TOR complex 1 (TORC1) is a potent anabolic regulator of cellular growth and metabolism. When cells have sufficient amino acids, TORC1 is active due to its lysosomal localization mediated via the Rag ...GTPases. Upon amino acid removal, the Rag GTPases release TORC1, causing it to become cytoplasmic and inactive. We show here that, upon amino acid removal, the Rag GTPases also recruit TSC2 to the lysosome, where it can act on Rheb. Only when both the Rag GTPases and Rheb are inactive is TORC1 fully released from the lysosome. Upon amino acid withdrawal, cells lacking TSC2 fail to completely release TORC1 from the lysosome, fail to completely inactivate TORC1, and fail to adjust physiologically to amino acid starvation. These data suggest that regulation of TSC2 subcellular localization may be a general mechanism to control its activity and place TSC2 in the amino-acid-sensing pathway to TORC1.
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•TSC2 is recruited by the Rag GTPases to lysosomes upon amino acid starvation•TSC2 is required for complete inactivation of TORC1 upon amino acid starvation•Loss of TSC2 causes Rheb-mediated lysosomal retention of mTOR upon aa starvation•TSC2 is required for the correct physiological response of cells to aa starvation
Localization of the TORC1 inhibitor TSC2 to the lysosomes facilitates inactivation of both Rheb and Rag GTPases and allows TORC1 to be released from the lysosome.
The insulin signalling pathway is highly conserved from mammals to Drosophila. Insulin signalling in the fly, as in mammals, regulates a number of physiological functions, including carbohydrate and ...lipid metabolism, tissue growth and longevity. In the present review, I discuss the molecular mechanisms by which insulin signalling regulates metabolism in Drosophila, comparing and contrasting with the mammalian system. I discuss both the intracellular signalling network, as well as the communication between organs in the fly.
Metabolites affect cell growth in two different ways. First, they serve as building blocks for biomass accumulation. Second, metabolites regulate the activity of growth-relevant signaling pathways. ...They do so in part by covalently attaching to proteins, thereby generating post-translational modifications (PTMs) that affect protein function, the focus of this Perspective. Recent advances in mass spectrometry have revealed a wide variety of such metabolites, including lipids, amino acids, Coenzyme-A, acetate, malonate, and lactate to name a few. An active area of research is to understand which modifications affect protein function and how they do so. In many cases, the cellular levels of these metabolites affect the stoichiometry of the corresponding PTMs, providing a direct link between cell metabolism and the control of cell signaling, transcription, and cell growth.
In this Perspective, Teleman and colleagues discuss how metabolites can regulate signaling pathways by covalently attaching to proteins and modifying their functions, providing direct evidence that cell metabolism can regulate cell signaling, transcription, and growth.
Translation regulation occurs largely during the initiation phase. Here, we develop selective 40S footprinting to visualize initiating 40S ribosomes on endogenous mRNAs in vivo. This reveals the ...positions on mRNAs where initiation factors join the ribosome to act and where they leave. We discover that in most human cells, most scanning ribosomes remain attached to the 5′ cap. Consequently, only one ribosome scans a 5′ UTR at a time, and 5′ UTR length affects translation efficiency. We discover that eukaryotic initiation factor 3B (eIF3B,) eIF4G1, and eIF4E remain bound to 80S ribosomes as they begin translating, with a decay half-length of ∼12 codons. Hence, ribosomes retain these initiation factors while translating short upstream open reading frames (uORFs), providing an explanation for how ribosomes can reinitiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.
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•Selective 40S footprinting visualizes regulation of mRNA translation initiation in vivo•Scanning ribosomes are cap-tethered in most human cells•Only one ribosome scans a 5′ UTR at a time in most human cells•Ribosomes retain eIFs during early translation, allowing reinitiation after uORFs
Bohlen et al. develop 40S selective ribosome footprinting to detect scanning 40S ribosomes in vivo and the position on mRNAs when initiation factors join or disengage from the ribosome. They discover that ribosomes remain attached to initiation factors and the mRNA 5′ cap throughout the scanning process and early elongation.
Abstract
Phosphorylation of Ribosomal Protein S6 (RPS6) was the first post-translational modification of the ribosome to be identified and is a commonly-used readout for mTORC1 activity. Although the ...cellular and organismal functions of RPS6 phosphorylation are known, the molecular consequences of RPS6 phosphorylation on translation are less well understood. Here we use selective ribosome footprinting to analyze the location of ribosomes containing phosphorylated RPS6 on endogenous mRNAs in cells. We find that RPS6 becomes progressively dephosphorylated on ribosomes as they translate an mRNA. As a consequence, average RPS6 phosphorylation is higher on mRNAs with short coding sequences (CDSs) compared to mRNAs with long CDSs. We test whether RPS6 phosphorylation differentially affects mRNA translation based on CDS length by genetic removal of RPS6 phosphorylation. We find that RPS6 phosphorylation promotes translation of mRNAs with short CDSs more strongly than mRNAs with long CDSs. Interestingly, RPS6 phosphorylation does not promote translation of mRNAs with 5′ TOP motifs despite their short CDS lengths, suggesting they are translated via a different mode. In sum this provides a dynamic view of RPS6 phosphorylation on ribosomes as they translate mRNAs and the functional consequence on translation.
mTORC1 promotes cell growth and is therefore inactivated upon unfavourable growth conditions. Signalling pathways downstream of most cellular stresses converge on TSC1/2, which serves as an ...integration point that inhibits mTORC1. The TSC1/2 complex was shown to translocate to lysosomes to inactivate mTORC1 in response to two stresses: amino-acid starvation and growth factor removal. Whether other stresses also regulate TSC2 localization is not known. How TSC2 localization responds to combinations of stresses and other stimuli is also unknown. We show that both amino acids and growth factors are required simultaneously to maintain TSC2 cytoplasmic; when one of the two is missing, TSC2 relocalizes to lysosomes. Furthermore, multiple different stresses that inhibit mTORC1 also drive TSC2 lysosomal accumulation. Our findings indicate that lysosomal recruitment of TSC2 is a universal response to stimuli that inactivate mTORC1, and that the presence of any single stress is sufficient to cause TSC2 lysosomal localization.
Recent work has brought to light many different mechanisms of translation initiation that function in cells in parallel to canonical cap‐dependent initiation. This has important implications for ...cancer. Canonical cap‐dependent translation initiation is inhibited by many stresses such as hypoxia, nutrient limitation, proteotoxic stress, or genotoxic stress. Since cancer cells are often exposed to these stresses, they rely on alternate modes of translation initiation for protein synthesis and cell growth. Cancer mutations are now being identified in components of the translation machinery and in cis‐regulatory elements of mRNAs, which both control translation of cancer‐relevant genes. In this review, we provide an overview on the various modes of non‐canonical translation initiation, such as leaky scanning, translation re‐initiation, ribosome shunting, IRES‐dependent translation, and m6A‐dependent translation, and then discuss the influence of stress on these different modes of translation. Finally, we present examples of how these modes of translation are dysregulated in cancer cells, allowing them to grow, to proliferate, and to survive, thereby highlighting the importance of translational control in cancer.
Cancer cells often rely on alternate modes of translation initiation for protein synthesis and growth. This review discusses how stressed cancer cells exploit non‐canonical translation initiation, highlighting the importance of translational control for tumorigenesis.
Abstract
Roughly half of animal mRNAs contain upstream open reading frames (uORFs). These uORFs can represent an impediment to translation of the main ORF since ribosomes usually bind the mRNA cap at ...the 5′ end and then scan for ORFs in a 5′-to-3′ fashion. One way for ribosomes to bypass uORFs is via leaky scanning, whereby the ribosome disregards the uORF start codon. Hence leaky scanning is an important instance of post-transcriptional regulation that affects gene expression. Few molecular factors regulating or facilitating this process are known. Here we show that the PRRC2 proteins PRRC2A, PRRC2B and PRRC2C impact translation initiation. We find that they bind eukaryotic translation initiation factors and preinitiation complexes, and are enriched on ribosomes translating mRNAs with uORFs. We find that PRRC2 proteins promote leaky scanning past translation start codons, thereby promoting translation of mRNAs containing uORFs. Since PRRC2 proteins have been associated with cancer, this provides a mechanistic starting point for understanding their physiological and pathophysiological roles.
Graphical Abstract
Graphical Abstract
PRRC2A, PRRC2B and PRRC2C proteins impact translation initiation by promoting leaky scanning at the start codons of upstream ORFs (uORFs), overlapping uORFs (oORFs) and main ORFs. In the absence of PRRC2 proteins, translation of mRNAs lacking uORFs or oORFs increases. In contrast, on mRNAs that have high initiation rates on uORFs or oORFs, the absence of PRRC2 proteins causes translation of the uORF or oORF to increase and as a consequence translation of the main ORF is reduced.
Abstract Translation initiation is a highly regulated step needed for protein synthesis. Most cell-based mechanistic work on translation initiation has been done using non-stressed cells growing in ...medium with sufficient nutrients and oxygen. This has yielded our current understanding of ‘canonical’ translation initiation, involving recognition of the mRNA cap by eIF4E1 followed by successive recruitment of initiation factors and the ribosome. Many cells, however, such as tumor cells, are exposed to stresses such as hypoxia, low nutrients or proteotoxic stress. This leads to inactivation of mTORC1 and thereby inactivation of eIF4E1. Hence the question arises how cells translate mRNAs under such stress conditions. We study here how mRNAs are translated in an eIF4E1-independent manner by blocking eIF4E1 using a constitutively active version of eIF4E-binding protein (4E-BP). Via ribosome profiling we identify a subset of mRNAs that are still efficiently translated when eIF4E1 is inactive. We find that these mRNAs preferentially release eIF4E1 when eIF4E1 is inactive and bind instead to eIF3d via its cap-binding pocket. eIF3d then enables these mRNAs to be efficiently translated due to its cap-binding activity. In sum, our work identifies eIF3d-dependent translation as a major mechanism enabling mRNA translation in an eIF4E-independent manner.
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