Abstract
Circadian rhythms of behavior and physiology are driven by the biological clock that operates endogenously but can also be entrained to the light-dark cycle of the environment. In mammals, the master circadian pacemaker is located in the suprachiasmatic nucleus (SCN), which is composed of individual cellular oscillators that are driven by a set of core clock genes interacting in transcriptional/translational feedback loops. Light signals can trigger molecular events in the SCN that ultimately impact on the phase of expression of core clock genes to reset the master pacemaker. While transcriptional regulation has received much attention in the field of circadian biology in the past, other mechanisms including targeted protein degradation likely contribute to the clock timing and entrainment process. In the present study, proteome-wide screens of the murine SCN led to the identification of ubiquitin protein ligase E3 component N-recognin 4 (UBR4), a novel E3 ubiquitin ligase component of the N-end rule pathway, as a time-of-day-dependent and light-inducible protein. The spatial and temporal expression pattern of UBR4 in the SCN was subsequently characterized by immunofluorescence microscopy. UBR4 is expressed across the entire rostrocaudal extent of the SCN in a time-of-day-dependent fashion. UBR4 is localized exclusively to arginine vasopressin (AVP)-expressing neurons of the SCN shell. Upon photic stimulation in the early subjective night, the number of UBR4-expressing cells within the SCN increases. This study is the first to identify a novel E3 ubiquitin ligase component, UBR4, in the murine SCN and to implicate the N-end rule degradation pathway as a potential player in regulating core clock mechanisms and photic entrainment.
Highlights
Mammals possess numerous peripheral clocks throughout the body that keep track of time and control rhythmic outputs of behavior and physiology [1]
To survey the murine suprachiasmatic nucleus (SCN) proteome for time-of-day-dependent fluctuations in protein expression, we conducted SILACbased quantitative mass spectrometry of murine SCN tissues harvested at 4 h intervals across the circadian cycle (Fig. 1A) (Chiang et al, manuscript describing mass spectrometry screen was submitted)
Out of 441 and 387 proteins whose expression were deemed to be time-of-day-dependent or lightinduced in the SCN based on these two studies, respectively, we focused our attention on one protein that was identified in both screens: ubiquitin protein ligase E3 component N-recognin 4 (UBR4)
Summary
Mammals possess numerous peripheral clocks throughout the body that keep track of time and control rhythmic outputs of behavior and physiology [1] These endogenous peripheral clocks are temporally coordinated by a central pacemaker located in the suprachiasmatic nucleus (SCN), a hypothalamic structure directly above the optic chiasm [2,3]. TTFLs are regulated by a wide array of post-transcriptional and posttranslational mechanisms such as phosphorylation and acetylation, which fine-tune the rhythmic expression of core clock genes [5]. As these molecular oscillations have an endogenous period that only approximates a 24 h day/night cycle, daily adjustments to the phase of the internal clock are required to align it precisely to the local environmental time. The retinohypothalamic tract (RHT) represents a direct path by which light information is transmitted from the retina to the SCN [7,8], where a series of molecular and cellular events are triggered that lead to transcription of immediate early genes and core clock genes [9] and resetting of the master pacemaker
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