Splicing by exon exclusion impaired by artificial box c/d RNA targeted to branch-point adenosine.
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%.
Semenov
Dmitry V
DV
Institute of Chemical Biology and Fundamental Medicine, Siberian Division, Russian Academy of Sciences, Novosibirsk, Russia. semenov@niboch.nsc.ru
Vratskih
Oksana V
OV
Kuligina
Elena V
EV
Richter
Vladimir A
VA
D000241:Adenosine / Q000378:metabolism*; D001483:Base Sequence; D045744:Cell Line, Tumor; D005091:Exons / Q000235:genetics; D018869:Heat-Shock Response / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*
D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D012342:RNA, Small Nuclear; D012313:RNA; D000241:Adenosine