Coordination of Mrna and Trna Methylations by TRMT10A
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Coordination of mRNA and tRNA methylations by TRMT10A R. Jordan Ontiverosa,b,1, Hui Shena,1, Julian Stoutea,b, Amber Yanasa,b, Yixiao Cuia, Yuyu Zhanga, and Kathy Fange Liua,b,2 aDepartment of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and bGraduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Edited by Lynne E. Maquat, University of Rochester School of Medicine and Dentistry, Rochester, NY, and approved February 25, 2020 (received for review August 21, 2019) The posttranscriptional modification of messenger RNA (mRNA) and by which deficiency of TRMT10A is linked to the disease transfer RNA (tRNA) provides an additional layer of regulatory phenotype remains enigmatic. complexity during gene expression. Here, we show that a tRNA Another major type of RNA, messenger RNA (mRNA), also methyltransferase, TRMT10A, interacts with an mRNA demethylase carries methylations. The most prevalent internal modification in FTO (ALKBH9), both in vitro and inside cells. TRMT10A installs N1- mRNA is N6-methyladenosine (m6A), which impacts nearly ev- methylguanosine (m1G) in tRNA, and FTO performs demethylation ery step of RNA processing, including reducing or extending 6 6 6 on N -methyladenosine (m A) and N ,2′-O-dimethyladenosine RNA half-life in cells (23–28). The methyltransferase heterodimer 6 6 (m Am) in mRNA. We show that TRMT10A ablation not only leads complex of METTL3 and METTL14 installs m AinmRNA(29– 1 6 to decreased m G in tRNA but also significantly increases m A levels 32).ALKBH5andFTO(alsoknownasALKBH9)aretheonly in mRNA. Cross-linking and immunoprecipitation, followed by high- known m6A demethylase enzymes (33, 34). FTO can also remove throughput sequencing results show that TRMT10A shares a signif- 6 6 N ,2′-O-dimethyladenosine (m Am) in mRNA (35, 36), and it is icant overlap of associated mRNAs with FTO, and these mRNAs have thus most appropriately referred to as an m6A/m6A demethylase. accelerated decay rates potentially through the regulation by a spe- m FTO was also reported performing demethylation on m1Ain cific m6A reader, YTHDF2. Furthermore, transcripts with increased mRNA and tRNA (37). The substrate selectivity of FTO has been m6A upon TRMT10A ablation contain an overrepresentation of 1 Gln(TTG) Arg(CCG) suggested to be dependent on its predominant subcellular locali- BIOCHEMISTRY m G9-containing tRNAs codons read by tRNA ,tRNA , 6 Thr(CGT) zation (37). The removal of m A by FTO can lead to differential and tRNA . These findings collectively reveal the presence of 6 coordinated mRNA and tRNA methylations and demonstrate a mech- regulation by m A readers, which specifically recognize the modi- anism for regulating gene expression through the interactions be- fication sites and bridge them to specific cellular complexes, such as tween mRNA and tRNA modifying enzymes. the splicing complex, translational machinery, and RNA decay machinery (25, 27, 38–42). Thus far, a handful of m6A readers have 6 tRNA | mRNA | modification been identified that all specifically recognize m A in mRNA, and yet can lead to different or even opposite cellular consequences o date, about 150 types of RNA chemical modifications on Talmost all RNA species have been described (1–4). These Significance modifications can dramatically expand the RNA alphabet by altering RNA structures, affinity to proteins, and base- It has been demonstrated that a diverse set of enzyme-mediated pairing ability, greatly impacting how RNA influences the chemical modifications are found within RNAs, and these modi- genetic flow of information from transcription to protein fications markedly influence the fate of RNAs in cells. Dysregu- synthesis (2, 5–9). While many of the enzymes that deposit lation of such RNA modifications is involved in the development and remove RNA modifications have been identified, the of a battery of human disorders. Studies to date have focused solely on individual modifications on isolated RNA; however, RNA details of how RNA modifications are regulated remain poorly modifications exist concurrently in multiple RNA species. Using understood. biochemistry and high-throughput sequencing techniques, we Among those RNA species, transfer RNA (tRNA) is the most revealed an interaction between a transfer RNA methyltransfer- extensively modified. tRNA methylations are known to be critical ase TRMT10A and a messenger RNA demethylase FTO, which for their stability and translational fidelity (1, 10, 11). TRMT10A influences the methylation levels on a subset of messenger RNAs. S – is an -adenosylmethionine dependent methyltransferase that This study unravels a regulatory mechanism by which methyla- 1 1 installs N -methylguanosine (m G) in tRNAs at the ninth position. tions across distinct RNA types are coordinated to impact gene 1 While previous studies have identified the m G9 sites in tRNAs in expression collaboratively. human cells (12, 13), TRMT10A is, so far, the only known methyltransferase that installs this modification in human tRNA. Author contributions: H.S. and K.F.L. designed research; R.J.O., H.S., J.S., A.Y., Y.C., Y.Z., The entire substrate repertoire of TRMT10A in human cells has and K.F.L. performed research; H.S., J.S., and K.F.L. analyzed data; and R.J.O., H.S., and K.F.L. wrote the paper. not been fully revealed, although tRNAGln and tRNAiMet were The authors declare no competing interest. shown as the substrates of human TRMT10A in pancreatic β-cells (14). Previous efforts in characterizing the tRNA substrates of This article is a PNAS Direct Submission. TRMT10A were mostly done in yeast (15–17). Human TRMT10A Published under the PNAS license. 1 Data deposition: The data reported in this paper have been deposited in the Gene Ex- was shown installing m G9 in tRNAs in vitro using yeast and pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession number human tRNAs as the substrates (18). Mutations in TRMT10A GSE146207). have been observed in patients with young-onset diabetes syn- 1R.J.O. and H.S. contributed equally to this work. drome and primary microcephaly-mild intellectual disability (14, 2To whom correspondence may be addressed. Email: [email protected]. – 19 22), and recent studies have suggested that deficiency of This article contains supporting information online at https://www.pnas.org/lookup/suppl/ TRMT10A can induce apoptosis in pancreatic cells, consistent doi:10.1073/pnas.1913448117/-/DCSupplemental. with the correlation with diabetes (14, 22). However, the mechanism www.pnas.org/cgi/doi/10.1073/pnas.1913448117 PNAS Latest Articles | 1of10 Downloaded by guest on October 6, 2021 (43–46). The functional significance of RNA-modifying enzymes and An unexpected, yet interesting observation is that many pro- reader proteins is further evidenced by the fact that dysregulation of teins identified from the TAP-MS experiments are involved in mRNA modification status or mutations in RNA-modifying enzymes the regulation of mRNA methylation status, including the m6A has been linked to a battery of human diseases, including cancer, demethylase FTO (Dataset S1). These results led us to investi- neurodegenerative disease, and diabetes (47–51). gate if TRMT10A is involved in the regulation of both mRNA Interestingly, previous studies have identified a few RNA- and tRNA methylations. Hence, further characterizations of this modifying enzymes that can install the same modification in potential interaction were executed. To validate the protein– different RNA species (52). For example, tRNA splicing ligase protein interactions between FTO and TRMT10A by an inde- also acts as a ligase for noncanonical mRNA splicing. Addi- pendent technique, we performed immunoprecipitation experi- tionally, tRNA pseudouridine synthases modify mRNAs (53–55). ments with endogenous TRMT10A as the bait protein followed In addition, the yeast tRNA methyltransferase Trm140 has been by Western blot. TRMT10A is shown in Fig. 1A interacting with shown to modify a noncanonical tRNA target only when asso- FTO in an RNA-facilitated manner. Adding Rnase A led to ciated with a seryl-tRNA synthetase (56). Considering these decreased binding between FTO and TRMT10A; however, it did examples of noncanonical substrate targeting by putatively type- not abolish their interactions. Reciprocally, we performed the specific enzymes, it is not surprising that FTO itself has been reverse immunoprecipitation using endogenous FTO to pull reported to perform its demethylation activity on both tRNAs down TRMT10A. As shown in Fig. 1B, endogenous FTO pulled and mRNAs (37). Furthermore, there are examples of the down TRMT10A. These experiments were reproducible with unusual collaboration of RNA modifications by proteins not two TRMT10A antibodies and two FTO antibodies (SI Appen- dix A B possessing modification activity (56) and coordination between , Fig. S2 and ). In addition, we showed that FLAG-tagged tRNA modifications and gene transcription (57). These studies TRMT10A can pull down FTO and FLAG-tagged FTO can SI Appendix C hint at an unusual mode of RNA modification regulation reciprocally pull down TRMT10A ( , Fig. S2 and D whereby the activity and selectivity of type-specific RNA- ). Furthermore, we carried out proximity ligation assays (PLA) modifying enzymes can change upon association with a partner to investigate the in situ interactions between FTO and TRMT10A. C protein. It stands to reason