HnRNP U Controls Circadian Rhythm Synchronization By Regulating Avp And Vip

Circadian rhythms control all kinds of behavioral and physiological processes in mammalian,such as ingestion and excretion,sleep and wakefulness,learning and forgetting,hormone synthesis and secretion.With robust circadian rhythm,animals are able to adapt to specific temporal-spatial niches.Suprachiasmatic nucleus(SCN)-enriched neuropeptides are critical for communication and coupling among SCN neurons which organize daily rhythms,but it is still unknown how these peptides are regulated.We found that heterogeneous nuclear ribonucleoprotein U(hnRNP U)was critical for SCN neurons synchronization.HnRNP U was reported as a RNA binding protein,participating in pre-mRNA alternative splicing,snRNA processing,and mature mRNA degrading.Additionally,it was reported that patients with 1q43 microdeletion,the location of human hnRNP U,had severe sleep disorders.But,direct evidence was lacking for hnRNP U to regulate animal biorhythms.Firstly,we constructed hnRNP U knockout mice.Homozygous mutant(Hnmpu-/-)mice were embryonic lethal,and heterozygous(Hnmpu+/-)mice exhibited seriously disrupted rhythms.Deleting only one copy of Hnmpu resulted in erratic and unconsolidated circadian rhythms,manifesting as fragmented and susceptible locomotor activity.Total locomotor activity was decreased in Hnmpu+/-mice.In 70%of Hnrnpu+/-mice,more movement than wild-type controls was detected in the daytime.Moreover,it was even totally arrhythmic for the other 30%.Consistently,energy metabolism of Hnrnpu+/-mice was erratic and disorganized.Oxygen consumption,carbon dioxide expiration,and thermogenesis were decreased significantly at night in Hnmpu+/-mice.While,food intake and respiratory exchange ratio(RER)exhibited a large phase shift.Core body temperature of Hnmpu+/-mice was higher in the daytime but lower at night than Hnrnpu+/+ mice,leading to a decreased temperature amplitude.Two possible factors influenced the stability of biorhythm:first,the robustness of molecular clock feedback loop in master pacemaker itself;second,the coupling extent between pacemaker and peripheral oscillators.On the one hand,we detected central clock genes,but no difference was detected between Hnrnpu+/-and Hnmpu+/+ mice.On the other hand,we determined 6 well-established SCN-enriched neuropeptides.Five of them:vasoactive intestinal polypeptide(Vip),arginine vasopressin(Avp),neuromedin S(Nms),enkephalin(Enk)and gastrin-releasing peptide(Grp)were suppressed in Hnmpu+/-mice,although the SCN size and boundary was preserved.In addition,Avp-promoter and Vip-promoter driving luciferase was suppressed in hnRNP U RNAi cells.Furthermore,we conducted light pulse induced phase shift experiment,and Hnrnpu+/-mice had difficulties to response to white light pulse at CT13-14.Then we microinjected Avp and Vip into SCN region,and Hnmpu+/-mice exhibited similar sensitivity to these neuropeptides as Hnrnpu+/+mice,which suggested that the downstream pathway was intact.We performed chromatin immunoprecipitation(ChIP)with hnRNP U,Bmal1,and Clock antibodies in high serum shock SCN2.2 cell line and found hnRNP U binding to Avp and Vip promoters in a phase-dependent manner.In addition,hnRNP U knockdown significantly inhibited Bmal1 and Clock binding to Avp and Vip E-boxes.Moreover,hnRNP U,Bmal1,and Clock formed a complex periodically,binding together at 12-20 hours after shock,separating away at 24-32 hours,and binding again at 36-44 hours.In conclusion,our findings demonstrated that hnRNP U regulated Avp and Vip expression by binding to their promoters together with Bmal1 and Clock.And then,SCN conferred accuracy and robustness to the rest of body.