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Bio Protoc. 2018 Feb 5;8(3). pii: e2717. doi: 10.21769/BioProtoc.2717.

Immunoprecipitation of Tri-methylated Capped RNA.

Author information

1
Department of Microbiology, Program in Cancer Cell Biology, WVU Cancer Institute, West Virginia University, Morgantown, WV, USA.
2
Department of Biochemistry & Molecular Biology, U.F. Health Cancer Center, University of Florida, Gainesville, FL, USA.
3
Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA.
4
Howard Hughes Medical Institute, Yale University, New Haven, CT 06536, USA.
5
Yale Cancer Center, New Haven, CT, USA.

Abstract

Cellular quiescence (also known as G0 arrest) is characterized by reduced DNA replication, increased autophagy, and increased expression of cyclin-dependent kinase p27Kip1. Quiescence is essential for wound healing, organ regeneration, and preventing neoplasia. Previous findings indicate that microRNAs (miRNAs) play an important role in regulating cellular quiescence. Our recent publication demonstrated the existence of an alternative miRNA biogenesis pathway in primary human foreskin fibroblast (HFF) cells during quiescence. Indeed, we have identified a group of pri-miRNAs (whose mature miRNAs were found induced during quiescence) modified with a 2,2,7-trimethylguanosine (TMG)-cap by the trimethylguanosine synthase 1 (TGS1) protein and transported to the cytoplasm by the Exportin-1 (XPO1) protein. We used an antibody against (TMG)-caps (which does not cross-react with the (m7G)-caps that most pri-miRNAs or mRNAs contain [Luhrmann et al., 1982]) to perform RNA immunoprecipitations from total RNA extracts of proliferating or quiescent HFFs. The novelty of this assay is the specific isolation of pri-miRNAs as well as other non-coding RNAs containing a TMG-cap modification.

KEYWORDS:

Pri-miRNA; RNA immunoprecipitation; TMG-cap RNA; Tri-methylated RNA; m2,2,7G-cap RNA

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