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.
Although tumor-infiltrating lymphocytes (TILs) maintain their ability to proliferate, persist, and eradicate tumors, they are frequently dysfunctional in situ. By performing both whole-genome CRISPR ...and metabolic inhibitor screens, we identify that nicotinamide phosphoribosyltransferase (NAMPT) is required for T cell activation. NAMPT is low in TILs, and its expression is controlled by the transcriptional factor Tubby (TUB), whose activity depends on the T cell receptor-phospholipase C gamma (TCR-PLCγ) signaling axis. The intracellular level of NAD+, whose synthesis is dependent on the NAMPT-mediated salvage pathway, is also decreased in TILs. Liquid chromatography-mass spectrometry (LC-MS) and isotopic labeling studies confirm that NAD+ depletion led to suppressed glycolysis, disrupted mitochondrial function, and dampened ATP synthesis. Excitingly, both adoptive CAR-T and anti-PD1 immune checkpoint blockade mouse models demonstrate that NAD+ supplementation enhanced the tumor-killing efficacy of T cells. Collectively, this study reveals that an impaired TCR-TUB-NAMPT-NAD+ axis leads to T cell dysfunction in the tumor microenvironment, and an over-the-counter nutrient supplement of NAD+ could boost T-cell-based immunotherapy.
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•Levels of NAD+ and NAMPT are decreased in TILs compared to those of other T cells•Tubby is a transcriptional factor for NAMPT in T cells•NAMPT-mediated NAD+ production is essential for T cell activation by generating ATP•NAD+ supplementation enhances the tumor-killing function of T cells
Wang et al. report that NAD+ biosynthesis through the TCR-PLCγ-TUB-NAMPT-mediated salvage pathway is important for T cell activation. Metabolically, NAD+ deficiency causes defects in aerobic glycolysis and oxidative phosphorylation. NAD+ supplementation rescues these defects and enhances the tumor-killing efficacy of T cells in both CAR-T and anti-PD1 mouse models.
Abstract R-loop-triggered collateral single-stranded DNA (ssDNA) nuclease activity within Class 1 Type I CRISPR-Cas systems holds immense potential for nucleic acid detection. However, the ...hyperactive ssDNase activity of Cas3 introduces unwanted noise and false-positive results. In this study, we identified a novel Type I-A Cas3 variant derived from Thermococcus siculi , which remains in an auto-inhibited state until it is triggered by Cascade complex and R-loop formation. This Type I-A CRISPR-Cas3 system not only exhibits an expanded protospacer adjacent motif (PAM) recognition capability but also demonstrates remarkable intolerance towards mismatched sequences. Furthermore, it exhibits dual activation modes—responding to both DNA and RNA targets. The culmination of our research efforts has led to the development of the Hyper-Active-Verification Establishment (HAVE, 惠父). This innovation enables swift and precise human papillomavirus (HPV) diagnosis in clinical samples, providing a robust molecular diagnostic tool based on the Type I-A CRISPR-Cas3 system. Our findings contribute to understanding type I-A CRISPR-Cas3 system regulation and facilitate the creation of advanced diagnostic solutions with broad clinical applicability.
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.
Long noncoding RNAs (lncRNAs) are key regulators of gene expression in diverse cellular contexts and biological processes. Given the surprising range of shapes and sizes, how distinct lncRNAs achieve ...functional specificity remains incompletely understood. Here, we identified a heat shock-inducible lncRNA,
, in mouse cells that acts as a transcriptional brake to restrain stress gene expression. Functional characterization reveals that
directly binds to heat shock transcription factor 1 (HSF1), thereby targeting stress genes in a trans-acting manner. Intriguingly,
is heavily methylated in the form of m
A. Although dispensable for HSF1 binding,
methylation is required for silencing stress genes to attenuate heat shock response. Consistently, m
A depletion results in prolonged activation of stress genes. Furthermore,
mediates these effects via the nuclear m
A reader YTHDC1, forming a transcriptional silencing complex for stress genes. Our study reveals a crucial role of nuclear epitranscriptome in the transcriptional regulation of heat shock response.
Long noncoding RNAs (lncRNAs) are key regulators of gene expression in diverse cellular contexts and biological processes. Given the surprising range of shapes and sizes, how distinct lncRNAs achieve ...functional specificity remains incompletely understood. Here, we identified a heat shock–inducible lncRNA, Heat, in mouse cells that acts as a transcriptional brake to restrain stress gene expression. Functional characterization reveals that Heat directly binds to heat shock transcription factor 1 (HSF1), thereby targeting stress genes in a trans-acting manner. Intriguingly, Heat is heavily methylated in the form of m6A. Although dispensable for HSF1 binding, Heat methylation is required for silencing stress genes to attenuate heat shock response. Consistently, m6A depletion results in prolonged activation of stress genes. Furthermore, Heat mediates these effects via the nuclear m6A reader YTHDC1, forming a transcriptional silencing complex for stress genes. Our study reveals a crucial role of nuclear epitranscriptome in the transcriptional regulation of heat shock response.
The amino acid cysteine and its oxidized dimeric form cystine are commonly believed to be synonymous in metabolic functions. Cyst(e)ine depletion not only induces amino acid response but also ...triggers ferroptosis, a non-apoptotic cell death. Here, we report that unlike general amino acid starvation, cyst(e)ine deprivation triggers ATF4 induction at the transcriptional level. Unexpectedly, it is the shortage of lysosomal cystine, but not the cytosolic cysteine, that elicits the adaptative ATF4 response. The lysosome-nucleus signaling pathway involves the aryl hydrocarbon receptor (AhR) that senses lysosomal cystine via the kynurenine pathway. A blockade of lysosomal cystine efflux attenuates ATF4 induction and sensitizes ferroptosis. To potentiate ferroptosis in cancer, we develop a synthetic mRNA reagent, CysRx, that converts cytosolic cysteine to lysosomal cystine. CysRx maximizes cancer cell ferroptosis and effectively suppresses tumor growth in vivo. Thus, intracellular nutrient reprogramming has the potential to induce selective ferroptosis in cancer without systematic starvation.
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•Cysteine depletion triggers adaptive ATF4 expression at the transcriptional level•Lysosomal cystine shortage induces ATF4 expression via the AhR signaling pathway•Attenuated cysteine stress response sensitizes ferroptosis upon cysteine starvation•CysRx elicits cancer cell ferroptosis via intracellular nutrient reprogramming
Swanda et al. reported that lysosomal cystine regulates the adaptative ATF4 expression, whereas cytosolic cysteine determines the ferroptosis sensitivity. By controlling the intracellular balance between cysteine and cystine, the synthetic CysRx reagent exhibits therapeutic potential by attenuating cysteine stress response and maximizing cancer cell ferroptosis.
RNA modification in the form of
N
6
-methyladenosine (m
6
A) regulates nearly all the post-transcriptional processes. The asymmetric m
6
A deposition suggests that regional methylation may have ...distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m
6
A modifications. Here we report the development of “m
6
A editing”, a powerful approach that enables m
6
A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single chain m
6
A methyltransferase that can be programmed with a guide RNA. The resultant m
6
A “writers” allow functional comparison of single site methylation in different mRNA regions. We further engineered m
6
A “erasers” by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m
6
A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
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