Format

Send to

Choose Destination
Bio Protoc. 2019 Apr 5;9(7). pii: e3200. doi: 10.21769/BioProtoc.3200.

Identification of RNase-sensitive LINE-1 Ribonucleoprotein Interactions by Differential Affinity Immobilization.

Author information

1
Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, USA.
2
Department of Pathology, Massachusetts General Hospital, Boston, USA.
3
Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, USA.
4
Moscow Institute of Physics and Technology, Dolgoprudny, Russia.

Abstract

Long Interspersed Nuclear Element-1 (LINE-1, L1) constitutes a family of autonomous, self-replicating genetic elements known as retrotransposons. Although most are inactive, copious L1 sequences populate the human genome. L1s proliferate in a 'copy-and-paste' fashion through an RNA intermediate; a full-length L1 transcript is ~6,000 nucleotides long and functions as a bicistronic mRNA that encodes and assembles in cis with two main polypeptides, ORF1p and ORF2p, forming a ribonucleoprotein (RNP); L1 RNPs also interact with a wide range of host factors in positive and negative regulatory capacities. The following protocol describes an approach to affinity enrich ectopically expressed L1 RNPs and, using RNases, release the fraction of protein that depends upon the presence of intact RNA for retention in the immobilized macromolecules.

KEYWORDS:

Affinity capture; L1 RNP; LINE-1; RNase treatment; Retrotransposon

Supplemental Content

Full text links

Icon for PubMed Central
Loading ...
Support Center