A ubiquitin-like protein encoded by the “noncoding” RNA TINCR promotes keratinocyte proliferation and wound healing
Fig 4
Generation of TUBL-deficient mice.
(A) Schematic representation of the WT TINCR allele, the single-stranded oligodeoxynucleotide (ssODN), and the mutant allele after homologous recombination. Exons are denoted by numbered boxes. The single guide RNA (sgRNA) for the CRISPR-Cas9 system and its protospacer adjacent motif (PAM) are indicated by contiguous black and red underlines, respectively. The TUBL ORF is represented by the gray shading in the box corresponding to exon 1 of TINCR. (B) Predicted secondary structure and minimal free energy for WT TINCR and the mutant form generated by the CRISPR-Cas9 system for establishment of Tubl−/− mice. The triangle indicates the 5’ end of the transcript. (C) PCR analysis of genomic DNA from the tail of mice of the indicated genotypes. The PCR products were digested with EcoRI before electrophoresis. (D) RT-qPCR analysis of TINCR in the epidermis of Tubl+/+ and Tubl−/− mice. Data are means ± SD (n = 3 independent experiments). ***p < 0.005 (Student’s t test). (E, F) Signal intensity of extracted ion chromatograms for mouse TUBL peptides in MRM analysis. The analysis detected two different peptides derived from mouse TUBL with the amino acid sequences AQLVGQGVSSWR (E) and DTLSDLR (F). “Standard” indicates an internal standard corresponding to stable isotope–labeled recombinant mouse TUBL. “Endogenous” indicates the endogenous TUBL peptides in epidermal lysates prepared from Tubl+/+ or Tubl−/− mice. Tryptic peptides derived from endogenous TUBL were mixed with tryptic peptides derived from the isotopically labeled recombinant protein and were then applied to MS analysis. Extracted ion chromatograms for each transition are shown in S1 Fig. (G) Hematoxylin-eosin staining of the skin of Tubl+/+ or Tubl−/− mice at 8 weeks of age. The boxed regions in the left panels of each set are shown at higher magnification in the right panels. Scale bars, 300 μm.