Abstract
Kti12 and PSTK are closely related and highly similar proteins implicated in different aspects of tRNA metabolism. Kti12 has been identified as an essential regulatory factor of the Elongator complex, involved in the modification of uridine bases in eukaryotic tRNAs. PSTK phosphorylates the tRNASec-bound amino acid serine, which is required to synthesize selenocysteine. Kti12 and PSTK have previously been studied independently in various organisms, but only appear simultaneously in some animalia, including humans. As Kti12- and PSTK-related pathways are clinically relevant, it is of prime importance to understand their biological functions and mutual relationship in humans. Here, we use different tRNA substrates to directly compare the enzymatic activities of purified human KTI12 and human PSTK proteins. Our complementary Co-IP and BioID2 approaches in human cells confirm that Elongator is the main interaction partner of KTI12 but additionally indicate potential links to proteins involved in vesicular transport, RNA metabolism and deubiquitination. Moreover, we identify and validate a yet uncharacterized interaction between PSTK and γ-taxilin. Foremost, we demonstrate that human KTI12 and PSTK do not share interactors or influence their respective biological functions. Our data provide a comprehensive analysis of the regulatory networks controlling the activity of the human Elongator complex and selenocysteine biosynthesis.
Highlights
In genome-wide mutational screens and subsequent complementa tion analyses, Kluyveromyces lactis Toxin Insensitive 12 (KTI12) was identified as a factor that mediates resistance to zymocin, a secreted fungal toxin that cleaves tRNAs [1,2]
As the discriminator base is a well-established determinant of tRNASec aminoacylation in archaea [48], we wondered, if its role is conserved in phosphoseryl tRNASec kinase (PSTK) and whether it influences the activity of KTI12
The ATPase activity of KTI12 is triggered by tRNASec without a discriminator and by other tRNAs, like tRNAArgACG
Summary
In genome-wide mutational screens and subsequent complementa tion analyses, Kluyveromyces lactis Toxin Insensitive 12 (KTI12) was identified as a factor that mediates resistance to zymocin, a secreted fungal toxin that cleaves tRNAs [1,2]. Kti was shown to act as a direct, but temporarily associated regulatory factor of the eukaryotic Elongator complex [3,4]. Multi-subunit complex [5,6] responsible for the 5-carboxy methyluridine (cm5) modification of uridine bases located in the tRNA wobble position (U34). This priming modification represents the initial step for the subsequent formation of 5-carbamoylmethyl-uridine (ncm5U34) and 5-methoxycarbonylmethyl-uridine (mcm5U34) by other modification enzymes [6,7]. Despite its clinical relevance and targetable active site, very little is known about KTI12 and its regulation of the Elongator complex in human cells
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