FIGURE 2.
GlnRSΔiABD was present in exosomes extruded from Jurkat cells. A, the consequence of the internal deletion in the ABD of GlnRS (to give GlnRSΔiABD) is modeled based on the structure of several lower homologs (Protein Data Bank (PDB) numbers 3tl4, 2hz7, and 1exd). The location of the splice junction in the structure is marked with a red arrow. B, The specific primers for GlnRSΔiABD were tested in a qPCR reaction with the templates of 106 copies of QRS1, 106 copies of GlnRSΔiABD, and cDNA generated from RNA of Jurkat cells. C, using RT-PCR, expression of GlnRSΔiABD was tested using cellular (cell) and exosomal (exo) RNA samples. (The notation − is for nonactivated Jurkat cells, and + is for activated Jurkat cells.) D, two SerRS splicing variants were present in the cells but not in the exosome. Using RT-PCR, expression of SRS1, SerRSΔE2, and SerRSΔE9 was tested using cellular (cell) and exosomal (exo) RNA samples. SerRSΔE2 and SerRSΔE9 are SerRS splicing variants with deletion of the second and ninth exons, respectively. E, the copy numbers of QRS1 and GlnRSΔiABD were determined by qRT-PCR and using a standard curve. The data represent mean ± S.E. of three independent experiments. ** designates p < 0.01 in a Student's t test. F, the tissue distribution across 22 samples of QRS1 (empty column) and GlnRSΔiABD (pink column) was determined by qRT-PCR, using primers that specifically target QRS1 or GlnRSΔiABD. The data are expressed as mean ± S.E. (n = 3). A light magenta dashed line marks 3 times the median (3M) from the expression levels in 22 samples. G, endogenous GlnRSΔiABD mRNA was detected in the polyribosomal fractions but not free mRNA fractions isolated from Jurkat cells. Fourteen fractions were obtained from across a 10–40% sucrose gradient. The mRNA for QRS1 and GlnRSΔiABD was detected with specific PCR primers. H, a recombinant gene encoding GlnRSΔiABD in pcDNA3.1 and an empty vector control (con) were subjected to a rabbit T7 RNA polymerase-coupled translation-transcription reticulocyte lysate system, and the products were analyzed by Western blotting. The black arrow points to rabbit QRS1, whereas the arrowhead marks human GlnRSΔiABD. I, The HEK 293T cells were transfected with a recombinant gene encoding GlnRSΔiABD in a pcDNA3.1 vector or with an empty vector (Con). Cell lysates were analyzed by Western blot analysis. The position of QRS1 is indicated by a black arrow, and an arrowhead marks the position of GlnRSΔiABD.