TCONS_00025035-miR-101-UROS is potentially involved in the regulation of heme synthesis pathway and influences mantle melanin deposition by targeting porphyrin in Manila clam (Ruditapes philippinarum).

Shell color is an important economic trait and one of the target traits in breeding and production. Non-coding RNA (ncRNA) refers to RNA molecules transcribed from the genome and do not encoding proteins, which can regulate the expression of target genes after transcription and participate in the regulation of many important traits, such as the formation of shell color and body color. In this study, we detected the porphyrins in the shells of three Manila clams with different shell colors, explored the expression pattern and function of Uroporphyrinogen III synthetase (UROS) in the shell color pigmentation of Ruditapes philippinarum, and found that it might be involved in the synthesis of porphyrins and potentially in the synthesis of melanin. The results showed that the expression levels of heme synthesis-related genes such as UROS, Uroporphyrinogen decarboxylase (UROD), Ferrochelatase (FECH), Hephaestin (HEPH), and pigment synthesis-related genes (Peroxidasin PXDN) in the positive group were significantly reduced compared with the control group after injection of UROS dsRNA, indicating that UROS plays a crucial role in the porphyrin synthesis pathway. Additionally, transmission electron microscopy and melanin extraction experiments also proved that it might participate in the synthesis of melanin. We further explored and verified the relationship between TCONS_00025035-miR-101-UROS and identified the changes in the expression level of UROS through RNA interference and injection of miR-101 antagomir, respectively. Our results imply that miR-101 antagonists affect the expression of UROS. Furthermore, dual-luciferase reporter gene experiments confirmed the relationship between TCON_00025035, miR-101, and UROS. The regulatory relationship between TCONS_00025035 and miR-101 is negative, and the regulatory relationship between miR-101 and UROS is also negative. In summary, we verified the function of UROS through RNA interference, qPCR, in situ hybridization, and melanin content detection. We speculated that there was a negative relationship between miR-101 and UROS, and there was also a negative relationship between TCONS_00025035 and miR-101. TCONS_00025035 might regulate UROS through the regulation of miR-101, and UROS might also regulate other pigmentation-related genes and affect the formation of pigments, thereby influencing porphyrin and melanin formation in Manila clam.