Splicing by exon exclusion impaired by artificial box c/d RNA targeted to branch-point adenosine

Ann N Y Acad Sci. 2008 Aug:1137:119-24. doi: 10.1196/annals.1448.037.

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%.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / metabolism*
  • Base Sequence
  • Cell Line, Tumor
  • Exons* / genetics
  • Heat-Shock Response / genetics
  • Humans
  • Molecular Sequence Data
  • Oligonucleotides, Antisense / genetics
  • Oligonucleotides, Antisense / metabolism
  • RNA / genetics
  • RNA / metabolism*
  • RNA Precursors / genetics
  • RNA Precursors / metabolism
  • RNA Splicing*
  • RNA, Small Nuclear / genetics
  • RNA, Small Nuclear / metabolism*

Substances

  • Oligonucleotides, Antisense
  • RNA Precursors
  • RNA, Small Nuclear
  • RNA
  • Adenosine