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PubMed1995

U24, a novel intron-encoded small nucleolar RNA with two 12 nt long, phylogenetically conserved complementarities to 28S rRNA.

Following computer searches of sequence banks, we have positively identified a novel intronic snoRNA, U24, encoded in the ribosomal protein L7a gene in humans and chicken. Like previously reported intronic snoRNAs, U24 is devoid of a 5'-trimethyl-cap. U24 is immunoprecipitated by an antifibrillarin antibody and displays an exclusively nucleolar localization by fluorescence microscopy after in situ hybridization with antisense oligonucleotides. In vertebrates, U24 is a 76 nt long conserved RNA which is metabolically stable, present at approximately 14,000 molecules per human HeLa cell. U24 exhibits a 5'-3' terminal stem-box C-box D structure, typical for several snoRNAs, and contains two 12 nt long conserved sequences complementary to 28S rRNA. It is, therefore, strikingly related to U14, U20 and U21 snoRNAs which also possess long sequences complementary to conserved sequences of mature 18S or 28S rRNAs. In 28S rRNA the two tracts complementary to U24 are adjacent to each other, they involve several methylated nucleotides and are surprisingly close, within the rRNA secondary structure, to complementarities to snoRNAs U18 and U21. Identification of the yeast Saccharomyces cerevisiae U24 gene directly confirms the outstanding conservation of the complementarity to 28S rRNA during evolution, suggesting a key role of U24 pairing to pre-rRNA during ribosome biogenesis, possible in the control of pre-rRNA folding. Yeast S.cerevisiae U24 is also intron-encoded but not in the same host-gene as in humans or chicken.

Animals↗
PubMed1998

Characterization of a novel trypanosomatid small nucleolar RNA.

Trypanosomes possess unique RNA processing mechanisms including trans- splicing of pre-mRNA and RNA editing of mitochondrial transcripts. The previous finding of a trimethylguanosine (TMG) capped U3 homologue in trypanosomes suggests that rRNA processing may be related to the processing in other eukaryotes. In this study, we describe the first trypanosomatid snoRNA that belongs to the snoRNAs that were shown to guide ribose methylation of rRNA. The RNA, identified in the monogenetic trypanosomatid Leptomonas collosoma, was termed snoRNA-2 and is encoded by a multi-copy gene. SnoRNA-2 is 85 nt long, it lacks a 5' cap and possesses the C and D boxes characteristic to all snoRNAs that bind fibrillarin. Computer analysis indicates a potential for base-pairing between snoRNA-2 and 5.8S rRNA, and 18S rRNA. The putative interaction domains obey the rules suggested for the interaction of guide snoRNA with its rRNA target for directing ribose methylation on the rRNA. However, mapping the methylated sites on the 5.8S rRNA and 18S rRNA indicates that the expected site on the 5.8S is methylated, whereas the site on the 18S is not. The proposed interaction with 5.8S rRNA is further supported by the presence of psoralen cross-link sites on snoRNA-2. GenBank search suggests that snoRNA-2 is not related to any published snoRNAs. Because of the early divergence of the Trypanosomatidae from the eukaryotic lineage, the presence of a methylating snoRNA that is encoded by a multi-copy gene suggests that methylating snoRNAs may have evolved in evolution from self-transcribed genes.

Animals↗
PubMed2000

The 28S-18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor.

In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from approximately 11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an approximately 55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A' pre-rRNA processing sites within the 5' external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5' ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C. fasciculata and Trypanosoma brucei involves 3'-terminal addition of three A residues that are not present in the corresponding DNA sequences.

Animals↗
PubMed2004

Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo.

Silencing gene expression by siRNAs is rapidly becoming a powerful tool for the genetic analysis of mammalian cells. However, the rapid degradation of siRNA and the limited duration of its action call for an efficient delivery technology. Accordingly, we describe here that Atelocollagen complexed with siRNA is resistant to nucleases and is efficiently transduced into cells, thereby allowing long-term gene silencing. Site-specific in vivo administration of an anti-luciferase siRNA/Atelocollagen complex reduced luciferase expression in a xenografted tumor. Furthermore, Atelocollagen-mediated transfer of siRNA in vivo showed efficient inhibition of tumor growth in an orthotopic xenograft model of a human non-seminomatous germ cell tumor. Thus, for clinical applications of siRNA, an Atelocollagen-based non-viral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo.

Animals↗
PubMed1990

Lethal and temperature-sensitive mutations and their suppressors identify an essential structural element in U2 small nuclear RNA.

U2 snRNA is an essential component of the splicing apparatus in eukaryotic cells. Three possible secondary structures for the highly conserved 5' half of U2 snRNA are consistent with U2 phylogenetic sequence variation. To distinguish among these models and to test the function of U2 structural elements, we made greater than 35 mutations in the yeast U2 snRNA gene. Some of the mutations were designed in pairs so that combinations could be made that would restore base-pairing to differentiate helix requirements from primary sequence requirements. The mutations identify an essential stem-and-loop structure adjacent to the branchpoint interaction region. A conserved complementarity to the loop just upstream of the Sm site and an additional conserved stem-loop are dispensable for U2 function, even in the background of a previously identified large internal deletion. Non-Watson-Crick base appositions at the 53-62 base pair in the essential stem lead to a variety of temperature and KCl-sensitive phenotypes, as well as an accumulation of unspliced precursors in vivo. Chemical structure probing of U2 RNA in vivo reveals that the bulk of U2 in a yeast cell adopts a structure in good agreement with that deduced from genetic results. We suggest that this stem-loop is not a binding site for an intrinsic U2 snRNP protein but may interact with other factors during spliceosome assembly or splicing.

Base Sequence↗
PubMed2004

Variants of U1 small nuclear RNA assemble into spliceosomal complexes.

In this study, the existence of additional U1 snRNA variants in the posterior silk gland of the Bombyx mori Nistari strain from India was investigated. Three new U1 variants were detected. One of the new isoforms (U1 SG1) was found to be preferentially assembled into high molecular weight spliceosomal complexes in comparison with the total cellular lysate RNA control. Structural and nucleotide differences were examined in these new isoforms and compared with the previously reported U1 variants. Free energy (Delta G) values for the entire U1 snRNA secondary structures as well as the individual stem/loops (I, II, III and IV) domains of the isoforms were estimated to determine their structural stability.

Animals↗
PubMed2006

Down-regulation of Sox10 with specific small interfering RNA promotes transdifferentiation of Schwannoma cells into myofibroblasts.

Neural crest-derived cells are noted for the long lasting plasticity during lineage commitment process and the potential for transdifferentiation into other neural crest derivatives. Schwann cells in particular have been reported to transdifferentiate into melanocytes and myofibroblasts. Detailed studies of transdifferentiation at the molecular level have been hampered by difficulty in isolating sufficient quantity of primary cells or cellular materials. Here, we describe a robust in vitro system in which Schwannoma cells undergo an apparent transdifferentiation into myofibroblasts. Importantly, we induce the transdifferentiation by down-regulating a single transcription factor, Sox10, thereby identifying a key molecular event in this process. Myofibroblasts thus generated showed carbachol-stimulated contraction and calcium transients and express several established myofibroblast-specific genes. These results suggest that generating desired cell types based on "knock-down" of critical genes may be a viable strategy.

Actins↗
PubMed1988

Three types of rat U1 small nuclear RNA genes with different flanking sequences are induced to express in vivo.

There are about 50 copies of U1 RNA genes/pseudogenes in the rat genome. To date, we have isolated so far 25 phage clones carrying a U1 RNA gene/pseudogene from two rat genomic libraries. The 12 clones were selected by hybridization with the U1 RNA coding region under a stringent condition, and were mapped and sequenced. Here, we report three types of U1 RNA genes with different flanking sequences, all of which were shown to be induced to express in vivo by transfection with their polylinker-inserted maxi U1 RNA genes into cultured rat cells. Although these three classes of U1 RNA genes have few homologous flanking sequences, they provide both upstream and downstream of the genes two conserved blocks, which may possibly play an important role in U1 RNA expression.

Animals↗
PubMed1982

Small nuclear RNA U2 is base-paired to heterogeneous nuclear RNA.

Eukaryotic cells contain a set of low molecular weight nuclear RNA's. One of the more abundant of these is termed U2 RNA. The possibility that U2 RNA is hydrogen-bonded to complementary sequences in other nuclear RNA's was investigated. Cultured human (HeLa) cells were treated with a psoralen derivative that cross-links RNA chains that are base-paired with one another. High molecular weight heterogeneous nuclear RNA was isolated under denaturing conditions, and the psoralen cross-links were reversed. Electrophoresis of the released RNA and hybridization with a human cloned U2 DNA probe revealed that U2 is hydrogen-bonded to complementary sequences in heterogeneous nuclear RNA in vivo. In contrast, U2 RNA is not base-paired with nucleolar RNA, which contains the precursors of ribosomal RNA. The results suggest that U2 RNA participates in messenger RNA processing in the nucleus.

Base Composition↗
PubMed2006

Small interfering RNA targeted to hepatitis C virus 5' nontranslated region exerts potent antiviral effect.

Hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma. Interferon alone or together with ribavirin is the only therapy for HCV infection; however, a significant number of HCV-infected individuals do not respond to this treatment. Therefore, the development of new therapeutic options against HCV is a matter of urgency. In the present study, we have examined vectors carrying short hairpin RNA (shRNA) targeting the 5' nontranslated conserved region of the HCV genome for inhibition of virus replication. Initially, three sequences were selected, and all three shRNAs (psh-53, psh-274, and psh-375) suppressed HCV internal ribosome entry site (IRES)-mediated translation to different degrees in Huh-7 cells. Next, we introduced siRNA into Huh-7.5 cells persistently infected with HCV genotype 2a (JFH1). The most efficient inhibition of JFH1 replication was observed with psh-274, targeted to the portion from subdomain IIId to IIIe of the IRES. Subsequently, Huh-7.5 cells stably expressing psh-274 further displayed a significant reduction in HCV JFH1 replication. The effect of psh-274 on cell-culture-grown HCV genotype 1a (H77) was also evaluated, and inhibition of virus replication and infectivity titers was observed. In the absence of a cell-culture-grown HCV genotype 1b, the effects of psh-274 on subgenomic and full-length replicons were examined, and efficient inhibition of genome replication was observed. Therefore, we have identified a conserved sequence targeted to the HCV genome that can inhibit replication of different genotypes, suggesting the potential of siRNA as an additional therapeutic modality against HCV infection.

5' Untranslated Regions↗
PubMed2006

Analysis of sequence and structural features that identify the B/C motif of U3 small nucleolar RNA as the recognition site for the Snu13p-Rrp9p protein pair.

The eukaryal Snu13p/15.5K protein binds K-turn motifs in U4 snRNA and snoRNAs. Two Snu13p/15.5K molecules bind the nucleolar U3 snoRNA required for the early steps of preribosomal processing. Binding of one molecule on the C'/D motif allows association of proteins Nop1p, Nop56p, and Nop58p, whereas binding of the second molecule on the B/C motif allows Rrp9p recruitment. To understand how the Snu13p-Rrp9p pair recognizes the B/C motif, we first improved the identification of RNA determinants required for Snu13p binding by experiments using the systematic evolution of ligands by exponential enrichment. This demonstrated the importance of a U.U pair stacked on the sheared pairs and revealed a direct link between Snu13p affinity and the stability of helices I and II. Sequence and structure requirements for efficient association of Rrp9p on the B/C motif were studied in yeast cells by expression of variant U3 snoRNAs and immunoselection assays. A G-C pair in stem II, a G residue at position 1 in the bulge, and a short stem I were found to be required. The data identify the in vivo function of most of the conserved residues of the U3 snoRNA B/C motif. They bring important information to understand how different K-turn motifs can recruit different sets of proteins after Snu13p association.

Base Pairing↗
PubMed1990

Structure and evolution of the U2 small nuclear RNA multigene family in primates: gene amplification under natural selection?

The organization of U2 genes was compared in apes, Old World monkeys, and the prosimian galago. In humans and all apes (gibbon, orangutan, gorilla, and chimpanzee), the U2 genes were organized as a tandem repeat of a 6-kb element; however, the restriction maps of the 6-kb elements in these divergent species differed slightly, demonstrating that mechanisms must exist for maintaining sequence homogeneity within this tandem array. In Old World monkeys, the U2 genes were organized as a tandem repeat of an 11-kb element; the restriction maps of the 11-kb elements in baboon and two closely related macaques, bonnet and rhesus monkeys, also differed slightly, confirming that efficient sequence homogenization is an intrinsic property of the U2 tandem array. Interestingly, the 11-kb monkey repeat unit differed from the 6-kb hominid repeat unit by a 5-kb block of monkey-specific sequence. Finally, we found that the U2 genes of the prosimian galago were dispersed rather than tandemly repeated, suggesting that the hominid and Old World monkey U2 tandem arrays resulted from independent amplifications of a common ancestral U2 gene. Alternatively, the 5-kb monkey-specific sequence could have been inserted into the 6-kb array or deleted from the 11-kb array soon after divergence of the hominid and Old World monkey lineages.

Animals↗
PubMed1994

Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

Promoter elements in the 5' flanking regions of vertebrate U6 RNA genes have been shown to be both necessary and sufficient for transcription by RNA polymerase III. We have recently isolated and characterized a variant human U6 gene (87U6) that can be transcribed by RNA polymerase III in vitro in the absence of any natural 5' or 3' flanking sequences. This gene contains 10 nucleotide differences from the previously characterized human U6 gene (wtU6) within the coding region but has no homology to wtU6 in the upstream promoter region. By constructing chimeras between these two genes, we have shown that mutation of as few as two nucleotides in the coding region of the human U6 RNA gene is sufficient to create an internal promoter that is functional in vitro. A T-to-C transition at position 57 and a single T deletion at position 52 produce an internal U6 promoter that is nearly as active in vitro as the external U6 polymerase III promoter utilized by wtU6. Neither of these residues is absolutely conserved during evolution, and both of these nucleotide changes occur within the previously noted A box homology. Deletion and linker scanning mutations within the coding region of this variant U6 gene suggest that, in addition to the central region including bp 52 and 57, sequences at the extreme 5' end of the gene are critical for efficient transcription. In contrast, flanking sequences have a minor effect on transcriptional efficiency. This arrangement is unique among internal RNA polymerase III promoters and may indicate unique regulation of this gene.

Base Sequence↗
PubMed1985

Human U1 small nuclear RNA genes: extensive conservation of flanking sequences suggests cycles of gene amplification and transposition.

The DNA immediately flanking the 164-base-pair U1 RNA coding region is highly conserved among the approximately 30 human U1 genes. The U1 multigene family also contains many U1 pseudogenes (designated class I) with striking although imperfect flanking homology to the true U1 genes. Using cosmid vectors, we now have cloned, characterized, and partially sequenced three 35-kilobase (kb) regions of the human genome spanning U1 homologies. Two clones contain one true U1 gene each, and the third bears two class I pseudogenes 9 kb apart in the opposite orientation. We show by genomic blotting and by direct DNA sequence determination that the conserved sequences surrounding U1 genes are much more extensive than previously estimated: nearly perfect sequence homology between many true U1 genes extends for at least 24 kb upstream and at least 20 kb downstream from the U1 coding region. In addition, the sequences of the two new pseudogenes provide evidence that class I U1 pseudogenes are more closely related to each other than to true genes. Finally, it is demonstrated elsewhere (Lindgren et al., Mol. Cell. Biol. 5:2190-2196, 1985) that both true U1 genes and class I U1 pseudogenes map to chromosome 1, but in separate clusters located far apart on opposite sides of the centromere. Taken together, these results suggest a model for the evolution of the U1 multigene family. We speculate that the contemporary family of true U1 genes was derived from a more ancient family of U1 genes (now class I U1 pseudogenes) by gene amplification and transposition. Gene amplification provides the simplest explanation for the clustering of both U1 genes and class I pseudogenes and for the conservation of at least 44 kb of DNA flanking the U1 coding region in a large fraction of the 30 true U1 genes.

Base Sequence↗