Role of HnRNP A1 in rhythmic synthesis of Nfil3 protein via IRES-mediated translation

Abstract

The circadian rhythm is observed from cyanobacteria to mammals. The mammalian circadian rhythm is tightly regulated by several regulation mechanisms in brain and peripheral tissues. Rhythmic expression of mRNA and protein is most remarkable feature of clock genes. Among them, Nuclear factor, interleukin 3, regulated (Nfil3) protein is a transcription factor that binds to DNA and generally represses target gene expression. In the circadian clock system, Nfil3 binds to a D box element residing in the promoter of clock genes and contributes to their robust oscillation. Until now, mechanisms of Nfil3 expression is focused on transcription. However, to explain fine-tuned regulation of Nfil3 and other D box containing clock genes expression, only transcriptional and posttranslational regulations are not enough. To generate robust oscillation of Nfil3 gene, its posttranscriptional regulation also needed. In this study, I show that the 5′-untranslated region (5’-UTR) of Nfil3 mRNA contains an internal ribosome entry site (IRES) and that the oscillation of IRES-mediated translation occurs in a phase-dependent manner. I demonstrate a cis-acting region as IRES in 5’-UTR. Additionally, RNA-binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) binds to cis-acting region and act as an IRES trans-acting factor (ITAF), it regulates IRES-mediated translation of Nfil3. Knockdown of hnRNP A1 reduces IRES activity and diminishes Nfil3 protein oscillation without changing mRNA oscillation. Moreover, I observed that intracellular calcium, which are closely related to bone formation, it could regulated by endogenous Nfil3 protein levels in osteoblast cell lines. This thesis on the regulation of Nfil3 suggests that relationship between cap-independent translation of Nfil3 mRNA and hnRNP A1 is provides extended new insight to rhythmic translation of circadian clock system.