Research Summary: Unexpected Accumulation of ncm5U and ncm5s2U in a trm9 Mutant Suggests an Additional Step in the Synthesis of mcm5U and mcm5s2U

ABSTRACT

Background

Transfer RNAs are synthesized as a primary transcript that is processed to
produce a mature tRNA. As part of the maturation process, a subset of the
nucleosides are modified. Modifications in the anticodon region often
modulate the decoding ability of the tRNA. At position 34, the majority of
yeast cytosolic tRNA species that have a uridine are modified to
5-carbamoylmethyluridine (ncm5U),
5-carbamoylmethyl-2′-O-methyluridine (ncm5Um),
5-methoxycarbonylmethyl-uridine (mcm5U) or
5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The
formation of mcm5 and ncm5 side chains involves a
complex pathway, where the last step in formation of mcm5 is a
methyl esterification of cm5 dependent on the Trm9 and Trm112
proteins.


Methodology and Principal Findings

Both Trm9 and Trm112 are required for the last step in formation of
mcm5 side chains at wobble uridines. By co-expressing a
histidine-tagged Trm9p together with a native Trm112p in E.
coli
, these two proteins purified as a complex. The presence of
Trm112p dramatically improves the methyltransferase activity of Trm9p
in vitro. Single tRNA species that normally contain
mcm5U or mcm5s2U nucleosides were
isolated from trm9Δ or trm112Δ
mutants and the presence of modified nucleosides was analyzed by HPLC. In
both mutants, mcm5U and mcm5s2U nucleosides
are absent in tRNAs and the major intermediates accumulating were
ncm5U and ncm5s2U, not the expected
cm5U and cm5s2U.


Conclusions

Trm9p and Trm112p function together at the final step in formation of
mcm5U in tRNA by using the intermediate cm5U as a
substrate. In tRNA isolated from trm9Δ and
trm112Δ strains, ncm5U and
ncm5s2U nucleosides accumulate, questioning the
order of nucleoside intermediate formation of the mcm5 side
chain. We propose two alternative explanations for this observation. One is
that the intermediate cm5U is generated from ncm5U by
a yet unknown mechanism and the other is that cm5U is formed
before ncm5U and mcm5U.

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Publisher: Public Library of Science

Date Published: 7-June-2011

Author(s): Chen C., Huang B., Anderson J., Byström A.

Link: https://doi.org/10.1371/journal.pone.0020783

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