Purification by affinity chromatography of H1 RNA-Binding Proteins from rat brain

Abstract

Post-transcriptional regulation of mRNA metabolism is involved in processes as different as cell fate specification in development and cell response to a large variety of environmental cues. Regulation of all steps of RNA metabolism depends on RNA-binding proteins (RBPs). By using a T1 RNase protection assay, we previously identified three H1° RNA-binding factors (p40, p70 and p110), highly expressed in the rat brain. Here we report enrichment of these factors from brain extracts, obtained by affinity chromatography of biotinylated H1° RNA-protein complexes on streptavidin-conjugated paramagnetic particles. The purified proteins maintain RNA-binding ability and preference for histone messages. Thanks to the efforts of a number of laboratories in the last decade, it is now clear that a central component in the control of gene expression involves post-transcriptional regulation of mRNA metabolism (reviewed in refs. 1-7). Post-transcriptional regulation of time and site in which a given mRNA must be translated and its duration depend on two main classes of RNA-binding proteins: i) RNA chaperones, that assist folding of RNA molecules (8), and ii) tertiary-structure-binding regulatory proteins, that recognize and stabilize specific tertiary structures (9). The two classes of proteins include both nonspecific and specific RNA-binding functions (10-13). In vivo, most mRNAs are actually present as large ribonucleoprotein complexes, the assembly of which probably requires a series of events including formation of RNA secondary structures and selective stabilization of specific tertiary structures by proteins (9,14-15). In the last few years we have been looking for proteins able to bind mRNAs encoding the histone variants H1° and H3.3 (16-19). In searching for such factors, we adopted and/or developed a series of different experimental approaches. In particular, by using a T1 RNase-protection assay, we previously identified three H1° RNA-binding factors (p40, p70 and p110), highly expressed in the rat brain (17). Herein we describe purification of H1° RNA-binding factors from rat brain by an affinity chromatography protocol that made use of an in vitro-transcribed, biotinylated H1° RNA and of streptavidin-conjugated paramagnetic particles.