Identification and functional analysis of 23 novel box C／D snoRNAs from Oryza sativa

In a cDNA library generated from rice small nuclear RNAs,30box C/D small nucleolar RNAs (snoRNAs) were identiffied through preliminary screen.Except 7 known snoRNAs such as U14,all snoRNAs were identified in rice for the first time experimentally.Among the 23 novel snoRNAs,11 snoRNAs appear rice-specific,6 snoRNAs are unique to plants,the remaining 6 snoRNAs have their counterparts in both Arabidopsis and yeast or mammals according to the conserved antisense sequencs that guide 2‘-O-ribose methylation of rRNA,17 of the 23 novel snoRNAs were predicted to guide 24 2‘‘-O-ribose methylations at the specificsites of rice 5.8S,18S,25S rRNAs,among which 19 methylated sites were determined by primer extension at low dNTP concentrations.The remaining 6 snoRNAs devoid of rRNA antisense elements may represent novel snoRNA species in rice.The results show that constructing a cDNA library from small nuclear RNAs is an effective experimental approach for novel snoRNA is identification.The novel snoRNAs are important in elucidating the genomic organization and expression of plant snoRNA genes and the mechanism through which 2‘‘-O-ribose methylations took place in rRNAs.     

Identification and characterization of two novel box HACA snoRNAs from Schizosaccharomyces pombe

Through constructing a specialized cDNA library based on small RNAs isolated from partial purified nuclei of Schizosaccharomyces pombe, two novel noncoding RNAs, termed Sp15-70 and Sp18-61, have been identified. Bioinformatics analysis reveals that both the novel RNAs possess a typical secondary structure of box HACA snoRNA and antisense elements to rRNAs. According to the relationship between the structure and function of box HACA snoRNA, Sp15-70 was predicted to direct pseudouridylation in 25S rRNA at U2401 and U2298; Sp18-61 was predicted to direct pseudouridylation in 18S rRNA at U208 and 25S rRNA at U2341. The four predicted pseudouridylation sites were all verified experimentally by the CMC-primer extension analysis. Both Sp15-70 and Sp18-61 were encoded by single copies which were located in the intergenic regions between the CDS of two protein-coding genes on chromosome Ⅰ and Ⅲ of S. pombe, respectively. Putative TATA-like elements can be found upstream from the 5′ end of these snoRNA genes, suggesting that they could be transcribed from their own promoters. Comparison of the two snoRNAs and their functional homologues in diverse organisms reveals that extensive recombinations among different snoRNAs have occurred during the evolution from their primitive progenitors.     

Identification and functional analysis of a novel box C／D snoRNA from Schizosaccharomyces pombe

By constructing and screening the Schizosaccharomyces pombe nuclear cDNA library, a novel small nucleolar RNAs (snoRNA) was identified. The novel snoRNA displays structural features typical of C/D box snoRNA family and possesses a 10-nt-long rRNA antisense element which is predicted to guide the 2‘-O-methylation of the fission yeast 25S rRNA at G940. As expected, the rRNA ribose-methyla- tion site predicted by the novel snoRNA was precisely mapped by a deoxynucleoside triphosphate concentration-dependent primer extension assay. The comparison of functional element of guide snoRNAs among eukaryotes reveals that the novel snoRNA is a partial counterpart of the budding yeast snR60 and was termed snR60-11, snR60-Ⅱ gene nested in the intron of a non-coding RNA gene with an unknown function, which is the first example of a yeast snoRNA encoded in an intron of a non-coding RNA gene. Furthermore, a number of yeast snR60 homologues were also identified from other fungi and fly. Our results reveal that snR60 exhibits diverse genomic organization in eukaryotes, implying the high mobility of snR60 gene in the course of evolution.     

Z25, a novel intronic snoRNA encoded in mammalian nucleolin gene

A novel intronic small nucleolar RNA ( snoRNA) , termed Z25, was identified from mammals by-computer analysis and experimental sequence methods. Z25 is a 69 nucleotides long RNA containing typical boxC/D motifs, terminal stem and an 11-nucleotide sequence complementary to 18S rRNA. In theory, Z25 functions as an RNA guide for the 2'-0-ribose methylation of adenine at position 1678 (human 18S rRNA coordinate) in 18S rRNA. Z25 snoRNA gene was found to be located in the fifth intron of nucleolin gene of human, mouse and rat, demonstrating that the mammalian nucleoline gene is a host gene encoding multiple snoRNAs.     

Effect of GbKTN1 from Gossypium barbadense on cell elongation of fission yeast（Schizosaccharomyces pombe）

The GbKTN1 gene was isolated from 10 DPA fiber cells of Gossypium barbadense using 5′RACE/3′RACE.Full-length cDNA of this gene is 2006 bp, including a 113 bp of 5′untranslated region, a 1563 bp of an open reading frame(ORF), and a 327 bp of 3′untranslated region (excluding the stop codon TAA). The ORF of GbKTN1 encodes a 521-amino acid protein with a predicted size of 55 kD. Near C-terminal of the deduced protein there is a putative ATP binding site between amino acid residues from 233 to 414. Southern blot analysis indicated that the GbKTN1 was a single copy gene in G barbadense. Combining semi-quantitative RT-PCR with Southern blot hybridization revealed that GbKTN1 expressed in all the organs detected such as roots, stems, leaves and fibers. However, the mRNA of GbKTN1 was the most abundant in fiber cells, while it was the lowest in leaves. The GbKTN1 cDNA was transformed into S. pombe to verify its function on cell elongation. Results showed that most yeast cells over expressing GbKTN1 gene were elongated dramatically with an average length increase of 2.18 times than that of the non-induced cells. Even the morphology of some yeast cells appeared irregularly. To the best of our knowledge this is the first evidence that KTN1 is correlated with cell elongation in vivo.     

New feature extraction in gene expression data for tumor classification

Using gene expression data to discriminate tumor from the normal ones is a powerful method. However, it is sometimes difficult because the gene expression data are in high dimension and the object number of the data sets is very small. The key technique is to find a new gene expression profiling that can provide understanding and insight into tumor related cellular processes. In this paper, we propose a new feature extraction method based on variance to the center of the class and employ the support vector machine to recognize the gene data either normal or tumor. Two tumor data sets are used to demonstrate the effectiveness of our methods. The results show that the performance has been significantly improved.     

Evolutionary Mode of Metallothioneins Inferred from Cd-MT Genes of Tetrahymena

From Tetrahymena thermophila （strain BF5）, the coding region of Cd-MT gene was cloned and sequenced. and identified as MTT1 isoform. A serial duplicate structure is discovered in its amino acid sequence, which separates the coding region into three parts （Part 1：7-61; Part 2：64-118; Part 3：122-162）. The alignments among them and comparison with the corresponding parts of MT1 isoform suggest that MT1 and MTT1 isoforms both come from the same ancient gene that is homologous to Part 1, and Cd-MTs of Tetrahymena are aroused by such ancient gene duplication. The prediction of secondary structures and the analysis of the disulfide-bonding state of cysteine show that there are a lot of differences between MT1 and MTT1 isoforms, which maybe relate to their function mechanism.     

Computer simulations on the gas-liquid phase diagram of Stockmayer fluids

Particle exchange molecular dynamics （PEMD） simulation technique is proposed to study the gas-liquid phase diagram of fluids. In the simulations, the fluid particles can be transferred between the two coupled boxes, which possess constant total number of particles and volume. The particle transfer is controlled by the difference of chemical potential in the respective simulation box. After equilibrium the two boxes have the same pressure, temperature and chemical potential. The method is further used to study the gas-liquid phase diagram of Stockmayer fluid. Increasing the dipole strength will enhance the critical temperature. The predicted critical points are in agreement with those from Gibbs ensemble Monte Carlo simulations, while the small systematic difference is attributed to the system size effects and the thermostat methods.     

Identification and characterization of two novel box H/ACA snoRNAs from Schizosaccharomyces pombe

Ineukaryotes,alargenumberofsmallnucleolar RNAs(snoRNAs)accumulatedwithinthenucleolus playimportantrolesinprecursorribosomalRNA(pre RNA)processingandmaturation[1].AllsnoR NAs,withtheexceptionofRNaseMRP,canbe broadlydividedintotwoexpendinggroups,boxC/D andH/ACAsnoRNAs,basedonconservedsequence elements[2].BoxC/DsnoRNAscontaintwocon servedshortsequencemotifs,boxC(UGAUGA)and boxD(CUGA),locatedonlyafewnucleotidesaway fromthe5′and3′ends,respectively,generallyas partofatypical5′3…     

Identification and evolutionary implication of four novel box H/ACA snoRNAs from Giardia lamblia

During the biogenesis of eukaryotic ribosomes, the nascent pre-rRNA undergoes a complex pattern of nu- cleotide modifications of its mature rRNA sequences, including 2′-O-ribose methylation and pseudouridyla- tion, which are critical for the biogenesis a…     

家蚕新的非编码RNA功能初探

构建了家蚕50～500 nt非编码RNA的cDNA文库,发现了189个新的ncRNA,其中一个小RNA-Bm-86在家蚕幼虫和蛹中的表达量显著高于卵和成虫.利用生物信息学软件对该ncRNA的特性及其与上下游基因的关系进行了深入分析,并利用Northern杂交和半定量RT-PCR技术对该ncRNA与其宿主基因在家蚕不同组织的表达情况进行了研究.结果发现,该ncRNA属于H/ACA box snoRNA家族,是由家蚕eIF5A基因的第二个内含子转录产生的,且在家蚕中没有明显的靶标位点.分析其上下游基因结构发现,在其上游263 bp处存在着一个可能的启动子位点,表明其有独立转录的倾向.进一步分析Bm-86与其宿主基因eIF5A在家蚕不同组织部位的表达情况发现,二者在表达上存在着负相关的关系,且该现象在丝腺中尤其明显.推测Bm-86可能通过影响eIF5A基因的表达参与到家蚕丝腺发育调控过程中.     

A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis

Although the U3 small nucleolar RNA (snoRNA), a member of the box C/D class of snoRNAs, was identified with the spliceosomal small nuclear RNAs (snRNAs) over 30 years ago, its function and its associated protein components have remained more elusive. The U3 snoRNA is ubiquitous in eukaryotes and is required for nucleolar processing of pre-18S ribosomal RNA in all organisms where it has been tested. Biochemical and genetic analyses suggest that U3 pre-rRNA base-pairing interactions mediate endonucleolytic pre-rRNA cleavages. Here we have purified a large ribonucleoprotein (RNP) complex from Saccharomyces cerevisiae that contains the U3 snoRNA and 28 proteins. Seventeen new proteins (Utp1 17) and Rrp5 were present, as were ten known components. The Utp proteins are nucleolar and specifically associated with the U3 snoRNA. Depletion of the Utp proteins impedes production of the 18S rRNA, indicating that they are part of the active pre-rRNA processing complex. On the basis of its large size (80S; calculated relative molecular mass of at least 2,200,000) and function, this complex may correspond to the terminal knobs present at the 5' ends of nascent pre-rRNAs. We have termed this large RNP the small subunit (SSU) processome.     

粟酒裂殖酵母SnoRNA snR90基因缺失株的筛选鉴定

snR90是在粟酒裂殖酵母中发现的一种单基因编码的box H/ACA类snoRNA.在利用遗传手段在粟酒裂殖酵母基因组中敲除了snR90基因后,通过选择性培养基、PCR、Northern杂交这一系列方法筛选鉴定出snR90基因缺失株.该基因缺失株ΔsnR90的成功构建对snR90功能的分析很有的意义.     

An early evolutionary origin for the minor spliceosome

The minor spliceosome is a ribonucleoprotein complex that catalyses the removal of an atypical class of spliceosomal introns (U12-type) from eukaryotic messenger RNAs. It was first identified and characterized in animals, where it was found to contain several unique RNA constituents that share structural similarity with and seem to be functionally analogous to the small nuclear RNAs (snRNAs) contained in the major spliceosome. Subsequently, minor spliceosomal components and U12-type introns have been found in plants but not in fungi. Unlike that of the major spliceosome, which arose early in the eukaryotic lineage, the evolutionary history of the minor spliceosome is unclear because there is evidence of it in so few organisms. Here we report the identification of homologues of minor-spliceosome-specific proteins and snRNAs, and U12-type introns, in distantly related eukaryotic microbes (protists) and in a fungus (Rhizopus oryzae). Cumulatively, our results indicate that the minor spliceosome had an early origin: several of its characteristic constituents are present in representative organisms from all eukaryotic supergroups for which there is any substantial genome sequence information. In addition, our results reveal marked evolutionary conservation of functionally important sequence elements contained within U12-type introns and snRNAs.     

Conservation between yeast and man of a protein associated with U5 small nuclear ribonucleoprotein

The process of nuclear pre-messenger RNA splicing is similar in Saccharomyces cerevisiae and metazoan cells in that the two-step mechanism is identical and the reaction occurs in a large ribonucleoprotein complex, the spliceosome. Little is known, however, about the degree of conservation of splicing factors other than of the small nuclear RNAs (snRNAs). Yeast counterparts of the metazoan spliceosomal snRNAs (U1, U2, U4, U5 and U6) have been identified but, with the exception of U6, the yeast snRNAs are larger and sequence similarity is limited to short regions. By using antibodies against the yeast PRP8 protein, a pre-mRNA splicing factor of relative molecular mass 280,000 (Mr280K) stably associated with U5 small nuclear ribonucleoproteins (snRNPs), we have now identified an immunologically related protein in HeLa cell nuclear extracts. The HeLa cell protein has an Mr greater than 200K and is associated with purified 20S U5 snRNPs. This is the first report of phylogenetic conservation between yeast and man of a protein splicing factor.