Internally deleted human tRNA synthetase suggests evolutionary pressure for repurposing - PubMed (original) (raw)

. 2012 Sep 5;20(9):1470-7.

doi: 10.1016/j.str.2012.08.001.

Zhiyi Wei, Jie J Zhou, Fei Ye, Wing-Sze Lo, Feng Wang, Ching-Fun Lau, Jingjing Wu, Leslie A Nangle, Kyle P Chiang, Xiang-Lei Yang, Mingjie Zhang, Paul Schimmel

Affiliations

Internally deleted human tRNA synthetase suggests evolutionary pressure for repurposing

Zhiwen Xu et al. Structure. 2012.

Abstract

Aminoacyl-tRNA synthetases (AARSs) catalyze aminoacylation of tRNAs in the cytoplasm. Surprisingly, AARSs also have critical extracellular and nuclear functions. Evolutionary pressure for new functions might be manifested by splice variants that skip only an internal catalytic domain (CD) and link noncatalytic N- and C-terminal polypeptides. Using disease-associated histidyl-tRNA synthetase (HisRS) as an example, we found an expressed 171-amino acid protein (HisRSΔCD) that deleted the entire CD, and joined an N-terminal WHEP to the C-terminal anticodon-binding domain (ABD). X-ray crystallography and three-dimensional NMR revealed the structures of human HisRS and HisRSΔCD. In contrast to homodimeric HisRS, HisRSΔCD is monomeric, where rupture of the ABD's packing with CD resulted in a dumbbell-like structure of flexibly linked WHEP and ABD domains. In addition, the ABD of HisRSΔCD presents a distinct local conformation. This natural internally deleted HisRS suggests evolutionary pressure to reshape AARS tertiary and quaternary structures for repurposing.

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Figures

Figure 1

Figure 1. Identification and Validation of a HisRS Splice Variant That Skips the Entire Catalytic Domain

(A) Schematic illustration of human HisRS protein and the identified exon-skipping splicing events. Human HisRS is composed of an N-terminal WHEP domain, a core catalytic aminoacylation domain and a C-terminal anticodon binding domain. The three conserved sequence motifs in its co re active site are indicated by green, blue and brown bars, respectively. The mRNA transcript of the gene for human HisRS is shown below and aligns with its encoded protein. Canonical exons are drawn in scale to the nucleotide length and are labeled consecutively. The splicing events identified by deep sequencing in the current study are illustrated by dashed lines to indicate non-canonical exon junctions. Novel exon junctions that were not annotated in the AceView database, including ΔE3–10 (HisRSΔCD), are indicated by red lines, and the previously reported ones are shown in grey. Targeting sites of the PCR primers are indicated by blue arrows and those of qPCR primers by green arrows. The primer sequences are shown in Table S2. (B) Validation by PCR of the splice variant that skips exons 3 to 10. PCR was performed using cDNA of IMR-32 neuronal cells and a pair of primers targeting 5’-UTR/Exon1 (FP) and 3’-UTR (RP) of the gene for HisRS. PCR products were separate by agarose gel electrophoresis. Lane 1: PCR by FP and RP, Lane 2: PCR by primers targeting GAPDH as control. (C) Sequencing result shows the novel Exon2–11 junction in the HisRSΔCD transcript. (D) Schematic of protein products of human HisRS FL and HisRSΔCD. The protein product of splice variant HisRSΔCD has the entire aminoacylation domain (aa61–398) removed due to skipping of exons 3 to 10 and therefore directly connects the WHEP domain to the ABD. (E) Detection of endogenous HisRSΔCD protein by western blot analysis. HisRSΔCD protein was detected in whole extracts of IMR-32 cells using antibodies against, separately, the N- and C-terminus of HisRS (N-mAb, monoclonal antibody against HisRS aa1–97; C-pAb, polyclonal antibody against HisRS C-terminus). Total lysates of 293T cells transiently transfected with a HisRSΔCD construct were run in parallel with IMR-32 cell extracts to serve as a control that shows the size of HisRSΔCD. The expected running position of the HisRSΔCD protein is indicated by an arrow. See also Figure S1, and Tables S1 and S2.

Figure 2

Figure 2. Structure Determination of Human HisRS by X-ray Crystallography

(A) Optimization of the boundary of human HisRS for high quality crystals. The amino acid range included in each mutant and the corresponding crystal resolutions are shown. The three conserved sequence motifs as well as the insertion domain in the aminoacylation domain are indicated by colored bars and labeled below. (B) Ribbon diagram of the 2.4 Å crystal structure of HisRS Δ1–53_507–509 (blue: CD, green: ABD). The secondary structure α-helices and β-strands are consecutively labeled, and the amino acids composing these structural elements are shown in detail in Figure S2D. (C) Structure comparison of HisRSs of different species including human, trypanosoma (T. brucei, PDB: 3HRI), archaea (T. acidophilum, 1WU7) and bacterium (S. aureus, 1QE0). (D) Superposition of the insertion domains of human, trypanosoma and archaea shows differences in the respective orientations of this domain. The α-helices in the insertion domain of human HisRS are labeled, and the corresponding amino acid sequence and the alignment among different species are shown in Figure S2D. See also Table 1 and Figure S2.

Figure 3

Figure 3. Structure Determination of the Splice Variant HisRSΔCD by NMR Spectroscopy

(A) Schematic of HisRSΔCD* (2C2S_W94Q) mutant employed for structural characterizations. The mutational sites are labeled in red and the corresponding C507, C509 and W432 residues in the native HisRS sequence are also indicated. (B) The 1H-15N HSQC spectrum of HisRSΔCD* used for structure determination. (C) Backbone superimposition of 20 calculated lowest-energy structures of the WHEP domain and the ABD of HisRSΔCD* are shown, and the HisRSΔCD* structure is presented below by ribbon diagram. WHEP domain (red) and ABD (green) are well folded and linked by a flexible loop. (D) Superposition of NMR structures of HisRSΔCD* and of the WHEP domain alone (PDB: 1X59). Structures are shown in ribbon diagram. Red: WHEP domain of HisRSΔCD*, pink: 1X59. (E) Superposition of human HisRS Δ1–53_507–509 crystal structure and HisRSΔCD* NMR structure. The W432 in HisRS FL was labeled in red. The circled area including helix α15 and the preceding loop had the most prominent differences (see also Figure S2D). Structures are shown in ribbon diagram. Orange: ABD of HisRSΔCD*, Green: ABD of HisRS FL, grey: CD of HisRS FL. See also Table 1 and Figures S2 and S3.

Figure 4

Figure 4. Possible Association of HisRSΔCD with IIM/ILD

Jo-1 antibodies from two different IIM patients react with recombinant human HisRS FL (hsHisRS) and HisRSΔCD, but not with E. coli HisRS (ecHisRS). The optical density at 450 nm was used to monitor the formation of antibody complexes in the ELISA. The “7B” stands for lot 7B04507 of Jo-1 antibodies and “4L” stands for lot 4L34811. The titration assay by ELISA was performed in duplicate and data are represented as mean ± SEM.

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