Mass Spectrometry Analysis of Protein Interactors for Pre-let-7 miRNA

Authors

• GC Lyons
• P Legault
• MK Meffert
• LM Jenkins

Abstract

RNA binding proteins (RBPs) play an important role in the regulation of RNA by binding in an RNA-sequence specific manner (1). MicroRNAs (miRNA), which are short non-coding RNAs, also play a role in the posttranscriptional regulation of mRNAs (2). Currently, the regulation of miRNAs and the role RBPs play in that regulation is not well understood. One common method to characterize specific interactors of miRNAs is to perform pulldowns followed by mass spectrometry to identify the bound proteins. Multiple methods have been used, such as biotinylation of RNA and immobilization on streptavidin sepharose. Here, we use the ARiBO method, a new purification technique that was developed to purify and isolate high yields of specific RNAs and their protein interactors (3), for the characterization of the interactors of members of the pre-let-7 family of miRNAs. The Let-7 family of miRNA have been found to be present during embryogenesis and brain development, suggesting that the miRNAs play a role in these processes (2). Over or under development of the brain has been one of the main signs for autism spectral disorders (ASD) (4). The ARiBO method was used to study the protein interactors of Let-7A1 miRNA and in order to better understand the role of the miRNA in ASD. First, the pulldown method was optimized using varying amounts of the whole cell lysate of control cells were examined to determine the optimum starting amount to identify the interactome. The method was combined with mass spectrometry to identify protein interactors of the pre-let-7A1 RNA. This study also investigated the protein interactors of pre-let-7A1 from both WT and FMR1KO synaptosomes, the latter being used as a model for autism. Thus, this study uses mass spectrometry to better define the functional role of let-7A1 miRNA in the brain and, in particular, in ASD.

Scientific Focus Area: Systems Biology

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