Abstract

Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongator's largest subunit (Elp1; also known as Iki3) was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine in vivo phosphorylation sites within Elp1 and show that four of these, clustered close to the Elp1 C-terminus and adjacent to a region that binds tRNA, are important for Elongator's tRNA modification function. Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable (alanine) and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase.

Highlights

  • Elongator is a conserved, multi-subunit protein complex containing six different polypeptides (Elp1-Elp6), first discovered in yeast in association with the elongating form of RNA polymerase II and initially proposed to play a role in transcriptional elongation [1,2]

  • When the tRNA contains a uridine residue in the ‘‘wobble position’’ of its anticodon, which base-pairs with purine residues in the third position of a cognate codon, it is almost always chemically modified and modification is required for efficient decoding

  • By blocking phosphorylation at these positions using mutations, we identified four phosphorylation sites that are important for Elongator’s role in tRNA modification

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Summary

Introduction

Multi-subunit protein complex containing six different polypeptides (Elp1-Elp6), first discovered in yeast in association with the elongating form of RNA polymerase II and initially proposed to play a role in transcriptional elongation [1,2]. Elongator is non-essential in yeast, knockout of the mouse IKBKAP gene encoding Elongator’s largest subunit leads to embryonic lethality and the protein is crucial for vascular and neural development [3]. Elongator in Caenorhabditis elegans is involved in neuronal function and development [6,7], while in plants it plays a role in proliferation during organ growth [8]. Elongator has been proposed to acetylate a-tubulin and the neuronal protein Bruchpilot [7,13,14] and has been implicated in paternal DNA demethylation in mouse zygotes [15]. Yeast Elongator has been implicated in transcriptional silencing, replication-coupled nucleosome assembly [17] and polarized secretion [19]

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