The Structure And Cellular Physiology Functions Analysis Of The 5′untranslated Region(5′UTR) Of RB1

RB gene is a retinoblastoma susceptibility gene, involved in regulation of cell cycle progression. Its encode proteins will promote the progression of many cancers if they are abnormal. However, there are few reports focused on the possible internal ribosome entry site(IRES) function of RB gene flanking sequence.In this study, we first analyzed sequence and second structure of 5′untranslated region(5′UTR) of RB family members and found that RB family members′ 5′UTR are accordance with the general characteristics of IRES element. Then, 5′UTR sequence of RB family members were tested by dual-luciferase reporter gene system and result showed that only RB1 5′UTR had IRES activity. Moreover, results of two experiments excluded the possible existence of cryptic promoter and splicing sites in 5′UTR of RB1, and thus they proved the IRES activity of detected sequence. Meanwhile, IRES activities of RB1 5′UTR were analyzed in different cell lines, including ovarian carcinoma cells A2780/WT, human cervical cancer cell HELA, murine fibroblast cells NIH3T3 and human embryonic kidney cells HEK293. Various IRES activities were shown in different cell lines.Several fators, including sequence of nucleic acid, second structure of RNA and IRES trans-acting factors(ITAFs), are essential for IRES activity. Therefore, previous works were focused on analyzing the core domain and ITAFs of IRES sequences. In this study, to detect the RB1 IRES core resion, the sequence of RB1 5′UTR was gradually truncated. The results showed that any truncation of RB1 5′UTR were harmful to its IRES activity, and two sequences, 5′ “1~25” and 3′end “131~166” sequence, were core sequences of RB1 5′UTR. Moreover, the bingding site of RB1 5′UTR for Y-box binding protein1 and polypyrimidine were predicted by Deeplearning system and located on 3′end. Meanwhile, the bingding site may also regulate the IRES activity of RB1 5′UTR.Effects of RB1 IRES activity on physiological function were also investigated in this study. Cell cycle was regulated by several conditions, including serum-starvation, paclitaxel and Adriamycin. Results showed that the highest IRES activity of RB1 was obtained under cell cycle of G1 phase. Meanwhile, effects of RB1 5′UTR mutated sequence in rentinoblastoma were analyzed and its IRES activity decreased nearly 50% compared with wild-type RB1 IRES. Threrefore, IRES activity of RB1 is associated with the regulation of cell cycle and retinoblastoma.In conclusion, RB1 function was connected with its IRES activity and the result of this study will be beneficial to supply a new target of cancer research.