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

Hua Jiang, Martin S. Taylor, Kelly R. Molloy, Ilya Altukhov, John LaCava*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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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 similar to 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.

Original languageEnglish
Article number3200
Number of pages31
JournalBio-Protocol
Volume9
Issue number7
DOIs
Publication statusPublished - 5-Apr-2019
Externally publishedYes

Keywords

  • Retrotransposon
  • LINE-1
  • L1 RNP
  • Affinity capture
  • RNase treatment
  • L1 RETROTRANSPOSITION
  • PROTEIN COMPLEXES
  • CELL-CULTURE
  • AMINO-ACIDS
  • PROTEOMICS
  • TETRACYCLINES
  • TRANSFECTION
  • GENE

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