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Series GSE112181 Query DataSets for GSE112181
Status Public on Apr 26, 2019
Title m7G methylation by METTL1 regulates let-7 microRNAs [BoRed-Seq & m7G-RIP-Seq A549]
Organism Homo sapiens
Experiment type Non-coding RNA profiling by high throughput sequencing
Summary Methylation of N7-methylguanosine (m7G) is found at mRNA caps and at defined internal positions within abundant tRNAs and rRNAs. However, its detection within low abundance mRNAs and microRNAs (miRNAs) has been hampered by lack of sensitive detection strategies. Here, we adapt a chemical reactivity assay to detect internal m7G in RNA from eukaryotic cells. Using this approach, alongside a confirmational RNA immunoprecipitation assay, we identify m7G within miRNAs inhibiting cell migration, and show that METTL1 mediates this m7G methylation. Using Let-7 as an example we demonstrate that METTL1 activity is necessary for correct processing, is required for Let-7 dependent gene regulation and consequently has a negative effect on cell migration. These results identify m7G modification as a new pathway for the regulation of miRNAs.
Overall design Identification of miRNAs harboring m7G modification. Total RNA from a human lung cancer cell line (A549 cells) was decapped, 5' end dephosphorylated, and treated with NaBH4 and low pH to generate abasic sites at positions harbouring m7G. These sites were exposed to a biotin-coupled aldehyde reactive probe (N-(aminooxyacetyl)-n'-(D-biotinoyl) hydrazine; ARP) that covalently binds to the abasic RNA sites. Modified RNAs were then pulled-down using streptavidin-beads, small RNA libraries were prepared and RNAs were identified by NGS. To confirm the validity of this approach and to provide an independent verification of m7G-methylated miRNAs, we performed a RIP-Seq experiment employing an antibody that recognizes m7G in RNA.
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Contributor(s) Pandolfini L, Barbieri I
Citation(s) 31031083
Submission date Mar 21, 2018
Last update date Jul 26, 2019
Contact name Luca Pandolfini
Organization name Istituto Italiano di Tecnologia - I
Department Center for Human Technologies
Street address Via Enrico Melen 83, Building B
City Genova
State/province Genova
ZIP/Postal code 16152
Country Italy
Platforms (2)
GPL18460 Illumina HiSeq 1500 (Homo sapiens)
GPL20301 Illumina HiSeq 4000 (Homo sapiens)
Samples (14)
GSM3059545 A549 BoRed-Seq Ctrl Rep 1
GSM3059546 A549 BoRed-Seq Ctrl Rep 2
GSM3059547 A549 BoRed-Seq Red Rep 1
This SubSeries is part of SuperSeries:
GSE112182 m7G methylation by METTL1 regulates let-7 microRNAs
BioProject PRJNA445178
SRA SRP136213

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Supplementary file Size Download File type/resource
GSE112181_TK_LP_A549_BoRed-Seq_RAW_miRNA_counts.txt.gz 13.3 Kb (ftp)(http) TXT
GSE112181_TK_LP_A549_RIP-Seq_RAW_miRNA_counts.txt.gz 26.5 Kb (ftp)(http) TXT
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Raw data are available in SRA
Processed data are available on Series record

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