Regulation and functional effects of localized RNAs

Division Of Basic Sciences - Nci

Investigators

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Abstract

The research program focuses on the role of localized RNAs, i.e. RNAs that are targeted to specific subcellular regions, and the consequences of local RNA translation in the processes of cell migration and cancer invasion. This is a markedly understudied area. The research from my group has spearheaded the study of RNAs localized at protrusions of non-neuronal migrating cells. Over the years we have made a number of contributions regarding the underlying molecular mechanisms used to transport RNAs to protrusions and to regulate their translation; the consequences of local RNA translation on protein function; and the relevance of these events to cancer invasion. We have developed methodologies to study protrusion-localized RNAs in 2D and 3D systems, with an emphasis both on the imaging as well as the generation of unbiased methods of image analysis and quantification (Stueland et al., 2019). We have shown that localization of RNAs at protrusions is directed through at least two distinct pathways which depend on different protein factors and are regulated differently by the mechanical properties of the cytoskeleton (Wang et al., 2017). Most of our work has focused on a subset of 70 RNAs which require the tumor suppressor APC for localization. We have shown that localization of APC-dependent RNAs is important for migration in 2D and 3D environments. We have identified cis RNA elements that direct accumulation at protrusions; protein factors that associate with these RNAs and control their transport or translation; and disease mutations that disrupt these processes (Mili et al., 2008; Yasuda et al., 2013; Yasuda et al., 2017; Moissoglu, Pichon et al., in submitted). We have further revealed novel mechanisms controlling localized transcripts, showing that APC-dependent RNAs require detyrosinated microtubules whose formation is promoted by the stiffness of the extracellular matrix, mechanical tension and actomyosin contractility (Wang et al., 2017). We have uncovered a novel mode of translational regulation of localized transcripts. Using state-of-the-art methodologies to visualize newly-synthesized endogenous proteins, or exogenous single-molecule translation imaging reporters, we have discovered that in dynamically migrating cells, translation of APC-dependent RNAs is not coordinated with cytoplasmic position, as the current paradigm suggests. Instead, we found that their translation is coordinated with specific peripheral cellular processes, being activated at extending protrusions/lamellipodia and suppressed upon protrusion retraction. Furthermore, silencing is coupled to a change in the physical state of the RNAs manifested by single RNAs clustering into heterogeneous granules at the tips of retracting protrusions (Moissoglu et al., 2019). These clusters are reminiscent of RNA granules formed by liquid-liquid phase separation. These findings have revealed a novel mode of spatial regulation of translation in dynamically polarized cells. They further suggest the existence of mechanisms that coordinate specific local cellular behaviors with the assembly/disassembly of phase-separated RNA granules. We have investigated the functional importance of RNA localization on cell migration and have made the significant discovery that the particular subcellular site of protein synthesis can affect the regulation and functional output of the encoded protein. Specifically, using the localized RAB13 RNA as a model, we have demonstrated that local translation of the RAB13 RNA at the periphery allows the co-translational association of the newly-synthesized RAB13 protein with its activator, the exchange factor RABIF. This peripheral association is required for directing RAB13 GTPase activity to promote cell migration and is governed primarily by the location of the RAB13 RNA (Moissoglu et al., 2020). These findings revealed that translation of the same RNA in different positions in the cytoplasm can direct the resulting protein to associate with different interacting networks and fulfil different functional outputs, with implications regarding protein regulation in general. The premise set forth by our work is that signaling pathways relevant to disease can be regulated, not at the protein, but rather at the RNA level through modulating the location of RNA translation. This insight offers the basis for the design of potential new therapeutic platforms. Indeed, we have exploited our knowledge of the underlying mechanisms to develop antisense oligonucleotides that interfere specifically with localization of particular endogenous transcripts. Using this methodology, which can be easily applied both in vitro and in vivo, we have demonstrated a requirement for specific localized RNAs not only during migration of individual cells, but also during collective invasion of 3-dimensional multicellular cancer spheroids. We are now extending our findings using xenograft mouse tumor models of cancer invasion (Chrisafis et al., 2020).