Developing a method to identify and study the transcriptome of miRNAs important in myogenesis

Abstract

microRNAs (miRNAs) play major roles in controlling gene expression, thereby regulating many cellular processes such as cell growth, cell death, and differentiation. Moreover, defects in miRNA expression and function have been implicated in many diseases. miRNAs elicit their effects by associating with specific mRNA targets and triggering mRNA degradation or preventing translation. In order to determine and understand the specific function of a miRNA, the direct bona fide mRNA targets of the miRNA must be identified. A means to concretely identify the “transcriptome” (i.e. the complete set of mRNA targets) for a given miRNA remains elusive. Current methods to identify the specific mRNA targets of miRNAs have met with mixed success. Computational programs that predict mRNA targets of miRNAs often result in a myriad of hits, many of which are false positives or false negatives. Experimental approaches often use cell‐altering tactics, such as overexpression or knockdown of a miRNA, followed by measuring global mRNA changes, which can identify indirect effects.The goals of this study are to develop a novel experimental method for identifying the endogenous mRNA targets of a select miRNA, and ultimately show that regulation of those mRNA targets is important for a particular biological pathway. We are developing an oligo‐affinity based purification method, termed crosslinking oligo purification (xOP). The xOP method is currently being optimized in both C2C12 cells (targeting miR‐206 and miR‐26a) and HeLa cells (targeting miR‐21 and miR‐26a). C2C12 cells are mouse skeletal muscle cells that highly express miR‐206 and miR‐26a during myogenesis, induced by low serum growth. HeLa cells highly express miR‐21, which has roles in cancer, and miR‐26a, which will likely have unique mRNA targets in HeLa cells compared to C2C12. Cells will be crosslinked with formaldehyde to generate a reversible network of miRNA, mRNA, and protein crosslinks. Cellular extracts will then be made and miRNA‐containing complexes will be captured and purified using a biotinylated DNA antisense oligo specific to the miRNA of interest (miR‐206, 26a, or 21) and Neutravidin beads. The mRNAs that co‐purify with a select miRNA after xOP will be identified via Illumina sequencing (xOP‐seq). We will validate a select set of newly identified mRNAs that specifically associate with miR‐206, miR‐26a, or miR‐21 after xOP‐seq as bona fide mRNA targets of the miRNAs.Recent work has shown that the xOP method specifically and efficiently captures the miRNA of interest in both cell types. For example in HeLa cells, miR‐21 is consistently depleted from miR‐21 xOP flow throughs ~15‐fold (and as high as 60‐fold). In addition, the levels of miR‐21 recovered in miR‐21 xOP eluates are around 70–80%. Current work is focused on validating recovery of mRNA targets using qRT‐PCR to detect the small handful of known targets for each miRNA. We are also working to decrease the level of background RNA before moving to sequencing.These studies will provide a novel experimental method to identify the bona fide endogenous mRNA targets of miRNAs, which could then be applied to any system to identify miRNA/mRNA interactions. This research will also provide an understanding of the function of newly identified miRNA targets of miR‐206 and miR‐26a in myogenesis, and miR‐21 and miR‐26a in a cancer cell line.Support or Funding InformationNIH R21 AR067826, NIH T32 GM008759