Affinity purification of mRNA-protein complexes

A functional mRNA never exists inside a cell in a naked form but it is always bound by proteins. RNA-binding proteins (RBPs) regulate every aspect of mRNA function, including splicing, export, translation and stability. However, the composition and function of many mRNA-protein complexes (mRNPs) is poorly understood. This is due to the complexity of mRNP biogenesis as well as to technical limitations.

We are establishing a novel method of mRNP affinity purification from Saccharomyces cerevisiae. In order to isolate the mRNA of interest the mRNA is tagged by inserting binding sites for MS2 coat protein (MS2CP) into the 3’ UTR. The tagged mRNA is co-expressed with MS2CP fused with Protein A, thus allowing the binding of the RNA tag to MS2CP inside cells. mRNP complexes are affinity-purified using IgG coated magnetic beads via Protein A-IgG interaction.

Our aim is to explore how the absence of RBPs such as RNA export factors influences mRNP formation. Changes in wt versus mutant strain will be studied on a quantitative level by using SILAC (stable isotope labeling by amino acids in cell culture).

We believe that mRNP affinity purification based on mRNA isolation is a good alternative to mRNP purification though tagging known mRNA-associated proteins. The advantage of isolating mRNA instead of protein is the possibility to purify mRNPs in all maturation states, thus enabling to identify all interaction partners of a certain mRNA.