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Method for the Enrichment of N6-Methyladenosine-Modified Cellular and HIV-1 RNA
Book chapter

Method for the Enrichment of N6-Methyladenosine-Modified Cellular and HIV-1 RNA

Tarun Mishra, Stacia Phillips and Li Wu
HIV Protocols, pp.195-208
Methods in molecular biology (Clifton, N.J.), v. 2807, Humana, Fourth edition
2024
DOI: 10.1007/978-1-0716-3862-0_14
PMCID: PMC11160405
PMID: 38743230
url
https://pmc.ncbi.nlm.nih.gov/articles/PMC11160405/pdf/nihms-1995699.pdfView
Open Access

Abstract

N6-methyladenosine (m6A) modification of RNA is an important area in studying viral replication, cellular responses, and host immunity. HIV-1 RNA contains multiple m6A modifications that regulate viral replication and gene expression. HIV-1 infection of CD4+ T-cells or HIV-1 envelope protein treatment upregulates m6A levels of cellular RNA. Changes in the m6A modification of cellular transcripts in response to HIV-1 infection provide new insights into the mechanisms of posttranscriptional gene regulation in the host cell. To better investigate the functions of m6A modification in HIV-1 infection and innate immune responses, it is helpful to standardize basic protocols. Here, we describe a method for the selective enrichment of m6A-modified RNA from HIV-1-infected primary CD4+ T-cells based on immunoprecipitation. The enriched RNA with m6A modifications can be used in a variety of downstream applications to determine the methylation status of viral or cellular RNA at resolution from transcript level down to single nucleotide.

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