Edelheit, S. | bioRxiv Science

bioRxiv2020

B Cell Division Capacity in Germinal Centers Depends on Myc Transcript Stabilization Through m6A mRNA Methylation and IGF2BP3 Functions

Long-lasting immunity from pathogens depends on the generation of protective antibodies through the germinal center (GC) reaction. The Myc gene produces highly short-lived transcripts which are essential for generation of high-affinity antibodies. mRNA lifetime is regulated by N6-methyladenosine (m6A)-modification of mRNAs through METTL3 activity; however, the role of this machinery in the GC remains unclear. Here, we find that m6A-modification of mRNAs is required for GC maintenance through Myc mRNA stabilization by the atypical m6A-interactor, IGF2BP3. MYC expression, activation of MYC transcriptional programs and cell-cycle progression were diminished in METTL3-deficient GC B cells. METTL3 attenuated Myc-transcript decay and overexpression of MYC in METTL3-deficient GC B cells restored the GC reaction. IGF2BP3 which was induced by CD40-signaling, reinforced MYC expression and MYC-related gene programs in GC B cells. Our findings explain how GC responses are maintained through regulation of Myc-transcript lifetime and expose new targets for manipulation in MYC-driven lymphoma. One Sentence SummaryGerminal centers depend on the m6A-machinery

immunology↗

bioRxiv2019

Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution

N6-methyladenosine (m6A) is the most abundant modification on mRNA, and is implicated in critical roles in development, physiology and disease. The ability to map m6A using immunoprecipitation-based approaches has played a critical role in dissecting m6A functions and mechanisms of action. Yet, these approaches are of limited specificity, unknown sensitivity, and unable to quantify m6A stoichiometry. These limitations have severely hampered our ability to unravel the factors determining where m6A will be deposited, to which levels (the m6A code), and to quantitatively profile m6A dynamics across biological systems. Here, we used the RNase MazF, which cleaves specifically at unmethylated RNA sites, to develop MASTER-seq for systematic quantitative profiling of m6A sites at 16-25% of all m6A sites at single nucleotide resolution. We established MASTER-seq for orthogonal validation and de novo detection of m6A sites, and for tracking of m6A dynamics in yeast gametogenesis and in early mammalian differentiation. We discover that antibody-based approaches severely underestimate the number of m6A sites, and that both the presence of m6A and its stoichiometry are hard-coded via a simple and predictable code within the extended sequence composition at the methylation sites. This code accounts for ~50% of the variability in methylation levels across sites, allows excellent de novo prediction of methylation sites, and predicts methylation acquisition and loss across evolution. We anticipate that MASTER-seq will pave the path towards a more quantitative investigation of m6A biogenesis and regulation in a wide variety of systems, including diverse cell types, stimuli, subcellular components, and disease states.

molecular biology↗