Heat-Shock Factor 1 (HSF1), master regulator of the heat-shock response, facilitates malignant transformation, cancer cell survival, and proliferation in model systems. The common assumption is that these effects are mediated through regulation of heat-shock protein (HSP) expression. However, the transcriptional network that HSF1 coordinates directly in malignancy and its relationship to the heat-shock response have never been defined. By comparing cells with high and low malignant potential alongside their nontransformed counterparts, we identify an HSF1-regulated transcriptional program specific to highly malignant cells and distinct from heat shock. Cancer-specific genes in this program support oncogenic processes: cell-cycle regulation, signaling, metabolism, adhesion and translation. HSP genes are integral to this program, however, many are uniquely regulated in malignancy. This HSF1 cancer program is active in breast, colon and lung tumors isolated directly from human patients and is strongly associated with metastasis and death. Thus, HSF1 rewires the transcriptome in tumorigenesis, with prognostic and therapeutic implications.

HSF1 Drives a Transcriptional Program Distinct from Heat Shock to Support Highly Malignant Human Cancers

Aberrant, misfolded and mislocalized proteins are often toxic to cells and result in many human diseases. All proteins and their mRNA templates are subject to quality control. There are several distinct mechanisms that control the quality of mRNAs and proteins during translation at the ribosome. mRNA quality control systems, nonsense-mediated decay (NMD), non-stop decay (NSD), and no-go decay (NGD) detect premature stop codons, the absence of a natural stop codon, and stalled ribosomes in translation, respectively, and degrade their mRNAs. Defective truncated polypeptide nascent chains generated from faulty mRNAs are degraded by ribosome-associated protein quality control pathways. Regulation of aberrant protein production (RAPP), a novel pathway, senses aberrant proteins by monitoring the status of nascent chain interactions during translation and triggers degradation of their mRNA. Here we review the current progress in understanding of the molecular mechanisms of mRNA and protein quality controls at the ribosome during translation.

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Lost in Translation: Ribosome-Associated mRNA and Protein Quality Controls.

Stem cells are characterized by their ability to self-renew and differentiate into many different cell types. Research has focused primarily on how these processes are regulated at a transcriptional level. However, recent studies have indicated that stem cell behaviour is strongly coupled to the regulation of protein synthesis by the ribosome. In this Review, we discuss how different translation mechanisms control the function of adult and embryonic stem cells. Stem cells are characterized by low global translation rates despite high levels of ribosome biogenesis. The maintenance of pluripotency, the commitment to a specific cell fate and the switch to cell differentiation depend on the tight regulation of protein synthesis and ribosome biogenesis. Translation regulatory mechanisms that impact on stem cell function include mTOR signalling, ribosome levels, and mRNA and tRNA features and amounts. Understanding these mechanisms important for stem cell self-renewal and differentiation may also guide our understanding of cancer grade and metastasis. Stem cells are well known to be controlled transcriptionally, but recent studies indicate that pluripotency, cell fate and differentiation depend on the regulation of translation and ribosome biogenesis by mTOR signalling, ribosome levels, and mRNA and tRNA features. Elucidating these stem cell regulatory mechanisms may increase our understanding of tumorigenesis.

Translational control of stem cell function.

Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule RNA fluorescence in situ hybridization (smFISH) is a widely used single-cell-single-molecule imaging technique enabling quantitative studies of gene expression and its regulatory mechanisms. Today, these methods are applicable at a large scale, which in turn come with a need for adequate tools for data analysis and exploration. Here, we present FISH-quant v2, a highly modular tool accessible for both experts and non-experts. Our user-friendly package allows the user to segment nuclei and cells, detect isolated RNAs, decompose dense RNA clusters, quantify RNA localization patterns and visualize these results both at the single-cell level and variations within the cell population. This tool was validated and applied on large-scale smFISH image data sets, revealing diverse subcellular RNA localization patterns and a surprisingly high degree of cell-to-cell heterogeneity.

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FISH-quant v2: a scalable and modular tool for smFISH image analysis

Protein domains of low sequence complexity do not fold into stable, three-dimensional structures. Nevertheless, proteins with these sequences assist in many aspects of cell organization, including assembly of nuclear and cytoplasmic structures not surrounded by membranes. The dynamic nature of these cellular assemblies is caused by the ability of low-complexity domains (LCDs) to transiently self-associate through labile, cross-β structures. Mechanistic studies useful for the study of LCD self-association have evolved over the past decade in the form of simple assays of phase separation. Here, we have used such assays to demonstrate that the interactions responsible for LCD self-association can be dictated by labile protein structures poised close to equilibrium between the folded and unfolded states. Furthermore, missense mutations causing Charcot-Marie-Tooth disease, frontotemporal dementia, and Alzheimer’s disease manifest their pathophysiology in vitro and in cultured cell systems by enhancing the stability of otherwise labile molecular structures formed upon LCD self-association. Description Keeping things dynamic Many proteins contain domains that do not adopt a stable folded structure but are involved in protein function, sometimes through self-association that can drive the formation of phase-separated structures inside cells. These regions are known as low complexity (LC) domains because they are composed of only a few different types of amino acids. Zhou et al. show that the LC domain of the RNA-binding protein TDP-43 associates transiently through a labile structure, and that this behavior is required for phase separation, which is key to its normal role of organizing mRNA (see the Perspective by Petsko and Small). Mutation of a proline in the LC domain caused aggregation by stabilizing the labile structure. In three other proteins, proline mutations linked to neuronal diseases were shown to similarly enhance the stability of self-association of LC domains. —VV Mutations that stabilize a labile protein structure cause a shift between dynamic self-association and aggregation. INTRODUCTION Most proteins fold into stable, morphologically distinct three-dimensional shapes. By contrast, between 10 and 20% of the proteome in eukaryotic cells does not adopt stable structures. Protein domains that exhibit intrinsic disorder are typified by unusual sequences consisting of a limited representation of amino acid residues known as low-complexity domain (LCD) sequences. Protein LCDs are important to cell biology. They line the permeability channel of nuclear pores, allow for the assembly of intermediate filaments, and assist in the assembly of a variety of nuclear and cytoplasmic puncta that are not surrounded by lipid membranes. Despite their lack of folding, LCDs self-associate in a manner that allows phase separation from aqueous solution. Assays of phase separation have emerged as useful tools with which to study LCD self-association and its potential relationship to LCD function in living cells. Such studies have yielded conflicting observations. The prevailing thinking is that LCDs self-associate in the absence of any form of protein structure. An alternative perspective posits that association of LCDs is initiated by the formation of labile structures, and that this structural order is facilitated by hydrogen bond networks, allowing for favorable interchain adhesion in the form of cross-β interactions, as described 70 years ago by Linus Pauling. RATIONALE To test whether LCDs use main-chain hydrogen bonding to facilitate phase separation, variant LCDs associated with the RNA-binding protein TDP-43 RNA were constructed without individual hydrogen bonds across a region known to assemble in a cross-β conformation. These constructs allowed for experimental assessment of the importance of main-chain hydrogen bonding to phase separation. The role of main-chain hydrogen bonds was further investigated in studies of the neurofilament light (NFL) chain protein, the microtubule-associated tau protein, and the heterogeneous nuclear RNPA2 (hnRNPA2) RNA-binding protein to determine whether mutations known to cause human neurological disease might create an extra hydrogen bond, thereby enhancing the avidity of cross-β interactions within the LCDs of these proteins. RESULTS We prepared 23 variants of the TDP-43 LCD lacking the capacity for forming individual hydrogen bonds across a region critical to self-association and phase separation. Nine contiguous variants impeded phase separation. The locations of these variants corresponded precisely with the location of nearly identical regions of the cross-β structure reported independently by two different cryo–electron microscopy reconstructions of self-associated TDP-43 protein. Mutant variants that replace a proline to allow an extra hydrogen bond within LCDs of the disease-associated NFL, tau, and hnRNPA2 proteins were found to specify formation of inordinately stable self-associations. Aberrant stability was reversed by chemical modifications blocking the ability of the peptide nitrogen associated with the variant amino acid residues to participate in main-chain hydrogen bonding. CONCLUSION The LCDs that we studied function by forming labile structures in the form of specific cross-β assemblies. These structures can be weakened by the removal of single, main-chain hydrogen bonds or strengthened by human mutations specifying the addition of single hydrogen bonds, suggesting that they are poised at the threshold of thermodynamic equilibrium. The labile nature of these structures offers a dynamic dimension to many aspects of cell organization, as well as the opportunity for regulation by many forms of posttranslational modification. The virtues of weakness: Protein structure poised at the edge of equilibrium. By using unnatural amino acids to manipulate the backbone hydrogen-bonding capacity, we reveal specific sites within LCDs that mediate self-association through the formation of labile cross-β structures. We found that multiple mutations causative of neurological disease target proline residues within the LCDs of functionally diverse proteins, and that these prolines serve to insulate labile cross-β structures and prevent aberrantly strong self-association. Denial of backbone-mediated hydrogen bonding at these mutation sites is sufficient to restore normal protein behavior.

Mutations linked to neurological disease enhance self-association of low-complexity protein sequences

Fine-tuning cellular physiology in response to intracellular and environmental cues requires precise temporal and spatial control of gene expression. High-resolution imaging technologies to detect mRNAs and their translation state have revealed that all living organisms localize mRNAs in subcellular compartments and create translation hotspots, enabling cells to tune gene expression locally. Therefore, mRNA localization is a conserved and integral part of gene expression regulation from prokaryotic to eukaryotic cells. In this Review, we discuss the mechanisms of mRNA transport and local mRNA translation across the kingdoms of life and at organellar, subcellular and multicellular resolution. We also discuss the properties of messenger ribonucleoprotein and higher order RNA granules and how they may influence mRNA transport and local protein synthesis. Finally, we summarize the technological developments that allow us to study mRNA localization and local translation through the simultaneous detection of mRNAs and proteins in single cells, mRNA and nascent protein single-molecule imaging, and bulk RNA and protein detection methods.

Intracellular mRNA transport and localized translation

The mTORC1/S6K/PDCD4/eIF4A Axis Determines Outcome of Mitotic Arrest.

During protein synthesis, nascent polypeptides emerge from ribosomes to fold into functional proteins. Misfolding of newly synthesized polypeptides (NSPs) at this stage leads to their aggregation. These misfolded NSPs must be expediently cleared to circumvent the deleterious effects of protein aggregation on cell physiology. To this end, a sizable portion of NSPs are ubiquitinated and rapidly degraded by the proteasome. This suggests the existence of co-translational mechanisms that play a pivotal role in the quality control of NSPs. It is generally thought that ribosomes play a central role in this process. During mRNA translation, ribosomes sense errors that lead to the accumulation of aberrant polypeptides, and serve as a hub for protein complexes that are required for optimal folding and/or proteasome-dependent degradation of misfolded polypeptides. In this review, we discuss recent findings that shed light on the molecular underpinnings of the co-translational quality control of NSPs.

https://www.tandfonline.com/doi/pdf/10.4161/trla.28109

Co-translational mechanisms of quality control of newly synthesized polypeptides.

The first sentence under the subheading ‘Regulating developmental stages’ has been updated to clarify that the early syncytial embryos referred to are from C. elegans, and that the smFISH and MS2–MCP studies mentioned extend previous observations summarized in reference 24. These changes have been made in the HTML and PDF versions of the article.

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Valentina Gandin

Papers

Intracellular mRNA transport and localized translation

The mTORC1/S6K/PDCD4/eIF4A Axis Determines Outcome of Mitotic Arrest.

Co-translational mechanisms of quality control of newly synthesized polypeptides.

Author Correction: Intracellular mRNA transport and localized translation.