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Mouse U14 snRNA is encoded in an intron of the mouse cognate hsc70 heat shock gene.

ABSTRACT: Mouse U14 snRNA (previously designated mouse 4.5S hybRNA) is an evolutionarily conserved eukaryotic low molecular weight RNA capable of intermolecular hybridization with both homologous and heterologous 18S rRNA (1). A single genomic fragment of mouse DNA containing the U14 snRNA gene(s) has been isolated from a Charon 4A lambda phage mouse genomic library and sequenced. Results have surprisingly revealed the presence of three U14 snRNA-homologous regions positioned within introns 5, 6, and 8 of the mouse cognate hsc70 heat shock gene. Comparative analysis with the previously reported rat and human cognate hsc70 genes revealed a similar positioning of U14 snRNA-homologous sequences within introns 5, 6 and 8 of the respective rat and human genes. The U14 sequences contained in all three introns of all three organisms are highly homologous to each other and well conserved with respect to the diverging intron sequences flanking each U14-homologous sequence. Comparison of the mouse U14 snRNA sequence with the U14 DNA sequences contained in the three mouse hsc70 introns indicates that intron 5 is utilized for U14 snRNA synthesis in normally growing mouse ascites cells. Analysis of the determined mouse, rat, and human U14-homologous sequences and the upstream and downstream flanking regions did not reveal the presence of any previously defined RNA polymerase I, II, or III binding sites. This suggests that either higher eukaryotic U14 snRNA is transcribed from a unique transcriptional promoter sequence, or alternatively, is generated by intron processing of the hsc70 pre-mRNA transcript.

SUBMITTER: Liu J

PROVIDER: S-EPMC332611 | biostudies-other | 1990 Nov

REPOSITORIES: biostudies-other

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Mouse U14 snRNA is encoded in an intron of the mouse cognate hsc70 heat shock gene.

Liu J J Maxwell E S ES

Nucleic acids research 19901101 22

Mouse U14 snRNA (previously designated mouse 4.5S hybRNA) is an evolutionarily conserved eukaryotic low molecular weight RNA capable of intermolecular hybridization with both homologous and heterologous 18S rRNA (1). A single genomic fragment of mouse DNA containing the U14 snRNA gene(s) has been isolated from a Charon 4A lambda phage mouse genomic library and sequenced. Results have surprisingly revealed the presence of three U14 snRNA-homologous regions positioned within introns 5, 6, and 8 of ...[more]

PMID: 2251119

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Project description:BackgroundHepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Because of the limited effectiveness of existing vaccines and drugs, development of novel antiviral strategies is urgently needed. Heat stress cognate 70 (Hsc70) is an ATP-binding protein of the heat stress protein 70 family. Hsc70 has been found to be required for HBV DNA replication. Here we report, for the first time, that combined siRNAs targeting viral gene and siHsc70 are highly effective in suppressing ongoing HBV expression and replication.MethodsWe constructed two plasmids (S1 and S2) expressing short hairpin RNAs (shRNAs) targeting surface open reading frame of HBV(HBVS) and one plasmid expressing shRNA targeting Hsc70 (siHsc70), and we used the EGFP-specific siRNA plasmid (siEGFP) as we had previously described. First, we evaluated the gene-silencing efficacy of both shRNAs using an enhanced green fluorescent protein (EGFP) reporter system and flow cytometry in HEK293 and T98G cells. Then, the antiviral potencies of HBV-specific siRNA (siHBV) in combination with siHsc70 in HepG2.2.15 cells were investigated. Moreover, type I IFN and TNF-α induction were measured by quantitative real-time PCR and ELISA.ResultsCotransfection of either S1 or S2 with an EGFP plasmid produced an 80%-90% reduction in EGFP signal relative to the control. This combinational RNAi effectively and specifically inhibited HBV protein, mRNA and HBV DNA, resulting in up to a 3.36 log10 reduction in HBV load in the HepG2.2.15 cell culture supernatants. The combined siRNAs were more potent than siHBV or siHsc70 used separately, and this approach can enhance potency in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells while forestalling escape by mutant HBV. The antiviral synergy of siHBV used in combination with siHsc70 produced no cytotoxicity and induced no production of IFN-α, IFN-β and TNF-α in transfected cells.ConclusionsOur combinational RNAi was sequence-specific, effective against wild-type and mutant drug-resistant HBV strains, without triggering interferon response or producing any side effects. These findings indicate that combinational RNAi has tremendous promise for developing innovative therapy against viral infection.

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Mouse U14 snRNA is encoded in an intron of the mouse cognate hsc70 heat shock gene.

Publications

Mouse U14 snRNA is encoded in an intron of the mouse cognate hsc70 heat shock gene.

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