Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA545736 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9171570.fastq.gz | Fastqsanger.gz | |||
| SRR9171571.fastq.gz | Fastqsanger.gz | |||
| SRR9171572.fastq.gz | Fastqsanger.gz | |||
| SRR9171563.fastq.gz | Fastqsanger.gz | |||
| SRR9171573.fastq.gz | Fastqsanger.gz |
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Project description:We engineered m6A methyltransferase or demethylases with CRISPR-Cas9 to achieve site-specific editing of m6A. The resultant m6A editors can be programmed with a guide RNA, allowing functional comparison of single site methylation in different mRNA regions.
2019-06-01 | GSE132051 | GEO
Project description:Programmable RNA N6-methyladenosine editing with CRISPR/dCas13a in plants
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Project description:N6-methyladenonsine (m6A) is the most prevalent modification on mRNA and plays critical roles in mRNA processing and metabolism. However, how the m6A modification on individual gene of interest regulates gene function and the links to phenotypic outcome in plants are mostly unknown. Here, we described the construction and characterization of programmable m6A editing tools by fusing the m6A writer, core catalytical domain of MTA and MTB complex, and eraser, ALKBH5, to catalytically dead Cas13a (dCas13a), respectively, for targeting methylation and demethylation of specific mRNA. We demonstrated that our m6A editors could efficiently and specifically add and remove the m6A modification on specific RNA transcript in both Nicotiana benthamiana and Arabidopsis. Moreover, targeting SHORT-ROOT (SHR) transcript with methylation editor could significantly increase its m6A levels with limited off-target effects and enhance its expression, giving rise to induced plant growth with enlarged leaf size and root, increased plant height, plant biomass and total grain weight in Arabidopsis. Collectively, these findings suggest that our programmable m6A editing tools can be applied to study m6A modification of specific genes in plants, and might also have great potential applications for crop improvement in future
2024-01-01 | GSE221143 | GEO
Project description:RNA modification in the form of N6-methyladenosine (m6A) regulates nearly all the post-transcriptional processes. The asymmetric m6A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m6A modifications. Here we report the development of 'm6A editing', a powerful approach that enables m6A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single-chain m6A methyltransferase that can be programmed with a guide RNA. The resultant m6A 'writers' allow functional comparison of single site methylation in different messenger RNA regions. We further engineered m6A 'erasers' by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m6A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.
| S-EPMC6702037 | biostudies-literature
Project description:Hypoxia as a crucial pathogenesis factor usually results in huge harmful effects on cardiac injury and dysfunction. In our previous study (PMID: 33294289), We observe a series of differential expressed genes between transcription and translation, which may be attributed to the hypoxia-specific binding affinity of Nuclear cap-binding subunit 3 (NCBP3) at 5’ un-translation region of target genes. But the underlying molecular mechanism of NCBP3 for gene translation modulation remains unclear. Here, we conducted RIP-seq of N6-Methyladenosine methylation in H9C2 cells with the conditions of normoxic, hypoxic and with additional NCBP3 knockdown.
2021-06-30 | E-MTAB-10682 | biostudies-arrayexpress
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
2015-06-09 | E-GEOD-63591 | biostudies-arrayexpress
Project description:Oxaliplatin as a first-line drug frequently causes the chemo-resistance on colorectal cancer (CRC). N6-methyladenosine (m6A) methylation has been largely acknowledged in multiple biological functions. However, the molecular mechanisms underlying the m6A methylation in modulating anticancer drug resistance in CRC are still obscure. In present study, RIP-seq was conducted to investigate the occupancy of N6-methyladenosine RNA binding protein 3 (YTHDF3) served as “readers” that can recognize m6A modification site in HCT116 cells with oxaliplatin resistance (HCT116R). Then, YTHDF3 was knockdown by siRNA in HCT116 cells with oxaliplatin resistance, and RIP-seq was further conducted to investigate m6A methylation of HCT116, HCT116R and HCT116R cells with YTHDF3 knockdown.
2022-02-04 | E-MTAB-11321 | biostudies-arrayexpress
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
2015-02-27 | E-GEOD-56010 | biostudies-arrayexpress
Project description:FMRP facilitates the nuclear export of N6-methyladenosine-containing mRNAs.
2019-11-21 | GSE133724 | GEO
Project description:N6-methyladenosine (m6A) is the most abundant internal modification in the messenger RNA (mRNA) of all higher eukaryotes. This modification has been shown to be reversible in mammals; it is installed by a methyltransferase heterodimer complex of METTL3 and METTL14 bound with WTAP, and reversed by iron(II)- and α-ketoglutarate-dependent demethylases FTO and ALKBH5. This modification exhibits significant functional roles in various biological processes. The m6A modification as a RNA mark is recognized by reader proteins, such as YTH domain family proteins and HNRNPA2B1; m6A can also act as a structure switch to affect RNA-protein interactions for biological regulation. In Arabidopsis thaliana, the methyltransferase subunit MTA (the plant orthologue of human METTL3, encoded by At4g10760) was well characterized and FIP37 (the plant orthologue of human WTAP) was first identified as the interacting partner of MTA. Here we report the discovery and characterization of reversible m6A methylation mediated by AtALKBH10B (encoded by At4g02940) in A. thaliana, and noticeable roles of this RNA demethylase in affecting plant development and floral transition. Our findings reveal potential broad functions of reversible mRNA methylation in plants. m6A peaks were identified from wild type Columbia-0 and atalkbh10b-1 mutant in two biological replicates
2016-03-24 | E-GEOD-79523 | biostudies-arrayexpress