Glioblastoma (GBM) is the most common type of adult malignant brain tumor, but its molecular mechanisms are not well understood. In addition, the knowledge of the disease-associated expression and ...function of YTHDF2 remains very limited. Here, we show that YTHDF2 overexpression clinically correlates with poor glioma patient prognosis. EGFR that is constitutively activated in the majority of GBM causes YTHDF2 overexpression through the EGFR/SRC/ERK pathway. EGFR/SRC/ERK signaling phosphorylates YTHDF2 serine39 and threonine381, thereby stabilizes YTHDF2 protein. YTHDF2 is required for GBM cell proliferation, invasion, and tumorigenesis. YTHDF2 facilitates m
A-dependent mRNA decay of LXRA and HIVEP2, which impacts the glioma patient survival. YTHDF2 promotes tumorigenesis of GBM cells, largely through the downregulation of LXRα and HIVEP2. Furthermore, YTHDF2 inhibits LXRα-dependent cholesterol homeostasis in GBM cells. Together, our findings extend the landscape of EGFR downstream circuit, uncover the function of YTHDF2 in GBM tumorigenesis, and highlight an essential role of RNA m
A methylation in cholesterol homeostasis.
RNA contains over 150 types of chemical modifications. Although many of these chemical modifications were discovered several decades ago, their functions were not immediately apparent. Discoveries of ...RNA demethylases, along with advances in mass spectrometry and high-throughput sequencing techniques, have caused research into RNA modifications to progress at an accelerated rate. Post-transcriptional RNA modifications make up an epitranscriptome that extensively regulates gene expression and biological processes. Here, we present an overview of recent advances in the field that are shaping our understanding of chemical modifications, their impact on development and disease, and the dynamic mechanisms through which they regulate gene expression.
Quantifying RNAs in their spatial context is crucial to understanding gene expression and regulation in complex tissues. In situ transcriptomic methods generate spatially resolved RNA profiles in ...intact tissues. However, there is a lack of a unified computational framework for integrative analysis of in situ transcriptomic data. Here, we introduce an unsupervised and annotation-free framework, termed ClusterMap, which incorporates the physical location and gene identity of RNAs, formulates the task as a point pattern analysis problem, and identifies biologically meaningful structures by density peak clustering (DPC). Specifically, ClusterMap precisely clusters RNAs into subcellular structures, cell bodies, and tissue regions in both two- and three-dimensional space, and performs consistently on diverse tissue types, including mouse brain, placenta, gut, and human cardiac organoids. We demonstrate ClusterMap to be broadly applicable to various in situ transcriptomic measurements to uncover gene expression patterns, cell niche, and tissue organization principles from images with high-dimensional transcriptomic profiles.
N6-methyladenosine (m6A) regulates mRNA metabolism and translation, serving as an important source of post-transcriptional regulation. To date, the functional consequences of m6A deficiency within ...the adult brain have not been determined. To achieve m6A deficiency, we deleted Mettl14, an essential component of the m6A methyltransferase complex, in two related yet discrete mouse neuronal populations: striatonigral and striatopallidal. Mettl14 deletion reduced striatal m6A levels without altering cell numbers or morphology. Transcriptome-wide profiling of m6A-modified mRNAs in Mettl14-deleted striatum revealed downregulation of similar striatal mRNAs encoding neuron- and synapse-specific proteins in both neuronal types, but striatonigral and striatopallidal identity genes were uniquely downregulated in each respective manipulation. Upregulated mRNA species encoded non-neuron-specific proteins. These changes increased neuronal excitability, reduced spike frequency adaptation, and profoundly impaired striatal-mediated behaviors. Using viral-mediated, neuron-specific striatal Mettl14 deletion in adult mice, we further confirmed the significance of m6A in maintaining normal striatal function in the adult mouse.
•Mettl14 is required for m6A mRNA methylation in the adult mammalian brain•m6A deficiency in both D1 and D2 striatal neurons downregulates neuronal mRNAs•m6A deficiency in striatal neurons impairs learning and performance•Striatal m6A deficiency alters firing properties without morphological changes
Koranda et al. demonstrate Mettl14 is indispensable for m6A mRNA methylation in the adult mammalian brain. Striatal Mettl14 deficiency decreases m6A tagging, altering the epitranscriptome profile. Mettl14 deficiency impairs behavior and alters intrinsic neuronal firing in the absence of morphological changes.
mRNA has recently been established as a new class of therapeutics, due to its programmability and ability to produce proteins of interest rapidly in vivo. Despite its demonstrated utility, mRNA as a ...protein expression platform remains limited by its translational capacity and RNA stability. Here, we introduce messenger-oligonucleotide conjugated RNAs (mocRNAs) to enable site-specific, robust, and modularized encoding of chemical modifications for highly efficient and stable protein expression. In mocRNA constructs, chemically synthesized oligonucleotides are ligated to the 3′ terminus of mRNA substrates to protect poly(A) tails from degradation, without compromising their potency in stimulating translation. As a proof-of-concept, mocRNAs modified by deadenylase-resistant oligonucleotides result in augmented protein production by factors of 2–4 in human HeLa cells and by 10-fold in primary rat cortical neuronal cultures. By directly linking enzymatic and organic synthesis of mRNA, we envision that the mocRNA design will open new avenues to expand the chemical space and translational capacity of RNA-based vectors in basic research and therapeutic applications.
Cellular RNAs are naturally decorated with a variety of chemical modifications. The structural diversity of the modified nucleosides provides regulatory potential to sort groups of RNAs for organized ...metabolism and functions, thus affecting gene expression. Recent years have witnessed a burst of interest in and understanding of RNA modification biology, thanks to the emerging transcriptome-wide sequencing methods for mapping modified sites, highly sensitive mass spectrometry for precise modification detection and quantification, and extensive characterization of the modification “effectors,” including enzymes (“writers” and “erasers”) that alter the modification level and binding proteins (“readers”) that recognize the chemical marks. However, challenges remain due to the vast heterogeneity in expression abundance of different RNA species, further complicated by divergent cell-type-specific and tissue-specific expression and localization of the effectors as well as modifications. In this review, we highlight recent progress in understanding the function of N6-methyladenosine (m6A), the most abundant internal mark on eukaryotic mRNA, in light of the specific biological contexts of m6A effectors. We emphasize the importance of context for RNA modification regulation and function.
RNA N6-methyladenosine (m6A) has emerged as a multifaceted controller for gene expression regulation, mediated through its effector proteins—writers, readers, and erasers. Shi et al. review recent advances in the mechanistic understandings of m6A effectors in various biological systems and cellular responses, emphasizing cellular and molecular contexts as important determinants of RNA modification functions.
Brain metastasis is a major cause of cancer mortality, but its molecular mechanisms are severely understudied. In addition, little is known regarding the role of m6A reader YTHDF3 in human diseases. ...Here, we show that YTHDF3 overexpression clinically correlates with brain metastases in breast cancer patients. YTHDF3 promotes cancer cell interactions with brain endothelial cells and astrocytes, blood-brain barrier extravasation, angiogenesis, and outgrow. Mechanistically, YTHDF3 enhances the translation of m6A-enriched transcripts for ST6GALNAC5, GJA1, and EGFR, all associated with brain metastasis. Furthermore, overexpression of YTHDF3 in brain metastases is attributed to increased gene copy number and the autoregulation of YTHDF3 cap-independent translation by binding to m6A residues within its own 5′ UTR. Our work uncovers an essential role of YTHDF3 in controlling the interaction between cancer cells and brain microenvironment, thereby inducing brain metastatic competence.
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•YTHDF3 overexpression clinically correlates with breast cancer brain metastases•Breast cancer brain metastases have YTHDF3 gene copy gain and autoregulation•YTHDF3 plays critical roles in multiple steps of brain metastasis cascade•YTHDF3 promotes translation of the key brain metastatic genes ST6GALNAC5 and GJA1
Chang et al. report that YTHDF3 overexpression associates with breast cancer brain metastasis and confers poor survival. Mechanistically, YTHDF3 enhances the expression of m6A-enriched transcripts that promote cancer cell-brain microenvironment interactions and brain metastasis.
N6-Methyladenosine (m6A) is the most abundant post-transcriptional mRNA modification in eukaryotes and exerts many of its effects on gene expression through reader proteins that bind specifically to ...m6A-containing transcripts. Fragile X mental retardation protein (FMRP), an RNA-binding protein, has previously been shown to affect the translation of target mRNAs and trafficking of mRNA granules. Loss of function of FMRP causes fragile X syndrome, the most common form of inherited intellectual disability in humans. Using HEK293T cells, siRNA-mediated gene knockdown, cytoplasmic and nuclear fractions, RNA-Seq, and LC-MS/MS analyses, we demonstrate here that FMRP binds directly to a collection of m6A sites on mRNAs. FMRP depletion increased mRNA m6A levels in the nucleus. Moreover, the abundance of FMRP targets in the cytoplasm relative to the nucleus was decreased in Fmr1-KO mice, an effect also observed in highly methylated genes. We conclude that FMRP may affect the nuclear export of m6A-modified RNA targets.
6
-methyladenosine (m
6
A), the most abundant internal modification on mRNAs in eukaryotes, play roles in adipogenesis. However, the underlying mechanism remains largely unclear. Here, we show that m
...6
A plays a critical role in regulating macroautophagy/autophagy and adipogenesis through targeting Atg5 and Atg7. Mechanistically, knockdown of FTO, a well-known m
6
A demethylase, decreased the expression of ATG5 and ATG7, leading to attenuation of autophagosome formation, thereby inhibiting autophagy and adipogenesis. We proved that FTO directly targeted Atg5 and Atg7 transcripts and mediated their expression in an m
6
A-dependent manner. Further study identified that Atg5 and Atg7 were the targets of YTHDF2 (YTH N6-methyladenosine RNA binding protein 2). Upon FTO silencing, Atg5 and Atg7 transcripts with higher m
6
A levels were captured by YTHDF2, which resulted in mRNA degradation and reduction of protein expression, thus alleviating autophagy and adipogenesis. Furthermore, we generated an adipose-selective fto knockout mouse and find that FTO deficiency decreased white fat mass and impairs ATG5- and ATG7-dependent autophagy in vivo. Together, these findings unveil the functional importance of the m
6
A methylation machinery in autophagy and adipogenesis regulation, which expands our understanding of such interplay that is essential for development of therapeutic strategies in the prevention and treatment of obesity.
3-MA: 3-methyladenine; ACTB: actin, beta; ATG: autophagy-related; Baf A1: bafilomycin A
1
; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; FABP4: fatty acid binding protein 4, adipocyte; FTO: fat mass and obesity associated; HFD: high-fat diet; LC-MS/MS: liquid chromatography-tandem mass spectrometry; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; m
6
A: N
6
-methyladenosine; MEFs: mouse embryo fibroblasts; MeRIP-qPCR: methylated RNA immunoprecipitation-qPCR; PPARG: peroxisome proliferator activated receptor gamma; RIP: RNA-immunoprecipitation; SAT: subcutaneous adipose tissue; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like kinase 1; VAT: visceral adipose tissue; WAT: white adipose tissue; YTHDF: YTH N6-methyladenosine RNA binding protein
N6-methyladenosine (m6A) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional m6A-dependent alternative splicing regulation is still ...poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an m6A reader protein that binds RNA through RRM and Arg-Gly-Gly (RGG) motifs. Here, we show that hnRNPG directly binds to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) using RGG motifs in its low-complexity region. Through interactions with the phosphorylated CTD and nascent RNA, hnRNPG associates co-transcriptionally with RNAPII and regulates alternative splicing transcriptome-wide. m6A near splice sites in nascent pre-mRNA modulates hnRNPG binding, which influences RNAPII occupancy patterns and promotes exon inclusion. Our results reveal an integrated mechanism of co-transcriptional m6A-mediated splicing regulation, in which an m6A reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing.
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•The m6A reader protein hnRNPG interacts with RNA polymerase II using an RGG region•hnRNPG binds to nascent m6A-modified pre-mRNA and regulates alternative splicing•m6A is enriched in exonic regions near splice sites of hnRNPG-regulated exons•hnRNPG affects RNA polymerase II occupancy around hnRNPG-regulated exons
Zhou et al. show that the m6A reader protein hnRNPG interacts with m6A-modified nascent pre-mRNA and the phosphorylated C-terminal domain of RNA polymerase II to regulate alternative splicing. These interactions depend on an RGG region in the low-complexity region of hnRNPG.
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