Small RNA, Big Impact: Probing miRNA pathways in living cells using single particle tracking

Abstract

MicroRNAs (miRNAs) are short non‐coding RNAs that control the expression of an estimated 30% of all human proteins post‐transcriptionally. Mature miRNA, typically 21 base pairs long, associates with several proteins to form a miRNA Induced Silencing Complex (miRISC). This complex associates with the Un‐Translated Region (UTR) of mRNAs in a sequence specific manner and forms an active miRISC that controls protein expression. While several different mechanisms have been proposed for miRNA mediated gene repression, they are highly debated. We are investigating the mechanism of miRNA action by observing the diffusive motions of miRISC‐mRNA complexes in living cells using single particle tracking microscopy (SPTM). To this end, we microinject fluorophore labeled miRNA probes into living cultured cells. The probe can bind to multiple sites on its target mRNA which results in an inherent fluorescent amplification and significantly aids in tracking the particle. Quantitative Real Time ‐ Polymerase Chain Reactions (qRT‐PCR) were performed to estimate the level of the HMGA2 mRNA in F9 cells and preliminary tests suggests that there are ~8 mRNA molecules/cell, which is a suitable target number for our SPT experiments. SPT experiments are being performed using which we can deduce whether a specific miRISC‐mRNA complex progresses through multiple mechanisms of repression.Funded by NIGMS.