Analysis of RNA:protein interactions in vivo: identification of RNA-binding partners of nuclear factor 90.
Ribonucleoprotein complexes (RNPs) perform a multitude of functions in the cell. Elucidating the composition of such complexes and unraveling their many interactions are current challenges in molecular biology. To stabilize complexes formed in cells and to preclude reassortment of their components during isolation, we employ chemical crosslinking of the RNA and protein moieties. Here we describe the identification of cellular RNAs bound to nuclear factor 90 (NF90), the founder member of a family of ubiquitous double-stranded RNA-binding proteins. Crosslinked RNA-NF90 complexes were immunoprecipitated from stable cell lines containing epitope-tagged NF90 protein isoforms. The bound RNA was released and identified through RNase H digestion and by various gene amplification techniques. We appraise the methods used by altering crosslinking conditions, and the binding profiles of different NF90 protein isoforms in synchronized and asynchronous cells are compared. This study discovers two novel RNA species and establishes NF90 as a multiclass RNA-binding protein, capable of binding representatives of all three classes of RNA.
Parrott
Andrew M
AM
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey, USA.
Walsh
Melissa R
MR
Mathews
Michael B
MB
D002478:Cells, Cultured; D000939:Epitopes; D006801:Humans; D047468:Immunoprecipitation; D050779:Nuclear Factor 90 Proteins / Q000502:physiology*; D011061:Poly A; D020033:Protein Isoforms / Q000302:isolation & purification; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000939:Epitopes; D050779:Nuclear Factor 90 Proteins; D020033:Protein Isoforms; D016601:RNA-Binding Proteins; D011061:Poly A