mRNPs take shape by CLIPPING and PAIRING

Abstract

The interaction of RNA-binding proteins (RBPs) with RNA is a crucial aspect of normal cellular metabolism. Yet, the diverse number of RBPs and RNA motifs to which they bind, the wide range of interaction strengths and the fact that RBPs associate in dynamic complexes have made it challenging to determine whether a particular RNA-binding protein binds a particular RNA. Recent work by three different laboratories has led to the development of new tools to query such interactions in the more physiological environs of cultured cells. The use of these methods has led to insights into (1) the networks of RNAs regulated by a particular protein, (2) the identification of new protein partners within messenger ribonucleoprotein particles and (3) the flux of RNA-binding proteins on an mRNA throughout its lifecycle. Here, I examine these new methods and discuss their relative strengths and current limitations.

Conformational-Dependent and Independent RNA Binding to the Fragile X Mental Retardation Protein

“…These include pull-down assays with homoribopolymers [15, 21, 49, 50], affinity capture using biotinylated RNA [21, 41, 51–53], affinity capture using immobilized protein [16], UV crosslinking [21, 49], filter-binding assays [22, 30, 41], electrophoretic mobility shift assays (EMSA) [26, 36, 40, 42, 47, 54], and agarose electrophoretic mobility shift assays (AGESA) [17, 41]. Each of these methods has its unique experimental advantages [55]. Acknowledging that binding between a nucleic acid and RNA-binding protein (RBP) can be affected by differences in posttranslational modification [56] and/or differences between different protein variants [57, 58] our working hypothesis is that given a particular RBP, a particular RNA and a defined buffer each of these methods should converge to produce a common answer.…”

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

“…The identification of RBPs associated with an mRNA could significantly improve our understanding of processes underlying their posttranscriptional regulation. Diverse genetic, microscopic, biochemical and bioinformatics methods have been used to identify proteins involved in mRNA regulation (reviewed in [9][10][11] ). However, only a few of these methods enable the identification of proteins associated with a particular target mRNA.…”