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J Vis Exp. 2017 Sep 28;(127). doi: 10.3791/56004.

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions.

Author information

1
Epigenetics Institute, University of Pennsylvania Perelman School of Medicine; Graduate Group in Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine.
2
Epigenetics Institute, University of Pennsylvania Perelman School of Medicine; Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine.
3
Epigenetics Institute, University of Pennsylvania Perelman School of Medicine; Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine.
4
Epigenetics Institute, University of Pennsylvania Perelman School of Medicine; Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine; rbon@mail.med.upenn.edu.

Abstract

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods. Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

PMID:
28994809
PMCID:
PMC5752346
DOI:
10.3791/56004
[Indexed for MEDLINE]
Free PMC Article

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