Poster abstracts
Poster number 64 submitted by Ameya Jalihal
Microscopically visible P-bodies contribute little to miRNA-mediated gene silencing
Sethu Pitchiaya (Department of Chemistry, University of Michigan, Ann Arbor, MI, USA), Ameya Jalihal (Department of Chemistry, University of Michigan, Ann Arbor, MI, USA), Marcio D.A. Mourao (Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA), Santiago Schnell (Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA), Nils G. Walter (Department of Chemistry, University of Michigan, Ann Arbor, MI, USA)
Abstract:
microRNA (miRNA) bound messenger RNAs (mRNAs) localize to processing bodies (PBs), which are sub-cellular, membrane-less foci enriched in RNA processing enzymes, as a cause or consequence of post-transcriptional gene silencing. To gain a deeper understanding of the hitherto unobserved dynamics of miRNA:PB interactions and the contribution of PBs to miRNA-mediated gene silencing, we used a modified version of our recently developed iSHiRLoC (intracellular Single molecule High-Resolution Localization and Counting) technique and combined the resulting data with molecular diffusion modeling. miRNAs and PBs were labeled with spectrally distinct fluorescent probes so that single functional miRNAs could be localized and tracked with respect to single PBs inside human cells with ~30 nm spatial accuracy at 100 ms time resolution. iSHiRLoC revealed that only a small fraction of miRNAs, independent of the abundance of their mRNA targets, localize to PBs, with the majority of PBs containing only one or two fluorophore labeled miRNA molecules. Surprisingly, higher mRNA target abundance led to a smaller fraction of miRNAs stably co-localizing with PBs, suggesting that PBs may play a significant role in sequestration of target-free miRNAs. Moreover, molecular diffusion modeling predicted that PB aggregation into fewer, but larger (microscopically visible) particles should diminish their contribution towards gene silencing. Supporting this observation, aggregation and immobilization of PBs in cultured cells via hypertonic treatment suppressed miRNA mediated gene silencing. Taken together, our data suggest that microscopically visible PBs minimally contribute to gene silencing and perhaps play a role in target-independent miRNA sequestration. Our single-molecule systems biology approach promises to map the dynamic interaction network of the RNA silencing pathway to reveal crucial mechanistic details and assess their impact on cellular gene expression.
Keywords: miRNA, Processing Bodies, Super Resolution Microscopy