{"doi":"10.1101/2022.05.23.493172","title":"Selective detection of m <sup>6</sup> A derived from mRNA using the Phospho-tag m <sup>6</sup> A assay","abstract":"ABSTRACT N6-methyladenosine (m 6 A) is a modified nucleotide found in mRNA, ribosome RNA (rRNA) and small nuclear RNA (snRNA). m 6 A in mRNA has important roles in regulating mRNA stability, splicing, and other processes. Numerous studies have described m 6 A as a dynamic modification using mass spectrometry-based quantification of m 6 A 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 m 6 A. 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 m 6 A from rRNA and snRNA being liberated as m 6 A monophosphate, while m 6 A from mRNA is mostly liberated as m6A nucleoside. m 6 A levels are normalized to transcript levels, using m 7 G 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 m 6 A detection and overcomes the 3’-end biases associated with mRNA purification. Overall, the Phospho-tag m 6 A provides a simple and efficient method for quantification of mRNA-derived m 6 A from total RNA samples.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":299234,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":4,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9593,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":232217,"name":"Nabeel Attarwala","orcid":"0000-0003-2112-9754","position":1,"is_corresponding":false},{"id":259684,"name":"Steven S. Gross","orcid":"0000-0001-8029-2338","position":2,"is_corresponding":false},{"id":259681,"name":"Qiuying Chen","orcid":"0000-0001-5909-3959","position":3,"is_corresponding":false},{"id":107966,"name":"Samie R. Jaffrey","orcid":"0000-0003-3615-6958","position":4,"is_corresponding":false},{"id":415476,"name":"Aashiq H. Mirza","orcid":"0000-0002-9655-5267","position":0,"is_corresponding":true}],"reference_count":56,"raw_metadata":null,"created_at":"2026-07-19T00:31:44.904250Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}