Isolation and characterization of rabbit anti-m3 2,2,7G antibodies.

Antibodies specific for intact 2,2,7-trimethylguanosine (m3 2,2,7G) were induced by immunization of rabbits with a nucleoside-human serum albumen (HSA) conjugate. Competition radioimmunoassay showed that the antibody distinguishes well between intact m3 2,2,7G and its alkali-hydrolysed form (m3 2,2,7G*). Antibody specificity is largely dependent on the presence of all three methyl groups in m3 2,2,7G: none of the less extensively methylated nucleosides m7G, m2G and m2 2,2G is able to compete efficiently with the homologous hapten. Little or no competition was observed with m1G, m1A, m6A, m5U and each of the four unmodified ribonucleosides. Binding studies with nucleoplasmic RNAs from Ehrlich ascites cells suggest that the antibody reacts specifically with the m3 2,2,7G-containing cap structure of the small nuclear U-RNAs (U-snRNAs). Thus the antibody should be a valuable tool for studying the role of the 5'-terminal regions of the U-snRNAs of eucaryotic cells.     

Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs.

Trypanosomes use trans splicing to place a common 39-nucleotide spliced-leader sequence on the 5' ends of all of their mRNAs. To identify likely participants in this reaction, we used antiserum directed against the characteristic U RNA 2,2,7-trimethylguanosine (TMG) cap to immunoprecipitate six candidate U RNAs from total trypanosome RNA. Genomic Southern analysis using oligonucleotide probes constructed from partial RNA sequence indicated that the four largest RNAs (A through D) are encoded by single-copy genes that are not closely linked to one another. We have cloned and sequenced these genes, mapped the 5' ends of the encoded RNAs, and identified three of the RNAs as the trypanosome U2, U4, and U6 analogs by virtue of their sequences and structural homologies with the corresponding metazoan U RNAs. The fourth RNA, RNA B (144 nucleotides), was not sufficiently similar to known U RNAs to allow us to propose an identify. Surprisingly, none of these U RNAs contained the consensus Sm antigen-binding site, a feature totally conserved among several classes of U RNAs, including U2 and U4. Similarly, the sequence of the U2 RNA region shown to be involved in pre-mRNA branchpoint recognition in yeast, and exactly conserved in metazoan U2 RNAs, was totally divergent in trypanosomes. Like all other U6 RNAs, trypanosome U6 did not contain a TMG cap and was immunoprecipitated from deproteinized RNA by anti-TMG antibody because of its association with the TMG-capped U4 RNA. These two RNAs contained extensive regions of sequence complementarity which phylogenetically support the secondary-structure model proposed by D. A. Brow and C. Guthrie (Nature [London] 334:213-218, 1988) for the organization of the analogous yeast U4-U6 complex.     

Synthesis of N2, N2, 7-trimethylguanosine cap derivatives.

Several derivatives of N2,N2-7-trimethylguanosine (m3(2,2,7G)-cap, which was found at the 5' ends of small nuclear RNAs, were synthesized by use of S-phenyl N2,N2,7-trimethylguanosine 5'-phosphorothioate (PhSpm3(2,2,7)G) as a key intermediate. This compound was activated by iodine in the presence of phosphoric acid and diphosphoric acid to give N2,N2,7-trimethylguanosine-5'-diphosphate (ppm3(2,2,7)G) and 5'-triphosphate (ppm3(2,2,7)G), respectively. Similar reactions of PhSpm3(2,2,7)G with ADP and GDP gave capped dinucleoside triphosphates, m3(2,2,7)G5'pppA and m3(2,2,7)G5'pppG, respectively.     

Ribonucleoprotein organization of eukaryotic RNA. XXXII. U2 small nuclear RNA precursors and their accurate 3' processing in vitro as ribonucleoprotein particles

Biosynthetic precursors of U2 small nuclear RNA have been identified in cultured human cells by hybrid-selection of pulse-labeled RNA with cloned U2 DNA. These precursor molecules are one to approximately 16 nucleotides longer than mature U2 RNA and contain 2,2,7-trimethylguanosine "caps". The U2 RNA precursors are associated with proteins that react with a monoclonal antibody for antigens characteristic of small nuclear ribonucleoprotein particles. Like previously described precursors of U1 and U4 small nuclear RNAs, the pre-U2 RNAs are recovered in cytoplasmic fractions, although it is not known if this is their location in vivo. The precursors are processed to mature-size U2 RNA when cytoplasmic extracts are incubated in vitro at 37 degrees C. Mg2+ is required but ATP is not. The ribonucleoprotein structure of the pre-U2 RNA is maintained during the processing reaction in vitro, as are the 2,2,7-trimethylguanosine caps. The ribonucleoprotein organization is of major importance, as exogenous, protein-free U2 RNA precursors are degraded rapidly in the in vitro system. Two lines of evidence indicate that the conversion of U2 precursors to mature-size U2 RNA involves a 3' processing reaction. First, the reaction is unaffected by a large excess of mature U2 small nuclear RNP, whose 5' trimethylguanosine caps would be expected to compete for a 5' processing activity. Second, when pre-U2 RNA precursors are first stoichiometrically decorated with an antibody specific for 2,2,7-trimethylguanosine, the extent of subsequent processing in vitro is unaffected. These results provide the first demonstration of a eukaryotic RNA processing reaction in vitro occurring within a ribonucleoprotein particle.     

5'-terminal caps of snRNAs are accessible for reaction with 2,2,7-trimethylguanosine-specific antibody in intact snRNPs

Immune precipitation assays with antibodies specific for 2,2,7-trimethylguanosine (m2,2,7(3)G) have been used to study the accessibility of the 5'-terminal m2,2,7(3)G-containing caps of eucaryotic small nuclear RNAs (snRNAs) either as naked RNAs or in intact small nuclear ribonucleoprotein (snRNPs). The antibody selectively precipitates snRNA species U1a, U1b, U2, U4, and U5 from total deproteinized RNA isolated from Ehrlich ascites cells. Binding by the antibody occurs via the m2,2,7(3)G moiety of the snRNAs' caps, since complex formation with the antibody can be completely abolished by excess nucleoside m2,2,7(3)G. The specificity of the antibody is further demonstrated by the complete absence of reaction with deproteinized snRNA species U6, the 5' terminus of which does not contain m2,2,7(3)G. Most importantly, the cap structures of the snRNAs U1a, U1b, U2, U4, and U5 are also accessible for anti-m2,2,7(3)G IgGs when intact snRNPs are reacted with the antibody. In this case, snRNP species U6 is coprecipitated, suggesting that there are intermolecular interactions between this and other snRNPs. Our data demonstrate that the 5'-terminal regions of the above snRNAs are not protected by the snRNP proteins. This finding is of special interest for snRNP species U1, and is discussed in terms of a model which proposes that the 5'-terminal region of U1 participates in the proper alignment of splice junctions in eucaryotic pre-mRNAs (Lerner, M. R., Boyle, J.A., Mount, S.M., Wolin, S.L., and Steitz, J. A. (1980) Nature (Lond.) 283, 220-224).     

Antibodies elicited by N2,N2-7-trimethylguanosine react with small nuclear RNAs and ribonucleoproteins

The 5' ends of U1, U2, U3, U4, and U5 small nuclear RNAs (snRNA) are capped by a structure which contains N2,N2-7-trimethylguanosine (m2,2,7 G). m2,2,7 G was used as hapten to raise antibodies in rabbits, and these antibodies were linked to Sepharose. When deproteinized RNA was passed through this antibody column, these snRNA species were retained by the column. Conversely, 4 S, 5 S, 5.8 S, U6, and 7 S RNA, whose 5' termini do not contain m2,2,7 G, were not recognized. After a nuclear extract was loaded on the column, U1 RNA and some U2 RNA were retained. Therefore, the 5' ends of at least U1 RNA are accessible when this RNA species is in small nuclear ribonucleoprotein particle (snRNP) form. This is of interest, since it has been proposed that the 5' terminus sequence of U1 RNA may hybridize with splice junctions in heterogeneous nuclear ribonucleoprotein particles (hnRNP) during mRNA splicing. The retention of m2,2,7 G-containing RNA species by these antibodies is not due to association of snRNAs or snRNPs with heterogeneous nuclear RNA (hnRNA) or hnRNP (and antibody recognition of 7-monomethylguanosine residues in hnRNA), since the reaction still occurs after removal of hnRNA or hnRNP by sucrose gradient centrifugation.     

U6 snRNA variants isolated from the posterior silk gland of the silk moth Bombyx mori

Five U6 small nuclear RNA (snRNA) isoforms were detected and characterized from the posterior silk gland (PSG) of the silk moth Bombyx mori (Nistari strain). Using the currently accepted U6 secondary structure model as a basis for comparison, the variants were analyzed for nucleotide differences across the sequence with a focus on known functional domains. Differences were observed primarily in single-stranded areas of which sixty percent were found in the highly conserved U4-U6 binding sites. In the Nistari strain, the U6A variant was found to be approximately four times more abundant as part of high molecular weight spliceosomal complexes when compared with U6A in the total unfractionated PSG cell lysate. Additionally, the European 703 B. mori strain total cell lysate U6 snRNA was analyzed and only the dominant U6A isoform initially identified in Nistari was found. Due to U6's essential role in pre-mRNA processing, variants may modulate assemblage of the catalytic core and in doing so potentially affect the rate of splicing. Phylogenetic analysis of the U6 snRNA sequences indicate an ancient divergence of U6 from the self-splicing group II intron module and a high degree of evolutionary conservation across species possibly due to functional constraints on the gene. Using in silico analysis, 35 full-length U6 variants were observed in the recently released Whole Genome Shotgun (WGS) database of the p50T strain. The consensus sequence of these U6 genes from p50T is identical to U6A identified in the Nistari strain. Furthermore p50T variant 1, which is represented in 14 genes, is equivalent to Nistari U6A.     

The box C/D motif directs snoRNA 5'-cap hypermethylation

The 5′-cap structure of most spliceosomal small nuclear RNAs (snRNAs) and certain small nucleolar RNAs (snoRNAs) undergoes hypermethylation from a 7-methylguanosine to a 2,2,7-trimethylguanosine structure. 5′-Cap hypermethylation of snRNAs is dependent upon a conserved sequence element known as the Sm site common to most snRNAs. Here we have performed a mutational analysis of U3 and U14 to determine the cis-acting sequences required for 5′-cap hypermethylation of Box C/D snoRNAs. We have found that both the conserved sequence elements Box C (termed C′ in U3) and Box D are necessary for cap hypermethylation. Furthermore, the terminal stem structure that is formed by sequences that flank Box C (C′ in U3) and Box D is also required. However, mutation of other conserved sequences has no effect on hypermethylation of the cap. Finally, the analysis of fragments of U3 and U14 RNAs indicates that the Box C/D motif, including Box C (C′ in U3), Box D and the terminal stem, is capable of directing cap hypermethylation. Thus, the Box C/D motif, which is important for snoRNA processing, stability, nuclear retention, protein binding, nucleolar localization and function, is also necessary and sufficient for cap hypermethylation of these RNAs.     

Characterization of the highly divergent U2 RNA homolog in the microsporidian Vairimorpha necatrix.

An RNA homologous to U2 RNA and a single copy gene encoding the RNA homolog have been characterized in the microsporidian, Vairimorpha necatrix. The RNA which is 165 nucleotides in length possesses significant similarity to U2 RNA, particularly in the 5' half of the molecule. The U2 homolog contains the highly conserved GUAGUA branch point binding sequence seen in all U2 RNAs except those of the trypanosomes. A U2 RNA sequence element implicated in a U2:U6 RNA intermolecular pairing is also present in the U2 homolog. The V. necatrix U2 RNA homolog differs at positions previously found to be invariant in U2 RNAs and appears to lack an Sm binding site sequence. The RNA can be folded into a secondary structure possessing three of the four principal stem-loops proposed for the consensus U2 RNA structure. A cis-diol containing cap structure is present at the 5' end of the U2 homolog. Unlike the cap structures seen in U-snRNAs and mRNAs it is neither 2,2,7-trimethylguanosine, gamma-monomethyl phosphate, nor 7-methylguanosine.     

Novel human autoantibodies reactive with 5'-terminal trimethylguanosine cap structures of U small nuclear RNA.

A class of RNA-containing particles, U small nuclear/nucleolar ribonucleoprotein particles (U snRNP), are well known to be targets for sera from patients with various autoimmune diseases. In the most cases the protein components carry the antigenic determinants. We have identified serum autoantibodies from three patients with systemic sclerosis that were directed against U1-U5 snRNA by immunoprecipitation of deproteinized 32PO4 labeled HeLa cell total RNA. By competitive radioimmunoprecipitation assays, an experimentally induced anti-2,2,7-trimethylguanosine (TMG) cap structure mAb inhibited the reaction of these antisera. In addition, IgG isolated from the antisera inhibited the anti-TMG mAb reaction to the U snRNA. Furthermore, a structural analog, 7-methylguanosine-triphosphate, competitively inhibited the reaction of the antisera to the U snRNA. Thus we concluded that the TMG cap structure of the U snRNA could be a target for serum autoantibodies.     

The nucleotide sequence of a major glycine transfer RNA from the posterior silk gland of Bombyx mori L.

The nucleotide sequence of tRNA1Gly isolated from the posterior silk gland of Bombyx mori has been determined. This transfer RNA is present in high amounts in the posterior silk gland during the fifth larval instar. It has a GCC anticodon, capable of decoding a major glycine codon in the fibroin messenger RNA, GGU. Structural features of Bombyx tRNA1Gly and its homology to other eukaryotic glycine tRNAs are discussed.     

家蚕丝素蛋白轻链基因（fib-L）启动子序列的克隆及其活性分析

家蚕Bombyx mori丝素蛋白轻链（fibroin light-chain, fib-L）基因fib-L具有在后部丝腺组织专一性、高效性表达的特点。为了利用其启动子构建能够表达外源基因的丝腺生物工厂,本实验对fib-L启动子活性进行了研究。通过PCR法克隆了fib-L启动子元件,序列分析显示fib-L启动子由位于-33 ～ -25处的TATA盒元件和位于－128～－121处的特征性序列GTCAATTT共同组成。用fib-L启动子控制报告基因DsRed进行家蚕BmN细胞和蚕体内的瞬时表达研究,结果表明fib-L启动子可以驱动DsRed报告基因在BmN细胞和家蚕后部丝腺组织中瞬时表达。     

家蚕 Fhx/P25 基因的一种新的转录模式分析研究

家蚕 Fhx/P25 蛋白是丝素蛋白的主要成分之一,过去报道只在家蚕后部丝腺特异的转录表达 . 通过对大规模的家蚕 EST 序列分析发现, Fhx/P25 基因不仅在家蚕后部丝腺高效转录,而且在家蚕幼虫五龄第三天的卵巢组织及其他组织也有转录;分析还发现 Fhx/P25 基因在丝腺和卵巢组织中有不同的转录起始位点,在卵巢组织中的转录起始位点比在丝腺中的至少要提前 115 bp 左右 . 用 RT-PCR 和 FQ-PCR 进一步验证,以上分析结果均正确 . 分析还发现 Fhx/P25mRNA 存在选择性拼接 . 以上结果表明 Fhx/P25 基因并不是组织特异转录基因,它的转录表达存在复杂的调控机制,可能还有其他功能 .     

家蚕Wnt信号通路下游关键基因Pangolin转录剪接体X3的克隆和分析

【目的】克隆家蚕Bombyx mori Wnt信号通路下游关键基因Pangolin isoforms A/H/I/S转录剪接体X3 (Pangolin X3),分析其序列和表达特征。【方法】从NCBI数据库检索家蚕Pangolin X3,根据其编码序列(coding sequence, CDS)设计引物,利用PCR从家蚕幼虫中肠和血淋巴中进行克隆并测序验证。利用SilkDB 3.0, SMART,多序列比对和系统发育树分析Pangolin X3的序列特征。利用qRT-PCR分析Pangolin X3在家蚕5龄第3天幼虫不同组织(头、血淋巴、体壁、性腺、中肠、前部丝腺、中部丝腺、后部丝腺、脂肪体和马氏管)中的相对表达水平。【结果】从家蚕幼虫中肠和血淋巴克隆了Pangolin X3(GenBank登录号：XM＿038020921)的CDS,其开放阅读框长1 560 bp,编码519个氨基酸残基,预测分子量为55.86 kD,预测等电点为7.53。Pangolin X3蛋白含有保守的β-catenin结合位点和HMG结构域,其氨基酸序列在不同的昆虫中比较保守,特别是与DNA结合的HMG结构域...     

Distribution of U3 small nucleolar RNA and fibrillarin during early embryogenesis in Caenorhabditis elegans

U3 small nucleolar RNA (snoRNA) is one of the members of the box C/D class of snoRNA and is essential for ribosomal RNA (rRNA) processing to generate 18S rRNA in the nucleolus. Although U3 snoRNA is abundant, and is well conserved from yeast to mammals, the genes encoding U3 snoRNA in C. elegans have long remained unidentified. A recent RNomics study in C. elegans predicted five distinct U3 snoRNA genes. However, characterization of these candidates for U3 snoRNA has yet to be performed. In this study, we isolated and characterized four candidate RNAs for U3 snoRNA from the immunoprecipitated RNAs of C. elegans using an antibody against the 2,2,7-trimethylguanosine (TMG) cap. The sequences were identical to the predicted U3 sequences in the RNomics study. Here, we show the several lines of evidence that the isolated RNAs are the true U3 snoRNAs of C. elegans. Moreover, we report the novel expression pattern of U3 snoRNA and fibrillarin, which is an essential component of U3 small nucleolar ribonucleoprotein complex, during early embryo development of C. elegans. To our knowledge, this is the first observation of the inconsistent localization U3 snoRNA and fibrillarin during early embryogenesis, providing novel insight into the mechanisms of nucleologenesis and ribosome production during early embryogenesis.     

Isolation and identification of the messenger RNA for silk fibroin from Bombyx mori

The messenger RNA for the protein silk fibroin has been isolated from the posterior silk gland of Bombyx mori and identified by partial sequence analysis. The sequence of mRNA could be predicted because the protein has a simple repetitious primary structure in which glycine residues comprise 45% of all residues and alternate predominantly with alanine and serine.     

Effect of estradiol-17beta on cell area, lumen area and trehalase activity of posterior silk gland of Bombyx mori L

Estradiol-17beta (E2) at the dose of 1 microg/g caused an increase in cell area, lumen area and the total (cell + lumen) area of posterior silk gland (PSG) in Bombyx mori indicating that exogenously applied estradiol-17beta has a regulatory influence on silk gland activity. A dose-dependent variation in trehalase activity of PSG was found on the 5th day after topical administration of estradiol on 1st and 2nd day of the fifth larval instar. Of all the doses of E2 used, 1 microg/g dose had maximum stimulatory effect on trehalase activity. Co-administration of each of a specific receptor antagonist for estradiol, the ICI-182780 and a protein biosynthetic blocker, cycloheximide with E2 suppressed the E2-induced increase in silk gland activity. The results suggest some specific metabolic action of E2 on silk gland and offer a promising way for future investigations regarding the physiological significance of vertebrate steroids in insects.