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.

Figure 1:

Biochemical elucidation of post-transcriptional gene silencing networks (A) A combined affinity purification, MS and nucleic acid sequencing approach to determine RISC protein factors, miRNAs and target mRNAs. Immunoprecipitations of endogenous or tagged proteins specific for various RISC components facilitate the isolation of RISCs. Protein components are identified by tandem MS. In parallel, RNA is extracted and analyzed by second-generation sequencing or by cDNA microarrays. The identified mRNA sequences are searched for corresponding miRNA seed matches by computational methods such as TargetScan, miRanda, PITA and RNA22 all available online. (B) A method to elucidate potential miRNA gene regulatory modules, one miRNA and one PTGS factor at a time. Cellular gene expression networks are perturbed by the introduction of a single miRNA and the mRNA targets are identified after immunoprecipitation as described earlier. Once the target mRNAs are identified, the miRNA’s effect on the mRNA’s abundance can be determined by quantification using cDNA microarrays or second-generation DNA sequencing. The miRNA’s effect on the corresponding protein levels can be interrogated by SILAC quantitative MS. The incorporation of many such experiments with various miRNAs allow for the determination of miRNA gene regulatory networks. Black circles in the center represent individual miRNAs and white/grey circles, target genes. White circle represents target genes whose translation is repressed without changes in mRNA levels, grey circle represents targets with decreased mRNA levels, and mixed white/grey circle represents targets with decreased mRNA levels and repressed translation. The large dashed circle represents a network module (a single miRNA and its target genes). The modules are linked by target genes, which are repressed by more than one miRNA.