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. 2014 Jun;92(6):1227-42.
doi: 10.1111/mmi.12624. Epub 2014 May 19.

A Conserved and Essential Basic Region Mediates tRNA Binding to the Elp1 Subunit of the Saccharomyces Cerevisiae Elongator Complex

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Free PMC article

A Conserved and Essential Basic Region Mediates tRNA Binding to the Elp1 Subunit of the Saccharomyces Cerevisiae Elongator Complex

Rachael Di Santo et al. Mol Microbiol. .
Free PMC article

Abstract

Elongator is a conserved, multi-protein complex discovered in Saccharomyces cerevisiae, loss of which confers a range of pleiotropic phenotypes. Elongator in higher eukaryotes is required for normal growth and development and a mutation in the largest subunit of human Elongator (Elp1) causes familial dysautonomia, a severe recessive neuropathy. Elongator promotes addition of mcm(5) and ncm(5) modifications to uridine in the tRNA anticodon 'wobble' position in both yeast and higher eukaryotes. Since these modifications are required for the tRNAs to function efficiently, a translation defect caused by hypomodified tRNAs may therefore underlie the variety of phenotypes associated with Elongator dysfunction. The Elp1 carboxy-terminal domain contains a highly conserved arginine/lysine-rich region that resembles a nuclear localization sequence (NLS). Using alanine substitution mutagenesis, we show that this region is essential for Elongator's function in tRNA wobble uridine modification. However, rather than acting to determine the nucleo-cytoplasmic distribution of Elongator, we find that the basic region plays a critical role in a novel interaction between tRNA and the Elp1 carboxy-terminal domain. Thus the conserved basic region in Elp1 may be essential for tRNA wobble uridine modification by acting as tRNA binding motif.

Figures

Fig. 1
Fig. 1
A highly conserved basic region in Elp1 is essential for Elongator function. A. Multiple sequence alignment of the Elp1 subunit of Elongator from various eukaryotic organisms showing a portion of the Elp1 C-terminal domain including the basic region to highlight its conservation. Alignment was carried out using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Sequences used were as follows with accession numbers in brackets; S. cerevisiae (Q06706), S. pombe (O59704), H. sapiens (O95163), M. musculus (Q7TT37), O. cuniculus (Q8WND5), R. norveigus (Q8VHU4), X. laevis (Q2TAQ1), D. melanogaster (Q9VGK7), A. thaliana (Q9FNA4). B. Schematic of Elp1 highlighting the Arg/Lys rich basic region (basic residues in bold) and alanine substitution mutations (boxed) made within this region. Mutations were made across the entire Elp1 basic region (elp1–KR9A) and within the first and second halves of the region (elp1–KR5A and elp1–KR4A respectively). For comparison the consensus sequence for a bipartite NLS is also shown. The indicated structural predictions were made using PSIPRED (Buchan et al., 2013). C. Zymocin sensitivity assays of strains containing GFP tagged wild-type ELP1 or elp1 basic region mutants at the ELP1 genomic locus. Wild-type (YRDS253) and elp1Δ (YRDS250) served as controls for zymocin sensitivity and resistance respectively. Left panel, Eclipse assays on YPAD medium measuring growth inhibition adjacent to a colony of a zymocin-secreting K. lactis strain. Centre and right panels, growth of equivalent 10-fold serial dilutions (indicating OD600) of the indicated strains containing pAE1, which expresses the zymocin γ subunit under control of a galactose inducible promoter. Strains were grown on YPARG (centre panel: zymocin induced) and YPAD (right panel: zymocin uninduced). D. Growth of equivalent 10-fold serial dilutions of the indicated strains containing chromosomal copies of ELP1 or elp1–KR9A together with the SUP4 ochre suppressor and either wild-type URA3 (as a control) or the ura3oc22 ochre allele. SUP4 can read through the premature stop codon in ura3oc22 to allow growth in the absence of uracil on DOA-Ura medium but efficient suppression requires mcm5 modification of the SUP4 tRNA at the wobble uridine position, as shown by comparing growth of the ELP1 and elp1Δ ura3oc22 strains. Growth of the same strains on synthetic complete (SC) medium without uracil selection is shown as a control.
Fig. 2
Fig. 2
The Elp1 C-terminal domain can drive nuclear import of GFP dependent on the conserved basic region. Representative images of cells expressing Nic96-4mCherry (red) to indicate the nuclear periphery (YRDS84) containing either pUG34 (GFP control) expressing GFP or its derivative plasmids expressing GFP fused at its C-terminus to residues 1181–1349 from wild-type (Elp1–CTD) or the mutant (Elp1–KR4A-CTD, Elp1–KR5A–CTD, Elp1–KR9A–CTD) versions of Elp1 shown in Fig. 1 (green). Cells were grown to log phase in DOA-Met-His medium to induce GFP expression and then fixed for imaging. Both GFP (27 kDa) and the fusion proteins (47 kDa) are expected to be present in both the nucleus and cytoplasm in the absence of active nuclear localization signals as they are small enough to enter the nucleus by diffusion. CTD, C-terminal domain; scale bar: 5 μm.
Fig. 3
Fig. 3
The nucleo-cytoplasmic distribution of Elp1 is unaffected by basic region mutations. A. Representative images of strains expressing Nic96-4mCherry (red) together with GFP-tagged wild-type Elp1, Elp1–KR9A, Elp1–NLS or Elp1–NLS4A (green). Elp1–NLS contains the NLS from Cbp80 and Elp1–NLS4A contains a mutant version containing 4 alanine substitutions, inserted in each case between the Elp1 and GFP sections of the fusion protein. Cells were grown to log phase in YPAD medium and then fixed for imaging. Scale bar: 5 μm. B. Zymocin sensitivity assays of the indicated strains as described in the legend to Fig. 2. Wild-type (WT, BY4741) and elp1Δ (YRDS250) served as controls for zymocin sensitivity and resistance respectively.
Fig. 4
Fig. 4
The elp1–KR9A mutation does not disrupt Elongator complex assembly but causes subtle differences in association with Elp5 and Kti12. A. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains expressing HA-tagged Elp3, Elp5 or Kti12. All samples were analysed with anti–GFP and anti-HA. Input samples were also analysed using anti-Cdc28 as a loading control. Arg4–GFP was confirmed to have similar pulldown efficiency to Elp1–GFP and was used as a control to demonstrate that interaction between Elp1 and the various HA tagged complex members was specific. All pulldown samples were analysed from the same blot. It should be noted that Elp1 is always observed by Western blot analysis as both a full-length and a faster migrating form (truncated at its N-terminus) that may be a degradation product or serve an as yet unknown function (Fichtner et al., 2003). B. Quantification of co-immunoprecipitation efficiency. Immunoprecipitation of HA-tagged proteins was quantified by densitometry of the HA tag signals and normalized using the Elp1–GFP signals across the indicated number of replicates (n), setting the value for the wild-type strain in each case to 1.0. Error bars represent the standard deviation of the mean and the significance of the differences was analysed using a one way ANOVA, with ‘*’ indicating a P-value of < 0.05 (significant) and ‘***’ indicating a P-value of < 0.001 (very highly significant). Any small differences in Elp3 association were not statistically significant when analysed in this manner.
Fig. 5
Fig. 5
Self-association of Elp1 is unaffected by the elp1–KR9A mutation and is independent of the assembly of the Elp4–6 hexamer. A. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains containing empty vector (EV), YCplac111–ELP1–6HA or YCplac111–elp1–KR9A–6HA expressing HA-tagged wild-type or mutant Elp1 respectively. All samples were analysed by Western blotting with anti–GFP and anti-HA. Inputs were also analysed using anti-Cdc28 as a loading control. B. Western blot analysis of inputs and GFP–Trap immunoprecipitates of Elp1–GFP and Elp1–KR9A–GFP from strains containing empty vector (EV), YCplac111–ELP1–6HA or YCplac111–elp1–KR9A–6HA expression plasmids either with or without deletion of the ELP5 gene (elp5Δ).
Fig. 6
Fig. 6
The Elp1 basic region is predicted to bind RNA and is required for formation of a complex with yeast tRNA. A. Analysis for predicted RNA binding sites within the S. cerevisiae and H. sapiens Elp1 protein sequence using BindN+ (http://bioinfo.ggc.org/bindn+/) showing the high confidence prediction of the basic region as a putative RNA binding region (confidence is scored on a scale of 1–9 with 9 as highest confidence). The locations of the Elp1 basic region and the elp1–KR9A alanine substitution mutations are shown for comparison. Overall conservation between the human and yeast sequences is also indicated (|, identity; •, similar residues). B. Recombinant Elp1 and Elp1–KR9A C-terminal domains (CTDs). Elp1 CTDs with N-terminal GST and C-terminal His6 tags were expressed and purified from E. coli along with GST-His6 as a control. The corresponding proteins were analysed on a 4–12% polyacrylamide gel and stained with instant blue. The GST–Elp1–CTD-His6 fusion proteins are indicated by an arrow. C. Electrophoretic mobility shift assay (EMSA) using ∼ 1 nM 32P-labelled yeast tRNA and the indicated concentrations of recombinant GST, GST–Elp1 and GST–Elp1–KR9A fusion proteins shown in (B). Binding reactions were separated on a 1.5% native agarose gel and analysed by autoradiography. A complex was formed between tRNA and the wild-type Elp1 CTD but not with the Elp1–KR9A CTD.
Fig. 7
Fig. 7
Binding of the Elp1 C-terminal domain to tRNA occurs with a dissociation constant in the low micromolar range and is not competed by poly(U) or ssDNA. A. Electrophoretic mobility shift assay (EMSA) using ∼ 1 nM 32P-labelled yeast tRNA, 0.4 μM recombinant Elp1 C-terminal domain and the indicated molar excess of unlabelled yeast tRNA, poly(U) or ssDNA. B. EMSA using ∼ 0.1 nM 32P-labelled yeast tRNA and the indicated concentrations of recombinant Elp1 C-terminal domain. Bound and free tRNA bands were quantified from three replicate experiments and used to estimate a dissociation constant (KD) of 0.55 μM for the interaction by non-linear curve fitting (f = a*xb/[cb + xb]). The Hill coefficient (b) for the fitted curve was 1.76 ± 0.19. All binding reactions were separated on a 1.5% native agarose gel and analysed by autoradiography.

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References

    1. Agarwalla S, Kealey JT, Santi DV, Stroud RM. Characterization of the 23 S ribosomal RNA m5U1939 methyltransferase from Escherichia coli. J Biol Chem. 2002;277:8835–8840. - PubMed
    1. Agris PF. Bringing order to translation: the contributions of transfer RNA anticodon-domain modifications. EMBO Rep. 2008;9:629–635. - PMC - PubMed
    1. Amberg DC, Burke D, Strathern JN. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. New York: Cold Spring Harbor Laboratory Press; 2005.
    1. Anderson SL, Coli R, Daly IW, Kichula EA, Rork MJ, Volpi SA, et al. Familial dysautonomia is caused by mutations of the IKAP gene. Am J Hum Genet. 2001;68:753–758. - PMC - PubMed
    1. Anton BP, Saleh L, Benner JS, Raleigh EA, Kasif S, Roberts RJ. RimO, a MiaB-like enzyme, methylthiolates the universally conserved Asp88 residue of ribosomal protein S12 in Escherichia coli. Proc Natl Acad Sci USA. 2008;105:1826–1831. - PMC - PubMed

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