TREX reveals proteins that bind to specific RNA regions in living cells.

Different regions of RNA molecules can often engage in specific interactions with distinct RNA-binding proteins (RBPs), giving rise to diverse modalities of RNA regulation and function. However, there are currently no methods for unbiased identification of RBPs that interact with specific RNA regions in living cells and under endogenous settings. Here we introduce TREX (targeted RNase H-mediated extraction of crosslinked RBPs)-a highly sensitive approach for identifying proteins that directly bind to specific RNA regions in living cells. We demonstrate that TREX outperforms existing methods in identifying known interactors of U1 snRNA, and reveals endogenous region-specific interactors of NORAD long noncoding RNA. Using TREX, we generated a comprehensive region-by-region interactome for 45S rRNA, uncovering both established and previously unknown interactions that regulate ribosome biogenesis. With its applicability to different cell types, TREX is an RNA-centric tool for unbiased positional mapping of endogenous RNA-protein interactions in living cells.

Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly.

Endothelial cell interactions with their extracellular matrix are essential for vascular homeostasis and expansion. Large-scale proteomic analyses aimed at identifying components of integrin adhesion complexes have revealed the presence of several RNA binding proteins (RBPs) of which the functions at these sites remain poorly understood. Here, we explored the role of the RBP SAM68 (Src associated in mitosis, of 68 kDa) in endothelial cells. We found that SAM68 is transiently localized at the edge of spreading cells where it participates in membrane protrusive activity and the conversion of nascent adhesions to mechanically loaded focal adhesions by modulation of integrin signaling and local delivery of ß-actin mRNA. Furthermore, SAM68 depletion impacts cell-matrix interactions and motility through induction of key matrix genes involved in vascular matrix assembly. In a 3D environment SAM68-dependent functions in both tip and stalk cells contribute to the process of sprouting angiogenesis. Altogether, our results identify the RBP SAM68 as a novel actor in the dynamic regulation of blood vessel networks.

The alternative matrisome: Alternative splicing of ECM proteins in development, homeostasis and tumor progression.

The extracellular matrix (ECM) is a fundamental component of the tissue of multicellular organisms that is comprised of an intricate network of multidomain proteins and associated factors, collectively known as the matrisome. The ECM creates a biophysical environment that regulates essential cellular processes such as adhesion, proliferation and migration and impacts cell fate decisions. The composition of the ECM varies across organs, developmental stages and diseases. Interestingly, most ECM genes generate transcripts that undergo extensive alternative splicing events, producing multiple protein variants from one gene thus enhancing ECM complexity and impacting matrix architecture. Extensive studies over the past several decades have linked ECM remodeling and expression of alternatively spliced ECM isoforms to cancer, and reprogramming of the alternative splicing patterns in cells has recently been proposed as a new hallmark of tumor progression. Indeed, tumor-associated alternative splicing occurs in both malignant and non-malignant cells of the tumor environment and growing evidence suggests that expression of specific ECM splicing variants could be a key step for stromal activation. In this review, we present a general overview of alternative splicing mechanisms, featuring examples of ECM components. The importance of ECM variant expression during essential physiological processes, such as tissue organization and embryonic development is discussed as well as the dysregulation of alternative splicing in cancer. The overall aim of this review is to address the complexity of the ECM by highlighting the importance of the yet-to-be-fully-characterized "alternative" matrisome in physiological and pathological states such as cancer.

Shaping Up the Tumor Microenvironment With Cellular Fibronectin.

Normal tissue homeostasis and architecture restrain tumor growth. Thus, for a tumor to develop and spread, malignant cells must overcome growth-repressive inputs from surrounding tissue and escape immune surveillance mechanisms that curb cancer progression. This is achieved by promoting the conversion of a physiological microenvironment to a pro-tumoral state and it requires a constant dialog between malignant cells and ostensibly normal cells of adjacent tissue. Pro-tumoral reprogramming of the stroma is accompanied by an upregulation of certain extracellular matrix (ECM) proteins and their cognate receptors. Fibronectin (FN) is one such component of the tumor matrisome. This large multidomain glycoprotein dimer expressed over a wide range of human cancers is assembled by cell-driven forces into a fibrillar array that provides an obligate scaffold for the deposition of other matrix proteins and binding sites for functionalization by soluble factors in the tumor microenvironment. Encoded by a single gene, FN regulates the proliferation, motile behavior and fate of multiple cell types, largely through mechanisms that involve integrin-mediated signaling. These processes are coordinated by distinct isoforms of FN, collectively known as cellular FN (as opposed to circulating plasma FN) that arise through alternative splicing of the FN1 gene. Cellular FN isoforms differ in their solubility, receptor binding ability and spatiotemporal expression, and functions that have yet to be fully defined. FN induction at tumor sites constitutes an important step in the acquisition of biological capabilities required for several cancer hallmarks such as sustaining proliferative signaling, promoting angiogenesis, facilitating invasion and metastasis, modulating growth suppressor activity and regulating anti-tumoral immunity. In this review, we will first provide an overview of ECM reprogramming through tumor-stroma crosstalk, then focus on the role of cellular FN in tumor progression with respect to these hallmarks. Last, we will discuss the impact of dysregulated ECM on clinical efficacy of classical (radio-/chemo-) therapies and emerging treatments that target immune checkpoints and explore how our expanding knowledge of the tumor ECM and the central role of FN can be leveraged for therapeutic benefit.