A combined immunoprecipitation, mass spectrometric and nucleic acid sequencing approach to determine microRNA-mediated post-transcriptional gene regulatory networks

Abstract

While initiation of transcription has attracted the most attention in the field of gene regulation, it has become clear that additional stages in the gene expression cascade including post-transcriptional events are under equally exquisite control. The seminal discovery that short RNAs (microRNA, small interfering RNA, Piwi-interacting RNA), play important roles in repressing gene expression has spurred a rush of new interest in post-transcriptional gene silencing mechanisms. The development of affinity tags and high-resolution tandem mass spectrometry (MS/MS) has greatly simplified the analysis of proteins that regulate gene expression. Further, the use of DNA microarrays and 'second generation' nucleic acid sequencing ('deep sequencing') technologies has facilitated the identification of their regulatory targets. These technological advancements mark a significant step towards a comprehensive understanding of gene regulatory networks. The purpose of this review is to highlight several recent reports that illustrate the value of affinity-purification (immunoprecipitation) followed by mass spectrometric protein analysis and nucleic acid analysis by deep sequencing (AP-MS/Seq) to examine mRNA after it has been transcribed. The ability to identify the direct nucleic acid targets of post-transcriptional gene regulatory machines is a critical first step towards understanding the contribution of post-transcriptional pathways on gene expression.