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.