecules and molecular substructures. Trees of molecules failed to group tRNA according to amino acid specificity and did not reveal the tripartite nature of life, probably due to loss of phylogenetic signal or because tRNA diversification predated organismal diversification. Trees of substructures derived from both structural and statistical characters support the origin of tRNA in the acceptor arm and the hypothesis that the top half domain composed of acceptor and pseudouridine (TΨC) arms is more ancient than the bottom half domain composed of dihydrouridine (DHU) and anticodon arms. This constitutes the cornerstone of the genomic tag hypothesis that postulates tRNAs were ancient telomeres in the RNA world. The trees of substructures suggest a model for the evolution of the major functional and structural components of tRNA. In this model, short RNA hairpins with stems homologous to the acceptor arm of present day tRNAs were extended with regions homologous to TΨC and anticodon arms. The DHU arm was then incorporated into the resulting three-stemmed structure to form a proto-cloverleaf structure. The variable region was the last structural addition to the molecular repertoire of evolving tRNA substructures. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cytosolic Accumulation of Small Nucleolar RNAs (snoRNAs) Is Dynamically Regulated by NADPH Oxidase

Small nucleolar RNAs (snoRNAs) guide nucleotide modifications of cellular RNAs in the nucleus. We previously showed that box C/D snoRNAs from the Rpl13a locus are unexpected mediators of physiologic oxidative stress, independent of their predicted ribosomal RNA modifications. Here we demonstrate that oxidative stress induced by doxorubicin causes rapid cytoplasmic accumulation of the Rpl13a snoRNAs through a mechanism that requires superoxide and a nuclear splice variant of NADPH oxidase. RNA-sequencing analysis reveals that box C/D snoRNAs as a class are present in the cytoplasm, where their levels are dynamically regulated by NADPH oxidase. These findings suggest that snoRNAs may orchestrate the response to environmental stress through molecular interactions outside of the nucleus.     

Grass Evolution Inferred from Chromosomal Rearrangements and Geometrical and Statistical Features in RNA Structure

The grasses (Poaceae) represent a monophyletic lineage that arose about 70 million years ago. The lineage contains about 10,000 species that differ widely in morphology and physiology. Species show striking differences in genome size, a feature important in the context of conservation of gene content and order (synteny and colinearity) and in the extension of genomic information directly from one grass species to another using comparative approaches. Grass diversification has been a contentious issue, as the exact branching order of the various subfamilies has been difficult to establish with standard methods. This motivated an evolutionary study of deep phylogenetic relationships based on the structure of coding and non-coding RNA molecules and on chromosomal rearrangements. Phylogenetic relationships in the grass family were inferred directly from the structure of RNA using cladistic principles and considerations in statistical mechanics. Coded attributes describing topological and thermodynamic information embedded in RNA molecules were treated as linearly ordered multi-state characters and were polarized by fixing the direction of character transformation toward molecular order. Intrinsically rooted phylogenies derived from the structure of signal recognition particle (SRP) RNA, the mRNA encoded by the early nodulation gene enod40, the small subunit of ribosomal RNA (rRNA), and the internal transcribed spacer ITS1 of rRNA established an order for the diversification of major grass lineages, suggesting a sister relationship of the Pooideae and the PACCAD clade. This same conclusion was reached when large-scale chromosomal rearrangements derived from the comparative genetic mapping of cereal genomes were studied. Chromosomal complements aligned in the most parsimonious manner allowed identification and coding of characters depicting chromosomal translocations, insertions, and linkage block arrangements and the reconstruction of phylogenetic trees based on large-scale chromosomal structure. Congruent reconstruction of deep branching relationships using geometrical and statistical features of RNA structure and orthology and large scale chromosomal recombination events support assumptions of polarization in character argumentation, and fail to falsify the claim that extant grass chromosomes can be considered combinations of linkage blocks of an ancestor of the rice genome. Congruence also suggests that the universal tendency toward order in RNA and the search for the most parsimonious organization of be genome architecture appear to be mutually supported drivers of molecular evolution. The study clarifies the relationship of major clades in the grasses, shows that phylogenetic history can be reconstructed effectively from the combinatorial exchange of chromosomal linkage blocks, and reveals considerable phylogenetic signal embedded in the structure of signal polypeptide-coding mRNA molecules, describing an instance where mRNA structure is the subject of strong evolutionary constraint.Reviewing Editor: Dr. David Pollock     

基于细胞核rDNA ITS片段的水青冈属的分子系统发育

对山毛榉科水青冈属6种、1亚种、1栽培变种的ITS区片段进行了测序和分析,并对其中2个具有ITS序列多态性的分类群进行了ITS区克隆。水青冈属ITS系统发育树聚成两支,位于基部的是分布于北美的大叶水青冈,另一分支则包括了欧洲和东亚的类群。在欧洲和东亚分支中,又包括两支,其中日本北部的波叶水青冈位于基部,台湾水青冈和欧亚大陆的水青冈形成另外一支。ITS区分析与现行的水青冈属基于形态学性状的属下分类系统有一定差异,而与本属现存物种的地理分布格局较为一致。各类群间TIS区序列差异较小,显示属内现存物种的分化时间不是太长。     

Evolution of the RNA polymerase B' subunit gene (rpoB') in Halobacteriales: a complementary molecular marker to the SSU rRNA gene

Many prokaryotes have multiple ribosomal RNA operons. Generally, sequence differences between small subunit (SSU) rRNA genes are minor (<1%) and cause little concern for phylogenetic inference or environmental diversity studies. For Halobacteriales, an order of extremely halophilic, aerobic Archaea, within-genome SSU rRNA sequence divergence can exceed 5%, rendering phylogenetic assignment problematic. The RNA polymerase B' subunit gene (rpoB') is a single-copy conserved gene that may be an appropriate alternative phylogenetic marker for Halobacteriales. We sequenced a fragment of the rpoB' gene from 21 species, encompassing 15 genera of Halobacteriales. To examine the utility of rpoB' as a phylogenetic marker in Halobacteriales, we investigated three properties of rpoB' trees: the variation in resolution between trees inferred from the rpoB' DNA and RpoB' protein alignment, the degree of mutational saturation between taxa, and congruence with the SSU rRNA tree. The rpoB' DNA and protein trees were for the most part congruent and consistently recovered two well-supported monophyletic groups, the clade I and clade II haloarchaea, within a collection of less well resolved Halobacteriales lineages. A comparison of observed versus inferred numbers of substitution revealed mutational saturation in the rpoB' DNA data set, particularly between more distant species. Thus, the RpoB' protein sequence may be more reliable than the rpoB' DNA sequence for inferring Halobacteriales phylogeny. AU tests of tree selection indicated the trees inferred from rpoB' DNA and protein alignments were significantly incongruent with the SSU rRNA tree. We discuss possible explanations for this incongruence, including tree reconstruction artifact, differential paralog sampling, and lateral gene transfer. This is the first study of Halobacteriales evolution based on a marker other than the SSU rRNA gene. In addition, we present a valuable phylogenetic framework encompassing a broad diversity of Halobacteriales, in which novel sequences can be inserted for evolutionary, ecological, or taxonomic investigations.     

Small nucleolar RNAs that guide modification in trypanosomatids: repertoire, targets, genome organisation, and unique functions

Small nucleolar RNAs constitute a family of newly discovered non-coding small RNAs, most of which function in guiding RNA modifications. Two prevalent types of modifications are 2'-O-methylation and pseudouridylation. The modification is directed by the formation of a canonical small nucleolar RNA-target duplex. Initially, RNA-guided modification was shown to take place on rRNA, but recent studies suggest that small nuclear RNA, mRNA, tRNA, and the trypanosome spliced leader RNA also undergo guided modifications. Trypanosomes contain more modifications and potentially more small nucleolar RNAs than yeast, and the increased number of modifications may help to preserve ribosome function under adverse environmental conditions during the cycling between the insect and mammalian host. The genome organisation in clusters carrying the two types of small nucleolar RNAs, C/D and H/ACA-like RNAs, resembles that in plants. However, the trypanosomatid H/ACA RNAs are similar to those found in Archaea and are composed of a single hairpin that may represent the primordial H/ACA RNA. In this review we summarise this new field of trypanosome small nucleolar RNAs, emphasising the open questions regarding the number of small nucleolar RNAs, the repertoire, genome organisation, and the unique function of guided modifications in these protozoan parasites.     

Molecular Evolution and Phylogenetic Utility of the Internal Transcribed Spacer 2 (ITS2) in Calyptratae (Diptera: Brachycera)

The resolution potential of internal transcribed spacer 2 (ITS2) at deeper levels remains controversial. In this study, 105 ITS2 sequences of 55 species in Calyptratae were analyzed to examine the phylogenetic utility of the spacer above the subfamily level and to further understand its evolutionary characteristics. We predicted the secondary structure of each sequence using the minimum-energy algorithm and constructed two data matrixes for phylogenetic analysis. The ITS2 regions of Calyptratae display strong A-T bias and slight variation in length. The tandem and dispersed repeats embedded in the spacers possibly resulted from replication slippage or transposition. Most foldings conformed to the four-domain model. Sequence comparison in combination with the secondary structures revealed six conserved motifs. Covariation analysis from the conserved motifs indicated that the secondary structure restrains the sequence evolution of the spacer. The deep-level phylogeny derived from the ITS2 data largely agreed with the phylogenetic hypotheses from morphologic and other molecular evidence. Our analyses suggest that the accordant resolutions generated from different analyses can be used to infer deep-level phylogenetic relations.     

Phylogenetic estimation and morphological evolution of Alsineae (Caryophyllaceae) shed new insight into the taxonomic status of the genus Pseudocerastium

Pseudocerastium is a monotypic genus in Caryophyllaceae endemic to China. The genus has been widely accepted since it was described in 1998, however its phylogenetic position within Caryophyllaceae has never been studied. In the present study, the whole plastid genome and nuclear ribosomal internal transcribed spacer (ITS) sequences of Pseudocerastium stellarioides was obtained through genome skimming, and the phylogenetic position of the species was studied for the first time. Plastid phylogenomic analysis of Caryophyllaceae revealed that Pseudocerastium is clustered within the tribe Alsineae with strong support. Phylogenetic analyses based on an enlarged taxon sampling of Alsineae using five DNA regions (matK, rbcL, rps16 intron, trnL-F and ITS) revealed that P. stellarioides was nested deeply within Cerastium with strong support. Analyses of morphological character evolution suggest that the ancestral states in Alsineae include three styles and a six-lobed capsule at the apex, while both Cerastium and Pseudocerastium have five styles and ten lobes at the apex of the capsule, further supporting their close relationship. The species Pseudocerastium stellarioides is similar to Cerastium wilsonii in morphology, but differs in having villous indumentum on the lower part of the filaments and compressed globose seeds. Therefore, based on the present molecular and morphological evidence, the generic name Pseudocerastium is reduced here as a new synonym of Cerastium and the species P. stellarioides is transferred to Cerastium as C. jiuhuashanense.     

蛋白质-RNA序列结构界面偏好性及用于对接打分统计势的构建

本文对来自PDB (Protein Data Bank)数据库的蛋白质-RNA复合物结构构建了非冗余非核糖体数据库(694个结构),并对此数据库统计了蛋白质和RNA序列及二级结构的界面偏好性.结果发现,蛋白质β折叠、3₁₀-helix和RNA未配对核苷酸,尤其是未配对中空间排列不规整的核苷酸,具有显著的界面偏好性.据此,对二级结构进行归类,建立了考虑序列和二级结构信息的60×12氨基酸-核苷酸成对偏好势,并将其作为打分函数用于蛋白质-RNA对接中近天然结构的筛选.结果表明,该60×12统计势的打分成功率为65.77%,优于考虑蛋白质或RNA二级结构信息的统计势,及我们小组之前在251个结构上构建的60×8^*统计势.该工作有助于加深对蛋白质-RNA特异性识别的理解,可推动复合物结构预测的进展.     

Sequence,proposed secondary structure,and phylogenetic analysis of the chloroplast 5S rRNA gene of the brown alga Pylaiella littoralis (L.) Kjellm

Summary The chloroplast 5S rRNA gene of the brown alga Pylaiella littoralis (L.) Kjellm has been cloned and sequenced. The gene is located 23 bp downstream from the 3 end of the 23S rRNA gene. The sequence of the gene is as follows: GGTCTTG GTGTTTAAAGGATAGTGGAACCACATTGAT CCATATCGAACTCAATGGTGAAACATTATT ACAGTAACAATACTTAAGGAGGAGTCCTTT GGGAAGATAGCTTATGCCTAAGAC. A secondary structure model is proposed, and compared to those for the chloroplast 5S rRNAs of spinach and the red alga Porphyra umbilicalis. Cladograms based on chloroplast and bacterial 5S rRNA and rRNA gene sequences were constructed using the MacClade program with a user-defined character transformation in which transitions and transversions were assigned unequal step values. The topology of the resulting cladogram indicates a polyphyletic origin for photosynthetic organelles.Offprint requests to: S. Loiseaux-de Goër     

Reconsideration of the Phylogenetic Positions of Five Peritrich Genera,Vorticella, Pseudovorticella,Zoothamnopsis, Zoothamnium,and Epicarchesium (Ciliophora,Peritrichia, Sessilida), Based on Small Subunit rRNA Gene Sequences

ABSTRACT. In order to re‐evaluate the systematics of sessilid peritrich ciliates, small subunit (SSU) rRNA gene sequences were determined for 12 species belonging to five genera: Vorticella, Pseudovorticella, Epicarchesium, Zoothamnium, and Zoothamnopsis. Phylogenetic trees were deduced using Bayesian inference, maximum parsimony, and maximum likelihood methods. The phylogenetic analyses suggest that (1) sessilids which have stalks with continuous myonemes that contract in a zig‐zag fashion form a separate clade from those which have stalks that contract independently and in a spiral fashion, supporting the separation of the family Zoothamniidae from the family Vorticellidae and (2) Epicarchesium and Pseudovorticella, both of which have reticulate silverline systems, are more closely related to each other than to other vorticellids, suggesting that differences in the silverline system (i.e. transverse vs. reticulate) may be the result of genuine evolutionary divergence among sessilid peritrichs. However, the newly sequenced Zoothamnopsis sinica, which has a reticulate silverline pattern, nests within the unresolved Zoothamnium species that have transverse silverline patterns. Thus, there were at least two evolutions of the reticulate silverline pattern character state from a plesiomorphic transverse state in the peritrichid ciliates. The molecular work demonstrates the genus Zoothamnium to be paraphyletic in relation to morphological studies, and suggests that Astylozoon, Opisthonecta, and Vorticella microstoma possibly share a SSU rRNA secondary structure in the helix E10‐1 region.     

Evolution of a pseudogene: exclusive survival of a functional mitochondrial nad7 gene supports Haplomitrium as the earliest liverwort lineage and proposes a secondary loss of RNA editing in Marchantiidae

Gene transfer from the mitochondrion into the nucleus is a corollary of the endosymbiont hypothesis. The frequent and independent transfer of genes for mitochondrial ribosomal proteins is well documented with many examples in angiosperms, whereas transfer of genes for components of the respiratory chain is a rarity. A notable exception is the nad7 gene, encoding subunit 7 of complex I, in the liverwort Marchantia polymorpha, which resides as a full-length, intron-carrying and transcribed, but nonspliced pseudogene in the chondriome, whereas its functional counterpart is nuclear encoded. To elucidate the patterns of pseudogene degeneration, we have investigated the mitochondrial nad7 locus in 12 other liverworts of broad phylogenetic distribution. We find that the mitochondrial nad7 gene is nonfunctional in 11 of them. However, the modes of pseudogene degeneration vary: whereas point mutations, accompanied by single-nucleotide indels, predominantly introduce stop codons into the reading frame in marchantiid liverworts, larger indels introduce frameshifts in the simple thalloid and leafy jungermanniid taxa. Most notably, however, the mitochondrial nad7 reading frame appears to be intact in the isolated liverwort genus Haplomitrium. Its functional expression is shown by cDNA analysis identifying typical RNA-editing events to reconstitute conserved codon identities and also confirming functional splicing of the 2 liverwort-specific group II introns. We interpret our results 1) to indicate the presence of a functional mitochondrial nad7 gene in the earliest land plants and strongly supporting a basal placement of Haplomitrium among the liverworts, 2) to indicate different modes of pseudogene degeneration and chondriome evolution in the later branching liverwort clades, 3) to suggest a surprisingly long maintenance of a nonfunctional gene in the presumed oldest group of land plants, and 4) to support the model of a secondary loss of RNA-editing activity in marchantiid liverworts.     

Analysis of the Secondary Structure of ITS1 in Pectinidae: Implications for Phylogenetic Reconstruction and Structural Evolution

It is at present difficult to accurately position gaps in sequence alignment and to determine substructural homology in structure alignment when reconstructing phylogenies based on highly divergent sequences. Therefore, we have developed a new strategy for inferring phylogenies based on highly divergent sequences. In this new strategy, the whole secondary structure presented as a string in bracket notation is used as phylogenetic characters to infer phylogenetic relationships. It is no longer necessary to decompose the secondary structure into homologous substructural components. In this study, reliable phylogenetic relationships of eight species in Pectinidae were inferred from the structure alignment, but not from sequence alignment, even with the aid of structural information. The results suggest that this new strategy should be useful for inferring phylogenetic relationships based on highly divergent sequences. Moreover, the structural evolution of ITS1 in Pectinidae was also investigated. The whole ITS1 structure could be divided into four structural domains. Compensatory changes were found in all four structural domains. Structural motifs in these domains were identified further. These motifs, especially those in D2 and D3, may have important functions in the maturation of rRNAs.     

狭义蛇葡萄属东亚支系的系统发生与生物地理演化

狭义蛇葡萄属(Ampelopsis s. str.)是葡萄科的落叶木质藤本植物,主要分布在北半球温带地区,特别是东亚为其重要的分布和演化中心。该研究选取了狭义蛇葡萄属15个种的37个样本,对其5个叶绿体基因片段(trnL-F、rps16、psbA-trnH、atpB-rbcL和trnK-petN)和2个核基因标记(ITS和GAI1)进行了分子测序;利用测序获得的分子数据探讨属内的系统发生关系,叶的性状演化以及生物地理起源演化。结果表明：(1)狭义蛇葡萄属是一个单系类群,属内东亚地区的物种聚成一支。(2)叶绿体基因数据分析结果表明,狭义蛇葡萄属东亚支系分为两支系,这两支系的分布范围大致与东亚植物区系的中国-日本森林和中国-喜马拉雅森林亚区范围相一致。(3)形态演化分析表明,掌状复叶为祖征,叶形态性状存在多次的独立起源和演化,其变化与系统进化没有明显的关系。(4)生物地理分析结果表明,狭义蛇葡萄属起源于北美,渐新世晚期至中新世早期迁移扩散至欧洲,中新世中期随着全球气温回暖迁移至东亚,并进一步在东亚地区快速分化形成多样化中心,这可能与中新世时期的气候温暖、受第四纪冰川影响较少以及东亚地区复杂...