LncRNA CA13/EFTUD2/CA13 caused crossed beak by regulating the mandibular calcification in chicken

Crossed beak is a beak deformity that reduces feeding and drinking in affected chickens, thereby diminishing chicken production performance. Previous studies have shown that long non-coding RNA CA13 (lncRNA CA13), as an upstream regulator of carbonic anhydrase 13 (CA13), exhibited down-regulated expression in the lateral mandibular condyles of the short bone branch in cross-beaked chickens along with CA13. The study investigated the expression characteristics of lncRNA CA13 and its regulatory relationship with CA13 and elongation factor Tu GTP-binding domain-containing protein 2 (EFTUD2), laying the groundwork for the molecular mechanisms of lncRNA CA13 in chicken mandibular mineralization. The length of lncRNA CA13, amplified by 5' and 3' rapid amplification of cDNA ends (RACE) technique, was 2380 bp. Expression of lncRNA CA13 was significantly higher in mandibular condylar tissues compared to other tissues (P < 0.01). Using a successfully constructed osteoblast-induced differentiation model, orange calcified nodules were observed under the microscope after alizarin red staining, and the expressions of CA13, lncRNA CA13, runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP) were also detected to be significantly higher than those before induction (P < 0.01; P < 0.05). Integrated RNA affinity pull-down assay (RNA pull-down) combined with multiple bioinformatic analysis methods, as well as RNA binding protein immunoprecipitation assay (RIP), identified EFTUD2 as a transcription factor involved in the cellular mineralization process. The oe-lncRNA CA13 vector was constructed with an overexpression efficiency of 95.85 %, and the sh-lncRNA CA13 vector had an interference efficiency of 54.58 %. Overexpression of lncRNA CA13 significantly upregulated EFTUD2 and downregulated CA13; simultaneous interference with lncRNA CA13 significantly downregulated EFTUD2 and upregulated CA13. Thus, the lncRNA CA13/EFTUD2/CA13 axis plays a key role in the development of crossed beak by regulating the calcification process in the chicken mandible.