Functional analysis of 3'-UTR hairpins supports a two-tiered model for posttranscriptional regulation of by METTL16.

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events, so cells need to carefully control SAM levels. encodes the only SAM synthetase expressed in the majority of human cells, and its 3'-UTR has six conserved regulatory hairpins (hp1-6) that can be methylated by the N6-methyladenosine methyltransferase METTL16. Hp1 begins 8 nt from the stop codon, whereas hp2-6 are clustered further downstream (∼800 nt). These hairpins have been proposed to regulate mRNA levels in response to intracellular SAM levels by regulating intron detention of the last intron of and by modulating the stability of the fully spliced mRNA. However, a dissection of these two posttranscriptional mechanisms has not been previously reported. Using a modular reporter system, we show that hp1 functions primarily when the detained intron is included in the reporter and when that intron has a suboptimal polypyrimidine tract. In contrast, the hp2-6 cluster modulates mRNA stability independent of the detained intron, although hp1 may make a minor contribution to the regulation of decay as well. Taken with previously published reports, these data support a two-tiered model for posttranscriptional regulation by METTL16 through its interactions with hp1 and hp2-6. In the upstream tier, hp1 and METTL16 control intron detention, whereas the second tier involves METTL16-dependent methylation of hp2-6 to control mRNA stability. Thus, cells use a similar set of molecular factors to achieve considerable complexity in the posttranscriptional regulation of SAM homeostasis.