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PMID: 20348447 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Regulation of pre-mRNA splicing in Xenopus oocytes by targeted 2'-O-methylation.

RNA (New York, N.Y.) ·Vol. 16 ·No. 5 ·2010-05-00 ·Pages 1078-85

Ge J, Liu H, Yu YT

Abstract

The 2'-OH group of the branch point adenosine is a key moiety to initiate pre-mRNA splicing. We use RNA-guided RNA modification to target the pre-mRNA branch point adenosine for 2'-O-methylation, with the aim of blocking pre-mRNA splicing in vertebrate cells. We show that, under certain conditions, injection of a branch point-specific artificial box C/D RNA into Xenopus oocytes effectively 2'-O-methylates adenovirus pre-mRNA at the target nucleotide. However, 2'-O-methylation at the authentic branch point activates a host of cryptic branch points, thus allowing splicing to continue. These cryptic sites are mapped, and mutated. Upon injection, pre-mRNA free of cryptic branch points fails to splice when the branch point-specific box C/D RNA is present. However, 2'-O-methylation at the branch point does not prevent pre-mRNA from being assembled into pre-catalytic spliceosome-like complexes prior to the first chemical step of splicing. Our results demonstrate that RNA-guided pre-mRNA modification can occur in the nucleoplasm of vertebrate cells, thus offering a powerful tool for molecular biology research.

MeSH Terms
Adenosine/chemistry Adenoviridae/genetics,metabolism Animals Base Sequence Female Gene Expression Humans In Vitro Techniques Methylation Models, Biological Nucleic Acid Conformation Oocytes/metabolism Plasmids/genetics RNA Precursors/chemistry,genetics,metabolism RNA Splicing RNA, Small Nucleolar/chemistry,genetics,metabolism RNA, Viral/genetics,metabolism Spliceosomes/metabolism Xenopus
Chemicals
RNA Precursors RNA, Small Nucleolar RNA, Viral Adenosine
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Ge Junhui
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642, USA.
Liu Huimin
Yu Yi-Tao
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1469-9001
DOI
10.1261/rna.2060210
Published
2010-05-00
Epub
2010-00-26
Pages
1078-85
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC2856880
Subset
IM
Grants
NIGMS NIH HHS · R01 GM062937 · United States
NIGMS NIH HHS · GM62937 · United States
External Links
PubMed source DOI: 10.1261/rna.2060210 PMC Full Text View journal details
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