Biology subjects

Jaffrey, S. R.

Publications and source records attributed to Jaffrey, S. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Reversible m6Am methylation of snRNA by FTO controls morphine reward and tolerance without altering analgesia

Mu opioids, such as morphine, are effective analgesics, but their reward and tolerance drive opioid use disorder. A major goal is to achieve analgesia without these harmful effects. Here we show that morphine reward and tolerance require the RNA demethylase FTO. Genetic depletion and pharmacologic inhibition of FTO each reduced morphine reward, measured by conditioned-place preference, and reduced antinociceptive tolerance to morphine and fentanyl, without altering analgesia. Although FTO is known to erase m6A on mRNA, we found no effect of FTO depletion on m6A sites, but markedly increased levels of m6Am on snRNA. The effects of FTO depletion were suppressed in mice that cannot make m6Am, supporting the role of m6Am in morphine reward and tolerance. We show that FTO depletion regulates a gene expression network linked to morphine signaling. FTO inhibitors may therefore provide useful adjuvants to mu opioids in pain management and treatment of opioid use disorder.

neuroscience↗

bioRxiv2026

m6A pathway suppression reprograms gene expression during the integrated stress response

The integrated stress response (ISR) orchestrates cellular adaptations through translational repression and upregulation of ATF4, a transcription factor. However, ATF4 only accounts for a minority of ISR-induced gene-expression changes. Here we show that the ISR reshapes the transcriptome by stabilizing N6-methyladenosine (m6A)-containing mRNAs that are normally unstable. We show that mRNA stabilization, not transcription activation, explains much of the transcriptome response during the ISR, and that this stabilization selectively occurs on m6A-mRNAs. m6A-mRNA degradation is a translation-dependent process and translation repression during the ISR stabilizes m6A-mRNAs. Consequently, the ISR leads to increased m6A-mRNA expression, thereby upregulating their protein output despite overall diminished translation. Notably, stabilization of m6A-mRNAs by m6A depletion is sufficient to induce transcriptomic and proteomic features of amino acid-depleted cells. Together, our study identifies the direct coordination of translation suppression and transcriptome reprogramming through m6A during the ISR.

molecular biology↗

bioRxiv2025

Establishing benchmarks for quantitative mapping of m6A by Nanopore Direct RNA Sequencing

Nanopore direct RNA sequencing (DRS) coupled with Dorado modification-aware basecalling enables mapping of epitranscriptomic modifications including N6-methyladenosine (m6A) at the level of individual RNAs. However, the sensitivity, specificity, and reproducibility of this method remain unclear and have only recently begun to be addressed through systematic benchmarking studies. Here, we aimed to establish a best-practice workflow for DRS-based epitranscriptomic analyses. Specifically, we evaluated multiple Dorado versions and models using RNA isolated from primary cells and unmodified in vitro transcribed RNAs. We further utilized an m6A methyltransferase inhibitor as a specificity control. We established that stringent filtering is necessary to reduce false-positive calls and found that Dorado predictions captured an increasing proportion of GLORI sites detected at high m6A/A proportions. Further, by applying DRS to human primary fibroblasts and HD10.6 neurons, we detected cell type-specific differences in the predicted m6A/A proportions at conserved sites. Our study thus presents the first systematic comparison of Dorado and GLORI from the same input RNA and expands characterization of the m6A epitranscriptome to fibroblasts and neurons.

bioinformatics↗

bioRxiv2025

FTO depletion does not alter m6A stoichiometry in AML mRNA: a reassessment using direct RNA nanopore sequencing

The RNA demethylase FTO has been proposed to promote acute myeloid leukemia (AML) by demethylating N6-methyladenosine (m6A) from oncogenic transcripts, especially MYC. However, the evidence that supports the idea that FTO demethylates m6A in AML relies on methods that are non-quantitative and unable to reveal m6A stoichiometry changes before or after FTO depletion. To directly test whether FTO regulates m6A in mRNA, we employed Oxford Nanopore direct RNA sequencing to map and quantify m6A at single-nucleotide resolution. We find that the stoichiometry of m6A sites throughout the transcriptome and especially at MYC-specific sites are unaffected despite depletion of FTO activity by knockout, knockdown, or pharmacologic inhibition. This pattern was seen in AML cell lines MONOMAC-6 and MOLM-13, as well as in the non-AML cell line HEK293T. We also find that the anti-leukemia effect of the small-molecule FTO inhibitor FB23-2 is not due to FTO inhibition since it remains cytotoxic to FTO-deficient cells. Instead of regulating m6A, we find that FTO depletion markedly increases N6,2-O-dimethyladenosine (m6Am) in snRNAs, consistent with m6Am in snRNA being a target of FTO. Overall, our findings do not support an m6A eraser role for FTO in AML cell lines under the conditions tested, and they suggest that the reported demethylation functions of FTO on m6A should be reinvestigated using quantitative m6A mapping methods.

molecular biology↗

bioRxiv2025

tRNA modifications are required for stress granule formation and melanoma metastasis

Metastasis is the leading cause of cancer related deaths, however therapies specifically targeting metastasis are lacking and remain a dire therapeutic need in the clinic. Metastasis is a highly inefficient process that is inhibited by extracellular stress. Therefore, metastasizing cells that ultimately survive and successfully colonize distant organs must undergo molecular rewiring to mitigate stress. Wobble uridine modifications, especially 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), have been implicated in stress response and poor prognosis of cancer patients. We use a patient derived xenograft (PDX) model of melanoma metastasis to study the role of the mcm5s2U34 modification in the stress response of metastasizing cells. We find that upon depletion of elongator acetyltransferase complex subunit 1 (ELP1)-- a component of the mcm5s2U34 pathway on [Formula], and [Formula] --codon-biased translation, migration, invasion, and metastatic burden in vivo is reduced. Further, we observe that stress granule components are enriched in a subset of codon-biased genes that are exclusively upregulated at the protein level in metastatic nodules compared to the primary tumor in our PDX model. Additionally, upon knockdown of ELP1, stress granule components have decreased protein expression with no significant change to their mRNA levels. Efficient translation, mediated by the carboxy-methylation arm of the mcm5s2U34 modification, is required for metastasizing cancer cells to withstand stress via stress granule formation and increase survival throughout the metastatic cascade. This makes the mcm5s2U34 machinery a potentially actionable therapeutic target, specific to metastatic disease.

cancer biology↗

bioRxiv2024

Decoding m6Am by simultaneous transcription-start mapping and methylation quantification

N6,2-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5 isoforms. Thus, gene levels annotations cannot capture the diversity of m6Am modification in the transcriptome. Here we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5 isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.

molecular biology↗

bioRxiv2024

RNA tertiary structure and conformational dynamics revealed by BASH MaP

The functional effects of an RNA can arise from complex three-dimensional folds known as tertiary structures. However, predicting the tertiary structure of an RNA and whether an RNA adopts distinct tertiary conformations remains challenging. To address this, we developed BASH MaP, a single-molecule dimethyl sulfate (DMS) footprinting method and DAGGER, a computational pipeline, to identify alternative tertiary structures adopted by different molecules of RNA. BASH MaP utilizes potassium borohydride to reveal the chemical accessibility of the N7 position of guanosine, a key mediator of tertiary structures. We used BASH MaP to identify diverse conformational states and dynamics of RNA G-quadruplexes, an important RNA tertiary motif, in vitro and in cells. BASH MaP and DAGGER analysis of the fluorogenic aptamer Spinach reveals that it adopts alternative tertiary conformations which determine its fluorescence states. BASH MaP thus provides an approach for structural analysis of RNA by revealing previously undetectable tertiary structures.

molecular biology↗

bioRxiv2023

Selenocysteine tRNA methylation promotes oxidative stress resistance in melanoma metastasis

Selenocysteine-containing proteins play a central role in redox homeostasis. Their translation is a highly regulated process, dependent upon two tRNASec isodecoders differing by a single 2-O-ribose methylation, called Um34. We characterized FTSJ1 as the Um34 methyltransferase and show that its activity is required for efficient selenocysteine insertion at the UGA stop codon during translation. Specifically, Loss of Um34 leads to ribosomal stalling and decreased UGA recoding. FTSJ1-deficient cells are more sensitive to oxidative stress and have decreased metastatic colonization in xenograft models of melanoma metastasis. We found that FTSJ1 mediates efficient translation of selenoproteins essential for the cellular antioxidant response. Our findings uncover a role for tRNASec Um34 modification in oxidative stress resistance and highlight FTSJ1 as a potential therapeutic target specific for metastatic disease.

cancer biology↗

bioRxiv2023

Highly efficient cellular expression of circular mRNA enables prolonged protein expression

A major problem with mRNA therapeutics is the limited duration of protein expression due to the short half-life of mRNA. New approaches for generating highly stable circular mRNA in vitro have allowed increased duration of protein expression. However, it remains difficult to genetically encode circular mRNAs in mammalian cells, which limits the use of circular mRNA in cell-derived therapeutics. Here we describe the adaptation of the Tornado (Twister-optimized RNA for durable overexpression) system to achieve in-cell synthesis of circular mRNAs. We identify the promoter and internal ribosomal entry site (IRES) that result in high levels of protein expression in cells. We then show that these circular mRNAs can be packaged into virus-like particles (VLPs) thus enabling prolonged protein expression. Overall, these data describe a platform for synthesis of circular mRNAs and how these circular mRNAs can markedly enhance the ability of VLPs to function as a mRNA delivery tool.

synthetic biology↗

bioRxiv2022

Selective detection of m6A derived from mRNA using the Phospho-tag m6A assay

N6-methyladenosine (m6A) is a modified nucleotide found in mRNA, ribosome RNA (rRNA) and small nuclear RNA (snRNA). m6A in mRNA has important roles in regulating mRNA stability, splicing, and other processes. Numerous studies have described m6A as a dynamic modification using mass spectrometry-based quantification of m6A in mRNA samples prepared from different cellular conditions. However, these results have been questioned based on the finding that the mRNA purification protocols often result in varying levels of rRNA contamination. Additionally, mRNA purification protocols disproportionately enrich for the 3 ends of mRNA, a region that is enriched in m6A. To address these problems, we developed the Phospho-tag m6A assay, a highly efficient method for quantifying m6A specifically from mRNA. In this assay, a series of selective RNase digestion steps is performed, which results in m6A from rRNA and snRNA being liberated as m6A monophosphate, while m6A from mRNA is mostly liberated as m6A nucleoside. m6A levels are normalized to transcript levels, using m7G monophosphate liberated by yDcpS decapping enzyme as a surrogate for mRNA levels. Notably, this approach uses total cellular RNA, rather than purified mRNA, which simplifies the steps for m6A detection and overcomes the 3-end biases associated with mRNA purification. Overall, the Phospho-tag m6A provides a simple and efficient method for quantification of mRNA-derived m6A from total RNA samples.

molecular biology↗

bioRxiv2022

m6a governs length-dependent enrichment of mRNAs in stress granules

Stress granules are biomolecular condensates composed of protein and mRNA. Long mRNAs are enriched in stress granules, which is thought to reflect the ability of long mRNAs to form multiple RNA-RNA interactions with other mRNAs. RNA-RNA interactions are thus thought to be critical for stress granule formation. Stress granule-enriched mRNAs also often contain multiple N6-methyladenosine (m6A) residues. YTHDF proteins bind m6A, creating mRNA-protein complexes that partition into stress granules. Here we determine the basis of length-dependent enrichment of mRNAs in stress granules. We show that depletion of m6A is sufficient to abrogate the length-dependent enrichment of mRNAs in stress granules. We show that the presence of m6A predicts which mRNAs are enriched. m6A formation is triggered by long exons, which are often found in long mRNAs, accounting for the link between m6A, length and stress granule enrichment. Thus, length-dependent enrichment of mRNAs in stress granules is driven by YTHDF-mRNA interactions.

molecular biology↗

bioRxiv2021

Understanding the source of METTL3-independent m6A in mRNA

N6-methyladenosine (m6A) is a highly prevalent mRNA modification which promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other pathways. METTL3 is the major methyltransferase that catalyzes the formation of m6A in mRNA. As 30-80% of m6A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m6A. Here, we re-examined the source of m6A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines which continue to have m6A in their mRNA after Mettl3 knockout. We show that these cells express alternatively spliced Mettl3 transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported METTL3 knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after METTL3 deletion, suggesting that reported METTL3 knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3s role in synthesizing m6A using a genomic deletion of Mettl3, and found that METTL3 is responsible for >95% of m6A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m6A in the transcriptome, and that remaining m6A in putative METTL3 knockout cell lines is due to the expression of altered but functional METTL3 isoforms.

molecular biology↗

bioRxiv2021

Cbln1 regulates axon growth and guidance in multiple neural regions

The accurate construction of neural circuits requires the precise control of axon growth and guidance, which is regulated by multiple growth and guidance cues during early nervous system development. It is generally thought that the growth and guidance cues that control the major steps of axon guidance have been defined. Here, we describe cerebellin-1 (Cbln1) as a novel cue that controls diverse aspects of axon growth and guidance throughout the central nervous system (CNS). Cbln1 has previously been shown to function in late neural development to influence synapse organization. Here we find that Cbln1 has an essential role in early neural development. Cbln1 is expressed on the axons and growth cones of developing commissural neurons and functions in an autocrine manner to promote axon growth. Cbln1 is also expressed in intermediate target tissues and functions as an attractive guidance cue. We find that these functions of Cbln1 are mediated by neurexin-2 (Nrxn2), which functions as the Cbln1 receptor for axon growth and guidance. In addition to the developing spinal cord, we further show that Cbln1 functions in diverse parts of the CNS with major roles in cerebellar parallel fiber growth and retinal ganglion cell axon guidance. Despite the prevailing role of Cbln1 as a synaptic organizer, our study discovers a new and unexpected function for Cbln1 as a general axon growth and guidance cue throughout the nervous system. Impact statementDespite the prevailing role of Cbln1 as a synaptic organizer, our study discovers a new and unexpected function for Cbln1 as a general axon growth and guidance cue throughout the nervous system.

neuroscience↗

bioRxiv2021

Mapping of m6A and Its Regulatory Targets in Prostate Cancer Reveals a METTL3-low Induction of Therapy Resistance

Recent evidence has highlighted the role of N6-methyladenosine (m6A) in the regulation of mRNA expression, stability and translation, supporting a potential role for post-transcriptional regulation mediated by m6A in cancer. Here we explore prostate cancer as an exemplar and demonstrate that low levels of N6-adenosine-methyltransferase (METTL3) is associated with advanced metastatic disease. To explore this relationship, we generated the first prostate m6A maps, and further examined how METTL3 regulates expression at the level of transcription, translation, and protein. Significantly, transcripts encoding extracellular matrix proteins are consistently upregulated with METTL3 knockdown. We also examined the relationship between METTL3 and androgen signaling and discovered the upregulation of a hepatocyte nuclear factor-driven gene signature that is associated with therapy resistance in prostate cancer. Significantly, METTL3 knockdown rendered the cells resistant to androgen receptor antagonists, implicating changes in m6A as a mechanism for therapy resistance in metastatic prostate cancer.

cancer biology↗

bioRxiv2020

N6-methyladenosine in poly(A) tails stabilize VSG transcripts

RNA modifications are important regulators of gene expression. In Trypanosoma brucei, transcription is polycistronic and thus most regulation happens post-transcriptionally. N6-methyladenosine (m6A) has been detected in this parasite, but its function remains unknown. Here we show that [~]50% of the m6A is located in the poly(A) tail of the monoallelically expressed Variant Surface Glycoprotein (VSG) transcript. m6A residues are removed from the VSG poly(A) tail prior to deadenylation and mRNA degradation. Using genetic tools, we identified a 16-mer motif in the 3UTR of VSG that acts as a cis-acting motif required for inclusion of m6A in the poly(A) tail. Removal of this motif from the VSG 3 UTR results in poly(A) tails lacking m6A, rapid deadenylation and mRNA degradation. To our knowledge this is the first identification of an RNA modification in the poly(A) tail of any eukaryote, uncovering a novel post-transcriptional mechanism of gene regulation.

molecular biology↗

bioRxiv2019

Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA

Chemical modification of transcripts with 5 caps occurs in all organisms. Here we report a systems-level mass spectrometry-based technique, CapQuant, for quantitative analysis of the cap epitranscriptome in any organism. The method was piloted with 21 canonical caps - m7GpppN, m7GpppNm, GpppN, GpppNm, and m2,2,7GpppG - and 5 \"metabolite\" caps - NAD, FAD, UDP-Glc, UDP-GlcNAc, and dpCoA. Applying CapQuant to RNA from purified dengue virus, Escherichia coli, yeast, mice, and humans, we discovered four new cap structures in humans and mice (FAD, UDP-Glc, UDP-GlcNAc, and m7Gpppm6A), cell- and tissue-specific variations in cap methylation, and surprisingly high proportions of caps lacking 2-O-methylation, such as m7Gpppm6A in mammals and m7GpppA in dengue virus, and we did not detect cap m1A/m1Am in humans. CapQuant accurately captured the preference for purine nucleotides at eukaryotic transcription start sites and the correlation between metabolite levels and metabolite caps. The mystery around cap m1A/m1Am analysis remains unresolved.

systems biology↗

bioRxiv2019

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

Many cellular mRNAs contain the modified base m6A, and recent studies have suggested that various stimuli can lead to changes in m6A. The most common method to map m6A and to predict changes in m6A 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 m6A 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 m6A 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.

bioinformatics↗

bioRxiv2019

Antibody cross-reactivity accounts for widespread appearance of m1A in 5 UTRs

N1-methyladenosine (m1A) was recently identified as a new mRNA modification based on its mapping to the 5 UTRs of thousands of mRNAs with an m1A-binding antibody. More recent studies have confirmed the prevalence of m1A, while others have questioned it. To address this discrepancy, we mapped m1A using ultra-deep RNA-Seq datasets based on m1A-induced misincorporations during reverse transcription. Using this approach, we find m1A only in the mitochondrial MT-ND5 transcript. In contrast, when we mapped m1A antibody-binding sites at single-nucleotide resolution, we found binding to transcription start nucleotides in mRNA 5 UTRs. Using different biochemical assays, we find that m1A is not present at these sites. Instead, we find that the m1A antibody exhibits m1A-independent binding to mRNA cap structures. We also tested a new and independently derived m1A antibody. We show that this m1A antibody lacks m7G cap-binding cross-reactivity, and notably does not map to 5 UTRs in the transcriptome. Our data demonstrate that high-stoichiometry m1A sites are rare in the transcriptome and that previous mapping of m1A to mRNA 5 UTRs are due to unintended binding of the m1A antibody to m7G cap structure in mRNA.

molecular biology↗