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 mRNAs, i.e. mRNAs that are targeted to specific subcellular regions, and on the consequences that local mRNA translation has on the function of the encoded proteins. Most studies focusing on these processes and the underlying mechanisms have been carried out mostly in large and stably polarized systems, such as oocytes and neurons. We are instead studying these events in small, dynamic cells such as mesenchymal and epithelial cells, in the context of cell migration and cancer invasion. This is a markedly understudied area. Specifically, the research from my group has spearheaded the study of mRNAs 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 mRNAs to protrusions and to regulate their translation; the consequences of local mRNA translation on protein function; and the relevance of these events to cancer invasion. 1) We have developed methodologies to study protrusion-localized mRNAs 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; Chrisafis et al., 2020; Moissoglu et al., in press). 2) We have shown that localization of mRNAs 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; Moriarty et al., 2022). Most of our work has focused on a subset of 70 mRNAs 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 mRNAs 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., 2021). 3) We have further revealed novel mechanisms controlling localized transcripts, showing that APC-dependent mRNAs require detyrosinated microtubules whose formation is promoted by the stiffness of the extracellular matrix, mechanical tension and actomyosin contractility (Wang et al., 2017; Moriarty et al., 2022). 4) We have uncovered a novel mode of translational regulation of localized transcripts. Specifically, 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 mRNAs 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 mRNAs manifested by single mRNAs 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. 5) 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 mRNA 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 mRNA (Moissoglu et al., 2020). These findings revealed that translation of the same mRNA 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 mRNA 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 (Chrisafis et al., 2020). We are now extending our findings using xenograft mouse tumor models of cancer invasion.