Abstract
BackgroundReversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations.MethodsHere we use quantitative mass spectrometry to determine the fate of chemically induced methylbases in the mRNA of human cells. Concomitant alteration in the mRNA binding proteome was analyzed by SILAC mass spectrometry.ResultsMMS induced prominent direct mRNA methylations that were chemically identical to endogenous methylbases. Transient loss of 40S ribosomal proteins from isolated mRNA suggests that aberrant methylbases mediate arrested translational initiation and potentially also no-go decay of the affected mRNA. Four proteins (ASCC3, YTHDC2, TRIM25 and GEMIN5) displayed increased mRNA binding after MMS treatment. ASCC3 is a binding partner of the DNA/RNA demethylase ALKBH3 and was recently shown to promote disassembly of collided ribosomes as part of the ribosome quality control (RQC) trigger complex. We find that ASCC3-deficient cells display delayed removal of MMS-induced 1-methyladenosine (m1A) and 3-methylcytosine (m3C) from mRNA and impaired formation of MMS-induced P-bodies.ConclusionsOur findings conform to a model in which ASCC3-mediated disassembly of collided ribosomes allows demethylation of aberrant m1A and m3C by ALKBH3. Our findings constitute first evidence of selective sanitation of aberrant mRNA methylbases over their endogenous counterparts and warrant further studies on RNA-mediated effects of chemical alkylators commonly used in the clinic.
Highlights
Reversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations
Our results suggest that cells exploit these properties of Activating signal cointegrator complex subunit 3 (ASCC3) to allow Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3 (ALKBH3)-mediated demethylation of aberrant m1A and m3C in the coding region of mRNA, whereas canonical methylbases remain unaffected
Each bar in C-F represents the mean of three biological replicates with SDs as indicated mediating a relative survival of 90% (Additional file 2: Figure S2A) but with a clear S-phase delay around 4 h after methyl methanesulfonate (MMS) treatment (Additional file 2: Figure S2B). 24 h after treatment, cells had returned to essentially normal asynchronous distribution
Summary
Reversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations. Enzymatic modification of nitrogen bases in mRNA regulates processing, transport, lifetime and translation of mRNA molecules. This influences cell differentiation, stress responses, immunity, cognition. 11 endogenous base modifications have been described in mammalian mRNA. These are 7-methylguanosine (m7G, residing in the mRNA cap structure [4] as well as internally [5,6,7]), pseudouridine (Ψ) [8], inosine (I) [9], N4-acetylcytidine (ac4C) [10], 5-methylcytidine (m5C) [11], 5-hydroxymethylcytidine (hm5C) [12], 3-methylcytidine (m3C) [13], 1-methyladenosine (m1A) [14, 15], 6-methyladenosine (m6A) [16, 17] and its derivatives 6-hydroxymethyladenosine (hm6A) and 6-formyladenosine (f6A) [18].
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