| Nearly all organisms possess circadian clocks that are able to anticipate the day/night cycles and are reset by the ambient light.The molecular mechanisms underlying this vital process are known to require light-responsive gene regulation,yet remain incompletely understood.Amongst the models in which to tackle this question is zebrafish,which represents a convenient research tool for studying key aspects of this circadian system in vertebrates.Unlike the situation in most species of mammals,the circadian clocks in zebrafish peripheral tissues,cells and in-vitro-cultured cell lines can be entrainable by direct exposure to light,thus providing unique insight into both function and mechanism of biological the functions affected by light.In this study,we used RNA sequence technologies to identify and screen 1365light-responsive m RNAs and 66 light-responsive mi RNAs.Functional enrichment analyses revealed that light-responsive m RNAs were strongly involved in oxidationreduction,visual perception,PPAR signaling pathway,phototransduction and peroxisome,while light-responsive mi RNAs were predicted to regulate many biological events including MAPK signaling pathway and peroxisome,which are closely related to the regulation of circadian rhythms.According to our K-mean clustering analysis and construction of a m RNA-mi RNA interaction network,we constructed a post-transcriptional regulatory network for the zebrafish circadian clock,and two light-responsive mi RNAs(mi R-204-3p and mi R-430a-3p),which are predicted to regulate cryptochrome(cry1a and cry1b)were focused for in-depth functional analysis.Specifically,luciferase reporter assays validated the ability of mi R-204-3p and mi R-430a-3p to directly bind the 3’-UTR of cry1 a and cry1 b,respectively.Subsequently,the functions of mi R-204-3p and mi R-430a-3p were investigated in vivo by injecting mi RNA mimics and inhibitors into zebrafish embryo.q RT-PCR tests analysis confirmed that mi R-204-3p and mi R-430a-3p significantly affected the expression of their target-m RNAs(cry1a and cry1b),and both two mi RNA may also regulate the overall core clock mechanism through indirectly affecting the expression of other core clock genes(bmal1b,clock1 a,per1b,per2,per3).Since rhythmic activity is the most important form of output of circadian clock,we hypothesized that mi R-204-3p and mi R-430a-3p might have the ability to affect the rhythmic activity of zebrafish.Therefore,we performed several behavioral tests on zebrafish larvae with overexpression or interference of mi RNA,and the results showed that mi R-204-3p and mi R-430a-3p could regulate the rhythmic locomotor of zebrafish under Light-Dark cycle and Free-Running period.Based on the whole transcriptome sequencing data,we screened and identified 330light-responsive lnc RNAs and 91 light-responsive circ RNAs.According to ce RNA mechanism,the lnc RNA-circ RNA-mi RNA-m RNA regulatory network was constructed,the ce RNA sub-network of clock genes(cry1a、cry1b、cry2、aanat1、dbpb、cry5、nfil3-6、tefa 和 nr1d2a)was screened out,and explored the non-coding RNA-mediated post-transcriptional regulation mechanism of circadian clock.These results are important for clarifying how light-responsive nc RNAs involved in the regulation of the circadian clock.Altogether,the light-responsive m RNA and mi RNA screening,identification and functional validation in zebrafish revealed that mi R-204-3p and mi R-430a-3p can affect the circadian clock system at the molecular and behavioral levels by targeting cry1 a and cry1 b.Meanwhile,bioinformatics analysis of non-coding RNAs verified an intricate multi-level regulation of the circadian clock by light.This study provides information for further understanding of the emerging roles for nc RNA-mediated posttranscriptional regulation in circadian,and laid the foundation for further mechanism research. |
