Methylation of tRNA-derived fragments regulates gene-silencing activity
in bladder cancer

Date:
May 9, 2022
Source:
University of Alabama at Birmingham
Summary:
Researchers describe a novel form of gene regulation that is altered
in bladder cancer, leading to the boosting of a gene pathway that
helps the cancer cells survive during rapid growth. The work focuses
on a 22-base fragment of transfer RNA, tRF-3b, which is modified
by the enzyme complex TRMT6/61A. In bladder cancer, the levels of
TRMT6/61A -- a methyltransferase -- are elevated. The methylation
modification prevents tRF-3bs from silencing the expression of
various genes in the unfolded protein response pathway in the
cancer cells.



FULL STORY ========================================================================== Anindya Dutta, MBBS, Ph.D., and colleagues have described a novel form of
gene regulation that is altered in bladder cancer, leading to the boosting
of a gene pathway that helps the cancer cells survive during rapid growth.


========================================================================== Their work focuses on a 22-base fragment of transfer RNAs known as a
tRF-3b, which is modified by an enzyme complex called TRMT6/61A. In
bladder cancer, the levels of TRMT6/61A -- a methyltransferase enzyme
that adds a methyl group onto the fourth base of the tRF-3bs --
are elevated. This modification prevents tRF- 3bs from silencing the
expression of various genes in the unfolded protein response pathway in
the cancer cells, resulting in increased expression of those genes.

"To the best of our knowledge, this is the first example of
microRNA-like gene silencing being regulated by the TRMT6/61 based on
an N1-methyladenosine modification, and our report provides a mechanism
by which the elevation of TRMT6/61A seen in cancers can impact gene expression," Dutta said. "Fast proliferating cancer cells synthesize and
fold many more proteins than normal cells and thus need to upregulate the unfolded protein response pathway to maintain protein homeostasis. We
find that one way bladder cancer cells activate the pro-survival
unfolded protein response to alleviate endoplasmic reticulum stress
is by preventing tRFs from silencing the expression of genes involved
in this unfolded protein response." "The unfolded protein response is
tightly linked to many aspects of cancer progression and has emerged as a promising therapeutic target," Dutta said. "It has been previously noted
that unfolded protein response-related genes are globally upregulated
in several cancer types, including bladder cancer, and so our results
suggest that inhibiting the TRMT6/61A enzyme may be a new approach to
treat bladder cancer." The study by Dutta and co-corresponding author
Rune Ougland, M.D., Ph.D., included analysis of bladder cancer tissue
obtained from patients undergoing transurethral resection of bladder
tumors. It is published in the journal Nature Communications.Dutta is
chair of the University of Alabama at Birmingham Department of Genetics,
and Ougland is a urologic surgeon and senior research investigator at
Oslo University Hospital Rikshospitalet, Oslo, Norway.

An important advance in the study was the workflow used to create
a library from human cells of small RNAs with an N1-methyladenosine modification, or m1A.

The workflow combined two independent approaches -- enrichment by
m1A-antibody, followed by small RNA-sequencing and m1A-induced mismatch signature by sequencing.



==========================================================================
The UAB researchers found that a reverse transcriptase enzyme,
ProtoScriptII, commonly used for short RNA sequencing, did a poor job of detecting small RNAs that contain m1A; but the use of two other reverse transcriptases in the workflow revealed that tRNA-derived fragments,
including tRF-3b, were enriched among short RNAs. This suggested that
small RNAs with an m1A modification are under-represented in most small RNA-sequence libraries that commonly have used ProtoScriptII.

With the improved workflow, the researchers found that the m1A
modification existed mostly on tRFs among the human small RNAs. They also
found that the m1A modification was highly specific and prevalent on both nuclear-encoded tRFs and mitochondria-encoded tRFs, and the m1A found on
tRF-3b from nuclear-encoded tRNAs was mediated by the TRMT6/61A complex.

How does the m1A-tRF-3b impede gene silencing? The answer involves an
even deeper dive into molecular genetics, but the key appears to be
that the N1- methyladenosine modification disrupts regular Watson-Crick
base pairing.

MicroRNAs are known to silence genes by binding to the RNA-induced
silencing complex, or RISC. There they act as a template to bind
complementary messenger RNAs, and the messenger RNA is then silenced
and degraded by RISC. Similar to microRNAs, tRF-3s have been found
in diverse biological pathways, in particular gene-silencing pathways
that rely on base pairing between the small RNAs, in this case tRF-3s,
and the target RNAs.

The researchers created a luciferase reporter assay and found that an unmodified tRF-3 triggered gene silencing, whereas the m1A-modified
tRF-3b abolished the gene silencing. "Since m1A interrupts canonical
base pairing, we hypothesize the weakened base pairing by m1A in the
tRF-3 with target messenger RNA explains the lowered gene-silencing
activity observed for m1A-containing tRF-3s," Dutta said.

Co-authors with Dutta and Ougland in the study, "TRMT6/61A-dependent base methylation of tRNA-derived fragments regulates gene-silencing activity
and the unfolded protein response in bladder cancer," are Zhangli Su,
UAB Department of Genetics; Ida Monshaugen and Arne Klungland, Oslo
University Hospital Rikshospitalet; and Briana Wilson and Fengbin Wang, University of Virginia.

Support came from National Institutes of Health grants CA044579, CA254134, AR067712 and CA259526; Vestre Viken Hospital (Gjettum, Norway) Trust
grant 25C003; and Norwegian Cancer Society grant 216115.


========================================================================== Story Source: Materials provided by
University_of_Alabama_at_Birmingham. Original written by Jeff
Hansen. Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Zhangli Su, Ida Monshaugen, Briana Wilson, Fengbin Wang, Arne
Klungland,
Rune Ougland, Anindya Dutta. TRMT6/61A-dependent base methylation
of tRNA-derived fragments regulates gene-silencing activity and the
unfolded protein response in bladder cancer. Nature Communications,
2022; 13 (1) DOI: 10.1038/s41467-022-29790-8 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/05/220509171056.htm

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