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
Summary
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]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have