DHX36 binding induces RNA structurome remodeling and regulates RNA abundance via m6A/YTHDF1
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ABSTRACT: RNA structure constitutes a new layer of gene regulatory mechanisms. RNA binding proteins can modulate RNA secondary structures, thus participating in post-transcriptional regulation. The DEAH-box helicase 36 (DHX36) is known to bind and unwind RNA G-quadruplex (rG4) structure but the transcriptome-wide RNA structure remodeling induced by DHX36 binding and the impact on RNA fate remain poorly understood. Here, we investigate the RNA structurome alteration induced by DHX36 depletion. Our findings reveal that DHX36 binding induces structure remodeling not only at the localized binding sites but also on the entire mRNA transcript most pronounced in3’UTR regions. DHX36 binding increase structural accessibility at 3’UTRs which is correlated with decreased post-transcriptional mRNA abundance. Further analyses and experiments uncover that DHX36 binding sites are enriched for N6-methyladenosine (m6A) modification and YTHDF1 binding; and DHX36 induced structural change may facilitate YTHDF1 binding to m6A sites leading to RNA degradation. Altogether, our findings uncover the structural remodeling effect of DHX36 binding and its impact on RNA abundance through regulating m6A dependent YTHDF1 binding.
Project description:RNA binding proteins can modulate RNA secondary structures, thus participating in post-transcriptional regulation. The DEAH-box helicase 36 (DHX36) has a remarkable ability to bind and unwind RNA G-quadruplex (rG4) and duplex. However, the transcriptome-wide RNA structure dynamic induced by DHX36 and how structure change subsequently influences RNA fate remain poorly understood. Here, we first identify the endogenous binding sites and specificity of DHX36 based on binding profiles. Next, we capture in vivo RNA structuromes to investigate the structure change of DHX36-bound mRNAs following DHX36 knockout. DHX36 induces structure remodeling on not only the localized binding sites but also the other sites across the entire mRNA especially in 3’UTR. DHX36-induced more accessible structures of 3’UTR are revealed to correlate with post-transcriptional mRNA decrease. Furthermore, we demonstrate that DHX36 binding sites are enriched for N6-methyladenosine (m6A) modification and YTHDF1 binding. Finally, we experimentally validate that YTHDF1 binding is repelled to DHX36 loss-induced structure inaccessibility and YTHDF1 loss-induced mRNA stabilization could be a source of DHX36 loss-induced mRNA increase. Altogether, our findings uncover the effect of DHX36 binding on in vivo mRNA structure and propose a plausible mechanism of how RNA secondary structure change involves in post-transcriptional regulation through orchestrating YTHDF1 binding.
Project description:RNA binding proteins can modulate RNA secondary structures, thus participating in post-transcriptional regulation. The DEAH-box helicase 36 (DHX36) has a remarkable ability to bind and unwind RNA G-quadruplex (rG4) and duplex. However, the transcriptome-wide RNA structure dynamic induced by DHX36 and how structure change subsequently influences RNA fate remain poorly understood. Here, we first identify the endogenous binding sites and specificity of DHX36 based on binding profiles. Next, we capture in vivo RNA structuromes to investigate the structure change of DHX36-bound mRNAs following DHX36 knockout. DHX36 induces structure remodeling on not only the localized binding sites but also the other sites across the entire mRNA especially in 3’UTR. DHX36-induced more accessible structures of 3’UTR are revealed to correlate with post-transcriptional mRNA decrease. Furthermore, we demonstrate that DHX36 binding sites are enriched for N6-methyladenosine (m6A) modification and YTHDF1 binding. Finally, we experimentally validate that YTHDF1 binding is repelled to DHX36 loss-induced structure inaccessibility and YTHDF1 loss-induced mRNA stabilization could be a source of DHX36 loss-induced mRNA increase. Altogether, our findings uncover the effect of DHX36 binding on in vivo mRNA structure and propose a plausible mechanism of how RNA secondary structure change involves in post-transcriptional regulation through orchestrating YTHDF1 binding.
Project description:The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader. PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) was applied to human YTHDC1 protein to identify its binding sites.
Project description:N6-methyladenosine (m6A) is the most abundant internal messenger (mRNA) modification in mammalian mRNA. This modification is reversible and non-stoichiometric, which potentially adds an additional layer of variety and dynamic control of mRNA metabolism. The m6A-modified mRNA can be selectively recognized by the YTH family “reader” proteins. The preferential binding of m6A-containing mRNA by YTHDF2 is known to reduce the stability of the target transcripts; however, the exact effects of m6A on translation has yet to be elucidated. Here we show that another m6A reader protein, YTHDF1, promotes ribosome loading of its target transcripts. YTHDF1 forms a complex with translation initiation factors to elevate the translation efficiency of its bound mRNA. In a unified mechanism of translation control through m6A, the YTHDF2-mediated decay controls the lifetime of target transcripts; whereas, the YTHDF1-based translation promotion increases the translation efficiency to ensure effective protein production from relatively short-lived transcripts that are marked by m6A. PAR-CLIP and RIP was used to identify YTHDF1 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF1 siRNA knock-down
Project description:We show that N6-methyladenosine (m6A), the most abundant internal modification in mRNA/lncRNA with still poorly characterized function, alters RNA structure to facilitate the access of RBM for heterogeneous nuclear ribonucleoprotein C (hnRNP C). We term this mechanism m6A-switch. Through combining PAR-CLIP with Me-RIP, we identify 39,060 m6A-switches among hnRNP C binding sites transcriptome-wide. We show that m6A-methyltransferases METTL3 or METTL14 knockdown decreases hnRNP C binding at 16,582 m6A-switches. Taken together, 2,798 m6A-switches of high confidence are identified to mediate RNA-hnRNP C interactions and affect diverse biological processes including cell cycle regulation. These findings reveal the biological importance of m6A and provide insights into the sophisticated regulation of RNA-RBP interactions through m6A-induced RNA structural remodeling. Measure the m6A methylated hnRNP C binding sites transcriptome-wide by PARCLIP-MeRIP; measure the differential hnRNP C occupancies upon METTL3/METTL14 knockdown by PAR-CLIP; measure RNA abundance and splicing level changes upon HNRNPC, METTL3 and METTL14 knockdown
Project description:To identify the YTHDF1 binding changes upon DHX36 loss, we conducted and analyzed YTHDF1 CLIP-seq data in WT and DHX36-KO HEK293T cells.
Project description:We show that N6-methyladenosine (m6A), the most abundant internal modification in mRNA/lncRNA with still poorly characterized function, alters RNA structure to facilitate the access of RBM for heterogeneous nuclear ribonucleoprotein C (hnRNP C). We term this mechanism m6A-switch. Through combining PAR-CLIP with Me-RIP, we identify 39,060 m6A-switches among hnRNP C binding sites transcriptome-wide. We show that m6A-methyltransferases METTL3 or METTL14 knockdown decreases hnRNP C binding at 16,582 m6A-switches. Taken together, 2,798 m6A-switches of high confidence are identified to mediate RNA-hnRNP C interactions and affect diverse biological processes including cell cycle regulation. These findings reveal the biological importance of m6A and provide insights into the sophisticated regulation of RNA-RBP interactions through m6A-induced RNA structural remodeling.
Project description:The N6-methyladenosine (m6A) mRNA modification and the mitochondrial respiratory chain (MRC) hold paramount importance in the advancement of MASLD. This study thoroughly investigates the relationship and impact of m6A mRNA modification and mitochondrial function in the progression of MASLD. Here we report that the mRNA and protein levels of mitochondrial respiratory chain (MRC) subunits showed inconsistent trends in vivo experiments. Abnormal m6A modification and mitochondrial dysfunction in MASLD were attributed to the upregulation of methyltransferase like 3 (Mettl3) and the downregulation of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) induced by high-fat foods. Mettl3 promoted the MRC's function. However, knockout of the reader protein YTHDF1, which plays a crucial role in the m6A modification process, counteracted the effect of Mettl3 and suppressed MRC. In MASLD, damage to the MRC may be regulated by the Mettl3-m6A-YTHDF1 complex axis, especially by the role of YTHDF1. Our research has offered a novel perspective on the involvement of m6A mRNA methylation in the pathogenesis of MASLD.
Project description:The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader.