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  • Title: Splicing by exon exclusion impaired by artificial box c/d RNA targeted to branch-point adenosine.
    Author: Semenov DV, Vratskih OV, Kuligina EV, Richter VA.
    Journal: Ann N Y Acad Sci; 2008 Aug; 1137():119-24. PubMed ID: 18837934.
    Abstract:
    Box C/D small nucleolar RNAs guide the site-specific 2'-O-ribose methylation of nucleotides in target rRNAs and snRNAs. In this study we used the ability of C/D box snoRNAs to guide modification of target RNAs with the aim of affecting the expression of predetermined human genes. We constructed an analogue of the human U24 box C/D RNA, the first antisense element of which was directed to induce 2'-O-ribose methylation of branch-point adenosine in the intron of the human heat-shock cognate protein (HSC8) pre-mRNA. The second antisense element was directed to induce methylation of G1702 in human 18S rRNA. Constructed artificial box C/D RNA was obtained by in vitro transcription by T7 RNA polymerase from a synthetic DNA template. It was shown that passive transfection of human adenocarcinoma MCF-7 cells with the artificial box C/D RNA, accompanied by heat shock, induced 2'-O-ribose methylation of targeted 18S rRNA guanosine G1702 and impaired splicing of the HSC8 pre-mRNA. Derangement of HSC8 splicing was the result of exclusion of the exon downstream to the targeted intron branch point. The total efficacy of pre-mRNA splicing impairment induced by artificial C/D box RNA was estimated as 6 to 10%.
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