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

通过差异亲和力固定法鉴定 RNase 敏感的 LINE-1 核糖核蛋白相互作用

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作者:Hua Jiang, Martin S Taylor, Kelly R Molloy, Ilya Altukhov, John LaCava

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.

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