TRMT6/61A-dependent base methylation of tRNA-derived fragments regulates gene-silencing activity and the unfolded protein response in bladder cancer

Abstract

RNA modifications are important regulatory elements of RNA functions. However, most genome-wide mapping of RNA modifications has focused on messenger RNAs and transfer RNAs, but such datasets have been lacking for small RNAs. Here we mapped N1-methyladenosine (m1A) in the cellular small RNA space. Benchmarked with synthetic m1A RNAs, our workflow identified specific groups of m1A-containing small RNAs, which are otherwise disproportionally under-represented. In particular, 22-nucleotides long 3′ tRNA-fragments are highly enriched for TRMT6/61A-dependent m1A located within the seed region. TRMT6/61A-dependent m1A negatively affects gene silencing by tRF-3s. In urothelial carcinoma of the bladder, where TRMT6/61A is over-expressed, higher m1A modification on tRFs is detected, correlated with a dysregulation of tRF targetome. Lastly, TRMT6/61A regulates tRF-3 targets involved in unfolded protein response. Together, our results reveal a mechanism of regulating gene expression via base modification of small RNA.

Characterization of novel small non-coding RNAs and their modifications in bladder cancer using an updated small RNA-seq workflow

“…High-quality total RNAs were used as input and first ligated with 3′ adaptor and 5’ adaptor. Ligated RNA was converted into cDNA by reverse transcriptase TGIRT, which has been shown to produce more mismatch (misincorporation) than hard-stop products at m 1 A modification sites than other reverse transcriptases ( Li et al, 2017 ; Su et al, 2022 ). When sequencing HEK293T RNAs, we noticed TGIRT protocol is better at capturing non-microRNA-small RNAs than the regular ProtoScriptII protocol ( Supplementary Figure S1A ).…”

“…How could these modifications alter in disease conditions and whether they have any impact on ncRNA functions will be an interesting prospective research topic. Recently we reported m 1 A impedes tRF-3 gene-silencing activity and is over-expressed in BLCA tumor, coinciding with over-expression of the writer enzyme proteins TRMT6/61A and dysregulation of the tRF-3 targetome ( Su et al, 2022 ). In addition to bladder cancer, TRMT6/61A is also over-expressed in liver cancer and glioma.…”

“…However, regular small RNA-seq protocol is known to suffer from the stalling of the reverse transcriptase at sites containing modifications that disrupt Watson-Crick base-pairing, including but not limited to m 1 A (N 1 -methyladenosine), m 1 G (N 1 -methylguanosine), and m 2 2 G (N 2 , N 2 -dimethylguanosine) ( Behrens et al, 2021 ; Shi et al, 2021 ). Recently we showed TGIRT (thermostable group II intron reverse transcriptase) can be used in small RNA-seq to overcome under-cloning of m 1 A-containing RNAs during regular small RNA-seq protocol, and further be used to identify the modification base position via mismatch ( Su et al, 2022 ). The under-representation of m 1 A-containing small RNAs and loss of quantitative mismatch ratio by a commonly used M-MuLV reverse transcriptase (ProtoScriptII) indicates the regular small RNA-seq pipeline is biased against m 1 A-modified small RNAs.…”

Transfer RNA-derived small RNAs and their potential roles in the therapeutic heterogeneity of sacubitril/valsartan in heart failure patients after acute myocardial infarction

“…Previous articles have reported that the above three pathways are intensively correlated to the inflammatory immune response, atherosclerosis and angiogenesis ( 36 , 37 ). Although tRNA was one of the highly modified RNAs in cells, a latest study found that unmodified tRF-3 could also result in gene silencing through luciferase reporting experiments ( 38 ). In this study, our group used unmodified tRF fragments for overexpression research and confirmed that tRF-60:76-Val-AAC-1-M5 might target Tnfrsf10b and Bcl2l1 to influence the therapeutic heterogeneity of sacubitril/valsartan through the lipid and atherosclerosis signaling pathway.…”

Methyladenosine Modification in RNAs: From Regulatory Roles to Therapeutic Implications in Cancer

“…Moreover, m 1 A methylation in a subset of tRNA is upregulated by TRMT6/TRMT61A, resulting in an increment of peroxisome-proliferator-activated receptor delta (PPARδ) translation, which in turn stimulates cholesterol synthesis and activates hedgehog signaling to promote the oncogenesis of HCC [59]. In bladder cancer, m 1 A upregulated by TRMT6/TRMT61A dysregulates the targetome of tRNA fragments, leading to the unfolded protein response and silencing of tumor suppressor genes [60].…”