Division of Newborn Medicine, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, NY 10065, USA; Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, 02-109 Warsaw, Poland.
Mol Cell. 2019 Aug 8;75(3):631-643.e8. doi: 10.1016/j.molcel.2019.06.006. Epub 2019 Jul 3.
mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N-methyladenosine (mA), found within mRNAs, and N,2'-O-dimethyladenosine (mAm), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates mAm. We find that PCIF1 binds and is dependent on the mG cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish mAm and mA. We find that mA and mAm misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain mAm that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of mAm-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize mAm's function.
mRNA 受到核苷酸修饰的调控，这些修饰影响它们的细胞命运。两种最丰富的修饰核苷酸是 N6-甲基腺苷（m6A），存在于 mRNA 中，以及 N2',O2-二甲基腺苷（mAm），它位于第一个转录的核苷酸上。在图谱研究中区分这些修饰一直很困难。在这里，我们鉴定并生化表征了 PCIF1，它是生成 mAm 的甲基转移酶。我们发现 PCIF1 结合并依赖于 mG 帽。通过耗尽 PCIF1，我们生成了全转录组图谱，区分了 mAm 和 mA。我们发现 mA 和 mAm 的错误注释来自具有替代转录起始位点（TSS）的 mRNA 亚型。这些亚型含有映射到“内部”位点的 mAm，增加了错误注释的可能性。我们发现耗尽 PCIF1 不会显著影响 mRNA 翻译，但与一组 mAm 注释的 mRNA 的稳定性降低有关。PCIF1 的发现和我们准确的图谱技术将有助于未来研究 mAm 的功能。
