Untranslated regions 를 통한 Period1과 Cryptochrome1 생체시계 유전자의 전사 후 조절에 관한 연구 : Rhythmic post-transcriptional regulation of Period1 and Cryptochrome1 via untranslated regions
저자
발행사항
포항 : 포항공과대학교 생명과학과, 2012
학위논문사항
Thesis(doctoral)-- 포항공과대학교 생명과학과 : 생명과학과 분자신경생리학 2012. 2
발행연도
2012
작성언어
영어
주제어
발행국(도시)
대한민국
형태사항
179 ; 26cm
일반주기명
지도교수:김경태 교수님
소장기관
Circadian rhythms exist in most living organisms, and the mammalian circadian rhythm is observed not only at the suprachiasmatic nucleus (SCN), a master pacemaker, but also throughout the peripheral tissues. Until now, researches in clock genes’ expression and working have mainly focused on transcription and post-translational modification. However, little is known about post-transcriptional regulation of these genes. Only transcription and post-translational modification cannot explain fine-tuned rhythmic mRNA and protein oscillation. To make rhythmic oscillation, in addition to the time-dependent transcription, rhythmic mRNA degradation, translation initiation, and modulated translational repression may be needed. Thus, the rhythmic post-transcriptional regulation, translation initiation, microRNA (miRNA)-mediated translation repression, and 3′-untranslated region (UTR)-mediated translation may be a novel and efficient mechanisms for controlling the circadian system.
In the first chapter, I will discuss about translational regulation of clock gene. The mouse PERIOD1 (mPER1) protein, along with other clock proteins, plays a crucial role in the maintenance of circadian rhythm. mPER1 also provides an important link between the circadian system and the cell cycle system. Here we show that the circadian expression of mPER1 is regulated by rhythmic translational control of mPer1 mRNA together with transcriptional modulation. This time-dependent translation is controlled by an internal ribosomal entry site (IRES) element in the 5′UTR of mPer1 mRNA along with the trans-acting factor, mouse heterogeneous nuclear ribonucleoprotein Q (HNRNPQ). Knockdown of HNRNPQ caused a decrease in mPER1 levels and a slight delay in mPER1 expression without changing mRNA levels. The rate of IRES-mediated translation exhibits phase-dependent characteristics through rhythmic interaction between mPer1 mRNA and HNRNPQ. Here, I demonstrate 5′UTR-mediated rhythmic mPer1 translation and provide evidence for posttranscriptional regulation of the circadian rhythmicity of core clock genes.
In the following chapter, I will describe the importance of HNRNPQ for Per1 mRNA translation with mathematical modeling. Translation of mPer1 is directed by both a cap-dependent process and cap-independent translation mediated by an IRES element in the 5′UTR. Here, I compared mPer1 IRES activity with other cellular IRESs and checked relative contribution of IRES-mediated translation in overall mPer1 translation. I also found critical region in mPer1 5′UTR for HNRNPQ binding. Deletion of HNRNPQ binding region markedly decreased IRES activity and disrupted rhythmicity. A mathematical model also suggests that rhythmic IRES-dependent translation is a key process in mPER1 oscillation. The IRES-mediated translation of mPer1 will help define the post-transcriptional regulation of the core clock genes.
Next, I will discuss about miRNA-mediated translational regulation of clock gene. I investigated the role of the 3'-untranslated region (UTR) of the mouse Cryptochrome 1 (Cry1) gene at the post-transcriptional level, especially miRNA-mediated regulation. Knockdown of Drosha, Dicer or Argonaute2 showed increase in the level of Cry1-3′UTR reporter. miRNA recognition element (MRE) of Cry1 which is important for miR-185 binding led to a decrease in protein level but not in mRNA quantity. Indeed, mutation of miR-185 binding region increased reporter protein level. Overexpression of miR-185 led to decrease in reporter activity. Cytoplasmic miR-185 levels were almost anti-phase to that of CRY1 protein levels and knockdown of miR-185 elevated the amplitude of CRY1 protein oscillation. My results suggest that miR-185 plays a role as a fine regulator, contributing to the Cry1 mRNA translation rate, of rhythmicity of CRY1 protein.
In the last chapter, I will describe an example of 3′UTR-mediated translational regulation of clock gene. CRY1 protein which is core clock gene shows circadian time-dependent expression, and inhibits the transcription of clock genes with cooperation of other factors. Rhythmic expression of Cry1 has been mainly explained by transcription and post-translational regulation. But how CRY1 protein expression is rhythmically regulated during circadian time is poorly defined. In the present study, I investigated the role of the 3'UTR of the mouse Cry1 gene at the post-transcriptional level, especially translational regulation. Previously, I reported that circadian amplitude of Cry1 is modulated by mRNA stability. mRNA stability of Cry1 was regulated via cytoplasmic HNRNPD oscillation. Interestingly, 3′UTR of Cry1 decreased its mRNA levels, but increased protein amounts. Inhibition of translation increased mRNA levels of Cry1. Knockdown of HNRNPD made decreased CRY1 protein levels. Expression of HNRNPD and binding between HNRNPD and Cry1-3′UTR was parallel to CRY1 protein profile. My results suggest that 3′UTR of Cry1 is important for rhythmic expression of mRNA and protein, and HNRNPD plays dual roles as a regulator contributing to the Cry1 mRNA turnover rate and modulator playing a part in translation activation.
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