Dynamic m<sup>6</sup>A methylation facilitates mRNA triaging to stress granules

Anders, Maximilian; Chelysheva, Irina; Goebel, Ingrid; Trenkner, Timo; Zhou, Jun; Mao, Yuanhui; Verzini, Silvia; Qian, Shu‐Bing; Ignatova, Zoya

doi:10.26508/lsa.201800113

Cited by 146 publications

(147 citation statements)

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“…2 or edgeR, Table 1) 30,31,[55][56][57] . In the mouse cortex and Huh7 cell data, we found that, similar to RNA-seq data 24,56,58 , the variance in read counts under peaks exceeded their mean, indicative of overdispersion ( Supplementary Fig. 6a).…”

Section: Detection Of Peaks Across Replicates Experiments and Cell mentioning

confidence: 57%

“…have reported changes to m 6 A with heat shock, microRNA expression, transcription factor expression, cancer, oxidative stress, human immunodeficiency virus (HIV) infection, Kaposi's sarcoma herpesvirus (KSHV) infection, and Zika virus infection, including hundreds to thousands of changes in enrichment at specific sites [20][21][22][23][24][25][26][27][28][29] . Statistical approaches to analysis have only recently been published and there have been no comprehensive evaluations of methods to detect changes in m 6 A based on MeRIP-seq data 30,31 .…”

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confidence: 99%

“…Thus, while studies have suggested that m 6 A shows widespread changes in response to diverse stimuli, they have applied inconsistent analysis methods to detect changes in m 6 A and often don't control for differences in RNA expression between conditions or typical variability in peak heights between replicates. In some cases, these studies have reported m 6 A changes based on simple differences in peak count 24,26,27,32 . However, others have applied statistical tests or thresholds for differences in immunoprecipitated (IP) over input fraction enrichment and visual analysis of coverage plots, and have reported fewer m 6 A changes or suggested that m 6 A is a relatively stable mark 33,34 .…”

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confidence: 99%

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Limits in the detection of m6A changes using MeRIP/m6A-seq

McIntyre

Gokhale

Cerchietti

et al. 2020

Sci Rep

156

122

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Many cellular mRNAs contain the modified base m 6 A, and recent studies have suggested that various stimuli can lead to changes in m 6 A. the most common method to map m 6 A and to predict changes in m 6 A between conditions is methylated RNA immunoprecipitation sequencing (MeRIP-seq), through which methylated regions are detected as peaks in transcript coverage from immunoprecipitated RNA relative to input RNA. Here, we generated replicate controls and reanalyzed published MeRIP-seq data to estimate reproducibility across experiments. We found that m 6 A peak overlap in mRNAs varies from ~30 to 60% between studies, even in the same cell type. We then assessed statistical methods to detect changes in m 6 A peaks as distinct from changes in gene expression. However, from these published data sets, we detected few changes under most conditions and were unable to detect consistent changes across studies of similar stimuli. Overall, our work identifies limits to MeRIP-seq reproducibility in the detection both of peaks and of peak changes and proposes improved approaches for analysis of peak changes. Methylation at the N6 position in adenosine (m 6 A) is the most common internal modification in eukaryotic mRNA. A methyltransferase complex composed of METTL3, METTL14, WTAP, VIRMA, and other cofactors catalyzes methylation at DRACH/DRAC motifs, primarily in the last exon 1,2. Most m 6 A methylation occurs during transcription 3. The modification then affects mRNA metabolism through recognition by RNA-binding proteins that regulate processes including translation and mRNA degradation 4-9. However, whether m 6 A is lost and gained in response to various cellular changes remains contentious 3,10-15. To assess the evidence for proposed dynamic changes in m 6 A, a reliable and reproducible method to detect changes in methylation as distinct from changes in gene expression is necessary. The first and most widely-used method to enable transcriptome-wide studies of m 6 A, MeRIP-seq or m 6 A-seq, involves the immunoprecipitation of m 6 A-modified RNA fragments followed by peak detection through comparison to background gene coverage 16,17. A second method was developed in 2015, miCLIP or m 6 A-CLIP, which involves crosslinking at the site of antibody binding to induce mutations during reverse transcription for single-nucleotide detection of methylated bases 2,18. MeRIP-seq is still more often used than miCLIP, despite less precise localization of m 6 A to peak regions of approximately 50-200 base pairs that can contain multiple DRAC motifs, since it follows a simpler protocol, requires less starting material, and generally produces higher coverage of more transcripts. Antibodies for m 6 A can also detect a second base modification, N 6 ,2′-O-dimethyladenosine (m 6 A m), found at a lower abundance than m 6 A and located at the 5′ ends of select transcripts 15,18. We thus refer to the base modifications detected through MeRIP-seq collectively as m 6 A (m) , although most are likely m 6 A. As of late 2018, over fifty studies used M...

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Section: Detection Of Peaks Across Replicates Experiments and Cell mentioning

confidence: 57%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Limits in the detection of m6A changes using MeRIP/m6A-seq

McIntyre

Gokhale

Cerchietti

et al. 2020

Sci Rep

156

122

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“…Moreover, m 6 A modifications in the 5′‐UTR of ATF4 mRNA (containing uORFs) regulate ribosome scanning and selection of start site under conditions of amino acid starvation. Additionally, a dose‐dependent response to sodium arsenite increases m 6 A signals at THE 5′‐UTR and 5′‐vicinity of CDS of mRNAs, which promotes their targeting to SGs via interaction with YTHDF3 protein . All this suggests that global and alternative translation is modulated by m 6 A modifications within mRNAs.…”

Section: Emerging Topics and Future Perspectivesmentioning

confidence: 96%

“…Additionally, a dose-dependent response to sodium arsenite increases m 6 A signals at THE 5′-UTR and 5′-vicinity of CDS of mRNAs, which promotes their targeting to SGs via interaction with YTHDF3 protein. [120] All this suggests that global and alternative translation is modulated by m 6 A modifications within mRNAs. We agree here that m 6 A (and likely other RNA modifications) broadly contribute to stress response by targeting "housekeeping" transcripts to SGs and stimulating alternative translation of pro-survival transcripts; we also speculate that it provides a dynamic synergy between tRNA pools and modified/unmodified mRNAs to rapidly alter proteome for translational response to stress.…”

Section: Emerging Topics and Future Perspectivesmentioning

confidence: 99%

Translational Control under Stress: Reshaping the Translatome

2019

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Adequate reprogramming of cellular metabolism in response to stresses or suboptimal growth conditions involves a myriad of coordinated changes that serve to promote cell survival. As protein synthesis is an energetically expensive process, its regulation under stress is of critical importance. Reprogramming of messenger RNA (mRNA) translation involves well‐understood stress‐activated kinases that target components of translation initiation machinery, resulting in the robust inhibition of general translation and promotion of the translation of stress‐responsive proteins. Translational arrest of mRNAs also results in the accumulation of transcripts in cytoplasmic foci called stress granules. Recent studies focus on the key roles of transfer RNA (tRNA) in stress‐induced translational reprogramming. These include stress‐specific regulation of tRNA pools, codon‐biased translation influenced by tRNA modifications, tRNA miscoding, and tRNA cleavage. In combination, signal transduction pathways and tRNA metabolism changes regulate translation during stress, resulting in adaptation and cell survival. This review examines molecular mechanisms that regulate protein synthesis in response to stress.

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Hypoxic Upregulation of IER2 Increases Paracrine GMFG Signaling of Endoplasmic Reticulum Stress‐CAF to Promote Chordoma Progression via Targeting ITGB1

Zhang,

Zheng,

Xia

et al. 2024

Advanced Science

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Currently, the oncogenic mechanism of endoplasmic reticulum stress‐CAF (ERS‐CAF) subpopulation in chordoma remains unknown. Here, single‐cell RNA sequencing, spatial transcriptomics, GeoMx Digital Spatial Profiler, data‐independent acquisition proteomics, bulk RNA‐seq, and multiplexed quantitative immunofluorescence are used to unveil the precise molecular mechanism of how ERS‐CAF affected chordoma progression. Results show that hypoxic microenvironment reprograms CAFs into ERS‐CAF subtype. Mechanistically, this occurrs via hypoxia‐mediated transcriptional upregulation of IER2. Overexpression of IER2 in CAFs promotes chordoma progression, which can be impeded by IER2 knockdown or use of ERS inhibitors. IER2 also induces expression of ERS‐CAF marker genes and results in production of a pro‐tumorigenic paracrine GMFG signaling, which exert its biological function via directly binding to ITGB1 on tumor cells. ITGB1 inhibition attenuates tumor malignant progression, which can be partially reversed by exogenous GMFG intervention. Further analyses reveal a positive correlation between ITGB1high tumor cell counts and SPP1+ macrophage density, as well as the spatial proximity of these two cell types. Clinically, a significant correlation of high IER2/ITGB1 expression with tumor aggressive phenotype and poor patient survival is observed. Collectively, the findings suggest that ERS‐CAF regulates SPP1+ macrophage to aggravate chordoma progression via the IER2/GMFG/ITGB1 axis, which may be targeted therapeutically in future.

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Dynamic m⁶A methylation facilitates mRNA triaging to stress granules

Abstract: m6A modification in the 5′ vicinity of the coding sequence of transcripts provides a selective mechanism for triaging mRNAs to stress granules and is mediated by the YTHDF3 “reader” protein.

Cited by 146 publications

References 50 publications

Limits in the detection of m6A changes using MeRIP/m6A-seq

Limits in the detection of m6A changes using MeRIP/m6A-seq

Translational Control under Stress: Reshaping the Translatome

Hypoxic Upregulation of IER2 Increases Paracrine GMFG Signaling of Endoplasmic Reticulum Stress‐CAF to Promote Chordoma Progression via Targeting ITGB1

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Dynamic m6A methylation facilitates mRNA triaging to stress granules

Abstract: m6A modification in the 5′ vicinity of the coding sequence of transcripts provides a selective mechanism for triaging mRNAs to stress granules and is mediated by the YTHDF3 “reader” protein.

Cited by 146 publications

References 50 publications

Limits in the detection of m6A changes using MeRIP/m6A-seq

Limits in the detection of m6A changes using MeRIP/m6A-seq

Translational Control under Stress: Reshaping the Translatome

Hypoxic Upregulation of IER2 Increases Paracrine GMFG Signaling of Endoplasmic Reticulum Stress‐CAF to Promote Chordoma Progression via Targeting ITGB1

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Dynamic m⁶A methylation facilitates mRNA triaging to stress granules