Don't shoot the messenger shoot the reader.

Einstein et al. (2021) uncover a novel role for the RNA-binding protein YTHDF2, one of the m6A reader proteins, in TNBC proliferation and survival. This study demonstrates the clinical potential of targeting a specific reader protein in the treatment of breast cancer.

System-wide analysis of RNA and protein subcellular localization dynamics.

Although the subcellular dynamics of RNA and proteins are key determinants of cell homeostasis, their characterization is still challenging. Here we present an integrative framework to simultaneously interrogate the dynamics of the transcriptome and proteome at subcellular resolution by combining two methods: localization of RNA (LoRNA) and a streamlined density-based localization of proteins by isotope tagging (dLOPIT) to map RNA and protein to organelles (nucleus, endoplasmic reticulum and mitochondria) and membraneless compartments (cytosol, nucleolus and cytosolic granules). Interrogating all RNA subcellular locations at once enables system-wide quantification of the proportional distribution of RNA. We obtain a cell-wide overview of localization dynamics for 31,839 transcripts and 5,314 proteins during the unfolded protein response, revealing that endoplasmic reticulum-localized transcripts are more efficiently recruited to cytosolic granules than cytosolic RNAs, and that the translation initiation factor eIF3d is key to sustaining cytoskeletal function. Overall, we provide the most comprehensive overview so far of RNA and protein subcellular localization dynamics.

Phosphorylation of the smooth muscle master splicing regulator RBPMS regulates its splicing activity.

We previously identified RBPMS as a master regulator of alternative splicing in differentiated smooth muscle cells (SMCs). RBPMS is transcriptionally downregulated during SMC dedifferentiation, but we hypothesized that RBPMS protein activity might be acutely downregulated by post-translational modifications. Publicly available phosphoproteomic datasets reveal that Thr113 and Thr118 immediately adjacent to the RRM domain are commonly both phosphorylated. An RBPMS T113/118 phosphomimetic T/E mutant showed decreased splicing regulatory activity both in transfected cells and in a cell-free in vitro assay, while a non-phosphorylatable T/A mutant retained full activity. Loss of splicing activity was associated with a modest reduction in RNA affinity but significantly reduced RNA binding in nuclear extract. A lower degree of oligomerization of the T/E mutant might cause lower avidity of multivalent RNA binding. However, NMR analysis also revealed that the T113/118E peptide acts as an RNA mimic which can loop back and antagonize RNA-binding by the RRM domain. Finally, we identified ERK2 as the most likely kinase responsible for phosphorylation at Thr113 and Thr118. Collectively, our data identify a potential mechanism for rapid modulation of the SMC splicing program in response to external signals during the vascular injury response and atherogenesis.

Post-transcriptional control of gene expression following stress: the role of RNA-binding proteins.

The ability of mammalian cells to modulate global protein synthesis in response to cellular stress is essential for cell survival. While control of protein synthesis is mediated by the regulation of eukaryotic initiation and elongation factors, RNA-binding proteins (RBPs) provide a crucial additional layer to post-transcriptional regulation. RBPs bind specific RNA through conserved RNA-binding domains and ensure that the information contained within the genome and transcribed in the form of RNA is exported to the cytoplasm, chemically modified, and translated prior to folding into a functional protein. Thus, this group of proteins, through mediating translational reprogramming, spatial reorganisation, and chemical modification of RNA molecules, have a major influence on the robust cellular response to external stress and toxic injury.

Yeast mRNA localization: protein asymmetry, organelle localization and response to stress.

The localization of mRNA forms a key facet of the post-transcriptional control of gene expression and recent evidence suggests that it may be considerably more widespread than previously anticipated. For example, defined mRNA-containing granules can be associated with translational repression or activation. Furthermore, mRNA P-bodies (processing bodies) harbour much of the mRNA decay machinery and stress granules are thought to play a role in mRNA storage. In the present review, we explore the process of mRNA localization in the yeast Saccharomyces cerevisiae, examining connections between organellar mRNA localization and the response to stress. We also review recent data suggesting that even where there is a global relocalization of mRNA, the specificity and kinetics of this process can be regulated.

Core Fermentation (CoFe) granules focus coordinated glycolytic mRNA localization and translation to fuel glucose fermentation.

Glycolysis is a fundamental metabolic pathway for glucose catabolism across biology, and glycolytic enzymes are among the most abundant proteins in cells. Their expression at such levels provides a particular challenge. Here we demonstrate that the glycolytic mRNAs are localized to granules in yeast and human cells. Detailed live cell and smFISH studies in yeast show that the mRNAs are actively translated in granules, and this translation appears critical for the localization. Furthermore, this arrangement is likely to facilitate the higher level organization and control of the glycolytic pathway. Indeed, the degree of fermentation required by cells is intrinsically connected to the extent of mRNA localization to granules. On this basis, we term these granules, core fermentation (CoFe) granules; they appear to represent translation factories, allowing high-level coordinated enzyme synthesis for a critical metabolic pathway.

Efficient recovery of the RNA-bound proteome and protein-bound transcriptome using phase separation (OOPS).

RNA-protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA-protein adducts in an unbiased way. OOPS avoids molecular tagging or the capture of polyadenylated RNA. Instead, it is based on sampling the interface of a standard TRIzol extraction to enrich RNA-binding proteins (RBPs) and their cognate bound RNA. OOPS specificity is achieved by digesting the enriched interfaces with RNases or proteases to release the RBPs or protein-bound RNA, respectively. Here we present a step-by-step protocol to purify protein-RNA adducts, free protein and free RNA from the same sample. We further describe how OOPS can be applied in human cell lines, Arabidopsis thaliana, Schizosaccharomyces pombe and Escherichia coli and how it can be used to study RBP dynamics.

The cell stress response: extreme times call for post-transcriptional measures.

Following cell stress, a wide range of molecular pathways are initiated to orchestrate the stress response and enable adaptation to an environmental or intracellular perturbation. The post-transcriptional regulation strategies adopted during the stress response result in a substantial reorganization of gene expression, designed to prepare the cell for either acclimatization or programmed death, depending on the nature and intensity of the stress. Fundamental to the stress response is a rapid repression of global protein synthesis, commonly mediated by phosphorylation of translation initiation factor eIF2α. Recent structural and biochemical information have added unprecedented detail to our understanding of the molecular mechanisms underlying this regulation. During protein synthesis inhibition, the translation of stress-specific mRNAs is nonetheless enhanced, often through the interaction between RNA-binding proteins and specific RNA regulatory elements. Recent studies investigating the unfolded protein response (UPR) provide some important insights into how posttranscriptional events are spatially and temporally fine-tuned in order to elicit the most appropriate response and to coordinate the transition from an early, acute stage into the chronic state of adaptation. Importantly, cancer cells are known to hi-jack adaptive stress response pathways, particularly the UPR, to survive and proliferate in the unfavorable tumor environment. In this review, we consider the implications of recent research into stress-dependent post-transcriptional regulation and make the case for the exploration of the stress response as a strategy to identify novel targets in the development of cancer therapies. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution Translation > Translation Mechanisms > Translation Regulation.

Translation factor mRNA granules direct protein synthetic capacity to regions of polarized growth.

mRNA localization serves key functions in localized protein production, making it critical that the translation machinery itself is present at these locations. Here we show that translation factor mRNAs are localized to distinct granules within yeast cells. In contrast to many messenger RNP granules, such as processing bodies and stress granules, which contain translationally repressed mRNAs, these granules harbor translated mRNAs under active growth conditions. The granules require Pab1p for their integrity and are inherited by developing daughter cells in a She2p/She3p-dependent manner. These results point to a model where roughly half the mRNA for certain translation factors is specifically directed in granules or translation factories toward the tip of the developing daughter cell, where protein synthesis is most heavily required, which has particular implications for filamentous forms of growth. Such a feedforward mechanism would ensure adequate provision of the translation machinery where it is to be needed most over the coming growth cycle.

Author Correction: Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS).

Existing high-throughput methods to identify RNA-binding proteins (RBPs) are based on capture of polyadenylated RNAs and cannot recover proteins that interact with nonadenylated RNAs, including long noncoding RNA, pre-mRNAs and bacterial RNAs. We present orthogonal organic phase separation (OOPS), which does not require molecular tagging or capture of polyadenylated RNA, and apply it to recover cross-linked protein-RNA and free protein, or protein-bound RNA and free RNA, in an unbiased way. We validated OOPS in HEK293, U2OS and MCF10A human cell lines, and show that 96% of proteins recovered were bound to RNA. We show that all long RNAs can be cross-linked to proteins, and recovered 1,838 RBPs, including 926 putative novel RBPs. OOPS is approximately 100-fold more efficient than existing methods and can enable analyses of dynamic RNA-protein interactions. We also characterize dynamic changes in RNA-protein interactions in mammalian cells following nocodazole arrest, and present a bacterial RNA-interactome for Escherichia coli. OOPS is compatible with downstream proteomics and RNA sequencing, and can be applied in any organism.

Trans-acting translational regulatory RNA binding proteins.

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms.

Granules harboring translationally active mRNAs provide a platform for P-body formation following stress.

The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems.