Glutathione peroxidase 4 (GPX4) utilizes glutathione (GSH) to detoxify lipid peroxidation and plays an essential role in inhibiting ferroptosis. As a selenoprotein, GPX4 protein synthesis is highly ...inefficient and energetically costly. How cells coordinate GPX4 synthesis with nutrient availability remains unclear. In this study, we perform integrated proteomic and functional analyses to reveal that SLC7A11-mediated cystine uptake promotes not only GSH synthesis, but also GPX4 protein synthesis. Mechanistically, we find that cyst(e)ine activates mechanistic/mammalian target of rapamycin complex 1 (mTORC1) and promotes GPX4 protein synthesis at least partly through the Rag-mTORC1-4EBP signaling axis. We show that pharmacologic inhibition of mTORC1 decreases GPX4 protein levels, sensitizes cancer cells to ferroptosis, and synergizes with ferroptosis inducers to suppress patient-derived xenograft tumor growth in vivo. Together, our results reveal a regulatory mechanism to coordinate GPX4 protein synthesis with cyst(e)ine availability and suggest using combinatorial therapy of mTORC1 inhibitors and ferroptosis inducers in cancer treatment.
Cell survival in changing environments requires appropriate regulation of gene expression, including translational control. Multiple stress signaling pathways converge on several key translation ...factors, such as eIF4F and eIF2, and rapidly modulate messenger RNA (mRNA) translation at both the initiation and the elongation stages. Repression of global protein synthesis is often accompanied with selective translation of mRNAs encoding proteins that are vital for cell survival and stress recovery. The past decade has seen significant progress in our understanding of translational reprogramming in part due to the development of technologies that allow the dissection of the interplay between mRNA elements and corresponding binding proteins. Recent genome-wide studies using ribosome profiling have revealed unprecedented proteome complexity and flexibility through alternative translation, raising intriguing questions about stress-induced translational reprogramming. Many surprises emerged from these studies, including wide-spread alternative translation initiation, ribosome pausing during elongation, and reversible modification of mRNAs. Elucidation of the regulatory mechanisms underlying translational reprogramming will ultimately lead to the development of novel therapeutic strategies for human diseases.
Precise control of protein synthesis by engineering sequence elements in 5' untranslated regions (5' UTRs) remains a fundamental challenge. To accelerate our understanding of the cis-regulatory code ...embedded in 5' UTRs, we devised massively parallel reporter assays from a synthetic messenger RNA library composed of over one million 5' UTR variants. A completely randomized 10-nucleotide sequence preceding an upstream open reading frame (uORF) and downstream GFP drives a broad range of translational outputs and mRNA stability in mammalian cells. While efficient translation protects mRNA from degradation, uORF translation triggers mRNA decay in a UPF1-dependent manner. We also identified translational inhibitory elements with G-quadruplexes as marks for mRNA decay in P-bodies. Unexpectedly, an unstructured A-rich element in 5' UTRs destabilizes mRNAs in the absence of translation, although it enables cap-independent translation. Our results not only identify diverse sequence features of 5' UTRs that control mRNA translatability, but they also reveal ribosome-dependent and ribosome-independent mRNA-surveillance pathways.
The most abundant mRNA post-transcriptional modification is N(6)-methyladenosine (m(6)A), which has broad roles in RNA biology. In mammalian cells, the asymmetric distribution of m(6)A along mRNAs ...results in relatively less methylation in the 5' untranslated region (5'UTR) compared to other regions. However, whether and how 5'UTR methylation is regulated is poorly understood. Despite the crucial role of the 5'UTR in translation initiation, very little is known about whether m(6)A modification influences mRNA translation. Here we show that in response to heat shock stress, certain adenosines within the 5'UTR of newly transcribed mRNAs are preferentially methylated. We find that the dynamic 5'UTR methylation is a result of stress-induced nuclear localization of YTHDF2, a well-characterized m(6)A 'reader'. Upon heat shock stress, the nuclear YTHDF2 preserves 5'UTR methylation of stress-induced transcripts by limiting the m(6)A 'eraser' FTO from demethylation. Remarkably, the increased 5'UTR methylation in the form of m(6)A promotes cap-independent translation initiation, providing a mechanism for selective mRNA translation under heat shock stress. Using Hsp70 mRNA as an example, we demonstrate that a single m(6)A modification site in the 5'UTR enables translation initiation independent of the 5' end N(7)-methylguanosine cap. The elucidation of the dynamic features of 5'UTR methylation and its critical role in cap-independent translation not only expands the breadth of physiological roles of m(6)A, but also uncovers a previously unappreciated translational control mechanism in heat shock response.
Conspectus Synthetic messenger RNA (mRNA), once delivered into cells, can be readily translated into proteins by ribosomes, which do not distinguish exogenous mRNAs from endogenous transcripts. Until ...recently, the intrinsic instability and immunostimulatory property of exogenous RNAs largely hindered the therapeutic application of synthetic mRNAs. Thanks to major technological innovations, such as introduction of chemically modified nucleosides, synthetic mRNAs have become programmable therapeutic reagents. Compared to DNA or protein-based therapeutic reagents, synthetic mRNAs bear several advantages: flexible design, easy optimization, low-cost preparation, and scalable synthesis. Therapeutic mRNAs are commonly designed to encode specific antigens to elicit organismal immune response to pathogens like viruses, express functional proteins to replace defective ones inside cells, or introduce novel enzymes to achieve unique functions like genome editing. Recent years have witnessed stunning progress on the development of mRNA vaccines against SARS-Cov2. This success is built upon our fundamental understanding of mRNA metabolism and translational control, a knowledge accumulated during the past several decades. Given the astronomical number of sequence combinations of four nucleotides, sequence-dependent control of mRNA translation remains incompletely understood. Rational design of synthetic mRNAs with robust translation and optimal stability remains challenging. Massively paralleled reporter assay (MPRA) has been proven to be powerful in identifying sequence elements in controlling mRNA translatability and stability. Indeed, a completely randomized sequence in 5′ untranslated region (5′UTR) drives a wide range of translational outputs. In this Account, we will discuss general principles of mRNA translation in eukaryotic cells and elucidate the role of coding and noncoding regions in the translational regulation. From the therapeutic perspective, we will highlight the unique features of 5′ cap, 5′UTR, coding region (CDS), stop codon, 3′UTR, and poly(A) tail. By focusing on the design strategies in mRNA engineering, we hope this Account will contribute to the rational design of synthetic mRNAs with broad therapeutic potential.
Translational control permits cells to respond swiftly to a changing environment. Rapid attenuation of global protein synthesis under stress conditions has been largely ascribed to the inhibition of ...translation initiation. Here we report that intracellular proteotoxic stress reduces global protein synthesis by halting ribosomes on transcripts during elongation. Deep sequencing of ribosome-protected messenger RNA (mRNA) fragments reveals an early elongation pausing, roughly at the site where nascent polypeptide chains emerge from the ribosomal exit tunnel. Inhibiting endogenous chaperone molecules by a dominant-negative mutant or chemical inhibitors recapitulates the early elongation pausing, suggesting a dual role of molecular chaperones in facilitating polypeptide elongation and cotranslational folding. Our results further support the chaperone “trapping” mechanism in promoting the passage of nascent chains. Our study reveals that translating ribosomes fine tune the elongation rate by sensing the intracellular folding environment. The early elongation pausing represents a cotranslational stress response to maintain the intracellular protein homeostasis.
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► Proteotoxic stress reduces global protein synthesis by influencing elongation ► Proteotoxic stress induces ribosome pausing on mRNAs in the first 50 codons ► Molecular chaperones facilitate translation elongation by binding to nascent chains ► Ribosomes fine tune elongation rate in response to proteotoxic stress
Decoding Human Cytomegalovirus Stern-Ginossar, Noam; Weisburd, Ben; Michalski, Annette ...
Science (American Association for the Advancement of Science),
11/2012, Letnik:
338, Številka:
6110
Journal Article
Recenzirano
Odprti dostop
The human cytomegalovirus (HCMV) genome was sequenced 20 years ago. However, like those of other complex viruses, our understanding of its protein coding potential is far from complete. We used ...ribosome profiling and transcript analysis to experimentally define the HCMV translation products and follow their temporal expression. We identified hundreds of previously unidentified open reading frames and confirmed a fraction by means of mass spectrometry. We found that regulated use of alternative transcript start sites plays a broad role in enabling tight temporal control of HCMV protein expression and allowing multiple distinct polypeptides to be generated from a single genomic locus. Our results reveal an unanticipated complexity to the HCMV coding capacity and illustrate the role of regulated changes in transcript start sites in generating this complexity.
Understanding translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. The recently developed ...ribosome-profiling technique enables global translation analysis, providing a wealth of information about both the position and the density of ribosomes on mRNAs. Here we present an approach, global translation initiation sequencing, applying in parallel the ribosome E-site translation inhibitors lactimidomycin and cycloheximide to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. This approach provides a view of alternative translation initiation in mammalian cells with single-nucleotide resolution. Systemic analysis of TIS positions supports the ribosome linear-scanning mechanism in TIS selection. The alternative TIS positions and the associated ORFs identified by global translation initiation sequencing are conserved between human and mouse cells, implying physiological significance of alternative translation. Our study establishes a practical platform for uncovering the hidden coding potential of the transcriptome and offers a greater understanding of the complexity of translation initiation.
RNA modification in the form of N
-methyladenosine (m
A) regulates nearly all the post-transcriptional processes. The asymmetric m
A deposition suggests that regional methylation may have distinct ...functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m
A modifications. Here we report the development of 'm
A editing', a powerful approach that enables m
A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single-chain m
A methyltransferase that can be programmed with a guide RNA. The resultant m
A 'writers' allow functional comparison of single site methylation in different messenger RNA regions. We further engineered m
A 'erasers' by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m
A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.
Rhenium tricarbonyl complexes have been recently investigated as novel anticancer agents. However, little is understood about their mechanisms of action, as well as the means by which cancer cells ...respond to chronic exposure to these compounds. To gain a deeper mechanistic insight into these rhenium anticancer agents, we developed and characterized an ovarian cancer cell line that is resistant to a previously studied compound Re(CO)3(dmphen)(ptolICN)+, where dmphen=2,9‐dimethyl‐1,10‐phenanthroline and ptolICN=para‐tolyl isonitrile, called TRIP. This TRIP‐resistant ovarian cancer cell line, A2780TR, was found to be 9 times less sensitive to TRIP compared to the wild‐type A2780 ovarian cancer cell line. Furthermore, the cytotoxicities of established drugs and other rhenium anticancer agents in the TRIP‐resistant cell line were determined. Notably, the drug taxol was found to exhibit a 184‐fold decrease in activity in the A2780TR cell line, suggesting that mechanisms of resistance towards TRIP and this drug are similar. Accordingly, expression levels of the ATP‐binding cassette transporter P‐glycoprotein, an efflux transporter known to detoxify taxol, were found to be elevated in the A2780TR cell line. Additionally, a gene expression analysis using the National Cancer Institute 60 cell line panel identified the MT1E gene to be overexpressed in cells that are less sensitive to TRIP. Because this gene encodes for metallothioneins, this result suggests that detoxification by this class of proteins is another mechanism for resistance to TRIP. The importance of this gene in the A2780TR cell line was assessed, confirming that its expression is elevated in this cell line as well. As the first study to investigate and identify the cancer cell resistance pathways in response to a rhenium complex, this report highlights important similarities and differences in the resistance responses of ovarian cancer cells to TRIP and conventional drugs.
TRIPping up resistance! A rhenium‐resistant ovarian cancer cell line was developed by prolonged treatment with a rhenium(I) tricarbonyl isonitrile complex. The cell line was characterized and found to overexpress the ACB transporters P‐glycoprotein and ABCC1, the metallothionein MT1E, and exhibit cross‐resistance with the known anticancer agents taxol and doxorubicin.
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