Probing microRNA Activity in vitro and inside Cells using Single Molecule Microscopy.

Non-coding RNAs (ncRNAs) outnumber their protein-coding counterparts, yet their presumably diverse functions are still ill-understood. This thesis reports the development of two novel single-molecule methods to probe the activity of microRNAs (miRNAs), a class of regulatory ncRNAs, in pursuit of their elusive mechanism of action. miRNAs associate with components of the RNA induced silencing complex (RISC) to assemble on messenger RNA (mRNA) targets and regulate protein expression in higher eukaryotes. Here, I describe a method for the intracellular single molecule, high resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore labeled, functional miRNAs were tracked within diffusing particles, a majority of which contained single miRNA molecules. Observed mobility and mRNA dependent assembly changes support a model of multiple target turnovers by miRNAs, revealing the dynamic nature of an important gene regulatory pathway and paving the way towards its single molecule systems biology. miRNAs accumulate in processing bodies (PBs), sub-cellular foci enriched in RNA processing enzymes, as a cause or consequence of post-transcriptional gene silencing. Despite numerous observations, quantitative analysis of miRNA localization within PBs has been lacking. iSHiRLoC of miRNAs revealed that only a small fraction of miRNAs localized to PBs and majority of PBs contained only one or two fluorophore labeled miRNA molecules. Moreover, miRNAs resided in PBs only for a few hundred milliseconds, suggesting the preponderance of unstable interactions. Heterogeneous distribution of miRNAs in PBs coupled with the observation that miRNAs docked onto PBs either stably or transiently suggests an underlying diversity in the composition of these two complexes. Some mRNAs contain multiple binding sites for a specific miRNA, presumably for enhanced regulation. To quantify the binding stoichiometry between miRNAs and such mRNAs, I developed a single-molecule in vitro assay based on the step-wise photobleaching of fluorescent probes. Our data, in two different cell extracts, showed that a majority of mRNAs are either bound by zero or a single miRNA under conditions of maximal repression. Computational mRNA structure analysis predicted low accessibility of miRNA binding sites. Together, these data suggest that a higher number of binding sites corresponds to a higher probability of binding, not multiple occupancy.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/91425/1/sethu_1.pd

Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102078/1/embr201285-sup-0001.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102078/2/embr201285.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102078/3/embr201285.reviewer_comments.pd

The lncRNA landscape of breast cancer reveals a role for DSCAM-AS1 in breast cancer progression.

Molecular classification of cancers into subtypes has resulted in an advance in our understanding of tumour biology and treatment response across multiple tumour types. However, to date, cancer profiling has largely focused on protein-coding genes, which comprise <1% of the genome. Here we leverage a compendium of 58,648 long noncoding RNAs (lncRNAs) to subtype 947 breast cancer samples. We show that lncRNA-based profiling categorizes breast tumours by their known molecular subtypes in breast cancer. We identify a cohort of breast cancer-associated and oestrogen-regulated lncRNAs, and investigate the role of the top prioritized oestrogen receptor (ER)-regulated lncRNA, DSCAM-AS1. We demonstrate that DSCAM-AS1 mediates tumour progression and tamoxifen resistance and identify hnRNPL as an interacting protein involved in the mechanism of DSCAM-AS1 action. By highlighting the role of DSCAM-AS1 in breast cancer biology and treatment resistance, this study provides insight into the potential clinical implications of lncRNAs in breast cancer

Invited Review Meeting Report: SMART Timing-Principles of Single Molecule Techniques Course at the University of Michigan 2014

ABSTRACT: Four days after the announcement of the 2014 Nobe

Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution

Summary: Regulation of microRNA (miRNA) localization and stability is critical for their extensive cytoplasmic RNA silencing activity and emerging nuclear functions. Here, we have developed single-molecule fluorescence-based tools to assess the subcellular trafficking, integrity, and activity of miRNAs. We find that seed-matched RNA targets protect miRNAs against degradation and enhance their nuclear retention. While target-stabilized, functional, cytoplasmic miRNAs reside in high-molecular-weight complexes, nuclear miRNAs, as well as cytoplasmic miRNAs targeted by complementary anti-miRNAs, are sequestered stably within significantly lower-molecular-weight complexes and rendered repression incompetent. miRNA stability and activity depend on Argonaute protein abundance, whereas miRNA strand selection, unwinding, and nuclear retention depend on Argonaute identity. Taken together, our results show that miRNA degradation competes with Argonaute loading and target binding to control subcellular miRNA abundance for gene silencing surveillance. Probing single cells for miRNA activity, trafficking, and metabolism promises to facilitate screening for effective miRNA mimics and anti-miRNA drugs. : Pitchiaya et al. describe tools to interrogate gene-regulatory microRNAs inside living cells at single-molecule resolution. They find that the RNA silencing machinery and RNA targets mediate gene silencing surveillance by modulating the abundance and subcellular location of microRNAs. These findings and tools promise to facilitate single-cell screening of microRNA activity. Keywords: microRNA, Argonaute, mRNA targets, anti-miRs, correlative counting analysis, single-molecule microscop

DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs

The DNA damage response (DDR) is a set of cellular events that follows the generation of DNA damage. Recently, site-specific small non-coding RNAs, also termed DNA damage response RNAs (DDRNAs), have been shown to play a role in DDR signalling and DNA repair. Dysfunctional telomeres activate DDR in ageing, cancer and an increasing number of identified pathological conditions. Here we show that, in mammals, telomere dysfunction induces the transcription of telomeric DDRNAs (tDDRNAs) and their longer precursors from both DNA strands. DDR activation and maintenance at telomeres depend on the biogenesis and functions of tDDRNAs. Their functional inhibition by sequence-specific antisense oligonucleotides allows the unprecedented telomere-specific DDR inactivation in cultured cells and in vivo in mouse tissues. In summary, these results demonstrate that tDDRNAs are induced at dysfunctional telomeres and are necessary for DDR activation and they validate the viability of locus-specific DDR inhibition by targeting DDRNAs

KRAS Engages AGO2 to Enhance Cellular Transformation

SummaryOncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we uncovered an interaction between RAS and Argonaute 2 (AGO2). Endogenously, RAS and AGO2 co-sediment and co-localize in the endoplasmic reticulum. The AGO2 N-terminal domain directly binds the Switch II region of KRAS, agnostic of nucleotide (GDP/GTP) binding. Functionally, AGO2 knockdown attenuates cell proliferation in mutant KRAS-dependent cells and AGO2 overexpression enhances KRASG12V-mediated transformation. Using AGO2−/− cells, we demonstrate that the RAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Mechanistically, oncogenic KRAS attenuates AGO2-mediated gene silencing. Overall, the functional interaction with AGO2 extends KRAS function beyond its canonical role in signaling