Biology subjects

Nachtergaele, S.

Publications and source records attributed to Nachtergaele, S..

3 recordsLinked to original sources

bioRxiv2025

Investigation of TRMT61B methyltransferase activity on mRNA and its effects on translation

Despite recent advances in technology to map RNA chemical modifications transcriptome-wide, the distribution of N{superscript 1}-methyladenosine (m{superscript 1}A) in mRNA remains contested, hindering a clear understanding of its function. Additionally, the enzyme(s) that installs the majority of reported mRNA m1A sites has yet to be identified. In this study, we characterized TRMT61B, an m1A methyltransferase known to methylate mitochondrial RNAs, but whose sequence preferences have been underexplored. By integrating cellular overexpression of TRMT61B and in vitro methylation of a synthetic pool of diverse human RNA sequences, we identified a preference for a YMRA consensus motif in single stranded RNA regions. In these experiments, TRMT61B methylated thousands of novel human mRNA sites, revealing activity on cytosolic mRNAs. We used these novel m1A-modifiable sequences to test the effects of m1A on translation of luciferase reporters and on ribosome recruitment to modified transcripts in the pool. We found that m1A addition can significantly affect translation and ribosome recruitment, but that these effects are vary by transcript. Taken together, our results can inform future studies of TRMT61B and mRNA, and emphasize that studies of m1A regulation of mRNA must be carried out and interpreted in a highly context-aware manner.

biochemistry↗

bioRxiv2025

N6-methyladenosine promotes temozolomide resistance through non-canonical regulation of mRNA stability in glioblastoma cells

N6-methyladenosine (m6A) is a critical regulator of mRNA processing and function, impacting nearly every step of the mRNA lifecycle. Changes in m6A status have been implicated in many different types of cancer, including glioblastoma where acquired resistance to temozolomide remains a major clinical challenge. We established glioblastoma cell culture models of acquired temozolomide resistance and analyzed the role of m6A in controlling resistance-associated pathways. We show that m6A stabilizes key genes and pathways promoting temozolomide resistance in glioblastoma, including MGMT, the enzyme that repairs primary temzolomide-induced DNA damage, and PI3K/Akt signaling, a major driver of chemoresistance. Pharmacological inhibition of the m6A methyltransferase METTL3 destabilizes MGMT and other critical mRNAs, restoring temozolomide sensitivity. Collectively, our results suggest that genes associated with temozolomide resistance are stabilized by m6A methylation, even as the majority of the transcriptome remains subject to m6A-mediated mRNA decay. Moreover, these data highlight METTL3 inhibition as a promising therapeutic approach to overcoming temozolomide resistance in glioblastoma.

molecular biology↗

bioRxiv2021

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates metabolism and development via translation

Ribosomal RNAs (rRNAs) have long been known to carry modifications, including numerous sites of 2O-methylation and pseudouridylation, as well as N6-methyladenosine (m6A), and N6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry m6A, and while ZCCHC4 has been identified as the methyltransferase responsible for the 28S rRNA m6A site, the methyltransferase responsible for the 18S rRNA m6A site has remained uncharacterized until recently. Here, we show that the METTL5-TRMT112 complex is the methyltransferase responsible for installing m6A at position 1832 of human 18S rRNA. TRMT112 is required for the metabolic stability of METTL5, and human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. Loss of METTL5 in human cancer lines alters the translation of transcripts associated with mitochondrial biogenesis and function. Mettl5 knockout mice display reduced body size and evidence of metabolic defects. This m6A site is located on the 3 end of 18S rRNA, which may become surface-exposed under some circumstances and thus may play a regulatory role in translation of specific transcripts. While recent work has focused heavily on m6A modifications in mRNA and its roles in mRNA processing and translation, deorphanizing putative methyltransferase enzymes is revealing previously unappreciated regulatory roles for m6A in noncoding RNAs.

biochemistry↗