Tae-Don Kim

Heterogeneous Nuclear Ribonucleoprotein C Modulates Translation of c-myc mRNA in a Cell Cycle Phase-Dependent Manner

ABSTRACT The c-myc proto-oncogene plays a key role in the proliferation, differentiation, apoptosis, and regulation of the cell cycle. Recently, it was demonstrated that the 5′ nontranslated region (5′ NTR) of human c-myc mRNA contains an internal ribosomal entry site (IRES). In this study, we investigated cellular proteins interacting with the IRES element of c-myc mRNA. Heterogeneous nuclear ribonucleoprotein C (hnRNP C) was identified as a cellular protein that interacts specifically with a heptameric U sequence in the c-myc IRES located between two alternative translation initiation codons CUG and AUG. Moreover, the addition of hnRNP C1 in an in vitro translation system enhanced translation of c-myc mRNA. Interestingly, hnRNP C was partially relocalized from the nucleus, where most of the hnRNP C resides at interphase, to the cytoplasm at the G2/M phase of the cell cycle. Coincidently, translation mediated through the c-myc IRES was increased at the G2/M phase when cap-dependent translation was partially inhibited. On the other hand, a mutant c-myc mRNA lacking the hnRNP C-binding site, showed a decreased level of translation at the G2/M phase compared to that of the wild-type message. Taken together, these findings suggest that hnRNP C, via IRES binding, modulates translation of c-myc mRNA in a cell cycle phase-dependent manner.

Mouse period 2 mRNA circadian oscillation is modulated by PTB–mediated rhythmic mRNA degradation

Circadian mRNA oscillations are the main feature of core clock genes. Among them, period 2 is a key component in negative-feedback regulation, showing robust diurnal oscillations. Moreover, period 2 has been found to have a physiological role in the cell cycle or the tumor suppression. The present study reports that 3′-untranslated region (UTR)-dependent mRNA decay is involved in the regulation of circadian oscillation of period 2 mRNA. Within the mper2 3′UTR, both the CU-rich region and polypyrimidine tract-binding protein (PTB) are more responsible for mRNA stability and degradation kinetics than are other factors. Depletion of PTB with RNAi results in mper2 mRNA stabilization. During the circadian oscillations of mper2, cytoplasmic PTB showed a reciprocal expression profile compared with mper2 mRNA and its peak amplitude was increased when PTB was depleted. This report on the regulation of mper2 proposes that post-transcriptional mRNA decay mediated by PTB is a fine-tuned regulatory mechanism that includes dampening-down effects during circadian mRNA oscillations.

Rhythmic Serotonin N-Acetyltransferase mRNA Degradation Is Essential for the Maintenance of Its Circadian Oscillation

ABSTRACT Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase [AANAT]) is the key enzyme in melatonin synthesis regulated by circadian rhythm. To date, our understanding of the oscillatory mechanism of melatonin has been limited to autoregulatory transcriptional and posttranslational regulations of AANAT mRNA. In this study, we identify three proteins from pineal glands that associate with cis-acting elements within species-specific AANAT 3′ untranslated regions to mediate mRNA degradation. These proteins include heterogeneous nuclear ribonucleoprotein R (hnRNP R), hnRNP Q, and hnRNP L. Their RNA-destabilizing function was determined by RNA interference and overexpression approaches. Expression patterns of these factors in pineal glands display robust circadian rhythm. The enhanced levels detected after midnight correlate with an abrupt decline in AANAT mRNA level. A mathematical model for the AANAT mRNA profile and its experimental evidence with rat pinealocytes indicates that rhythmic AANAT mRNA degradation mediated by hnRNP R, hnRNP Q, and hnRNP L is a key process in the regulation of its circadian oscillation.

Pharmacological characterization of adenosine receptors in PGT‐β mouse pineal gland tumour cells

The adenosine receptor in mouse pinealocytes was identified and characterized using pharmacological and physiological approaches. Expression of the two adenosine receptor subtypes A2B and A3 was detected in mouse pineal glands and PGT‐β cells by polymerase chain reaction and nucleotide sequencing. Adenosine and 5′‐N‐ethylcarboxamidoadenosine (NECA) evoked cyclic AMP generation but the A2A‐selective agonist 2‐(4‐(2‐carboxyethyl)phenylethylamino)adenosine‐5′‐N‐ethylcarboxamideadenosine (CGS 21680) and the A1‐specific agonists R‐N6‐(2‐phenylisopropyl)adenosine (R‐PIA) and N6‐cyclopentyladenosine (CPA) had little effect on intracellular cyclic AMP levels. The A2B receptor selective antagonists alloxazine and enprofylline completely blocked NECA‐mediated cyclic AMP accumulation. Treatment of cells with the A3‐selective agonist N6‐(3‐iodobenzyl)‐5′‐(N‐methylcarbamoyl)adenosine (IB‐MECA) inhibited the elevation of the cyclic AMP level induced by NECA or isoproterenol in a concentration‐dependent manner with maximal inhibition of 40 – 50%. These responses were blocked by the specific A3 adenosine receptor antagonist MRS 1191. Pretreatment of the cells with pertussis toxin attenuated the IB‐MECA‐induced responses, suggesting that this effect occurred via the pertussis toxin‐sensitive inhibitory G proteins. IB‐MECA also caused a concentration‐dependent elevation in [Ca2+]i and IP3 content. Both the responses induced by IB‐MECA were attenuated by treatment with U73122 or phorbol 12‐myristate 13‐acetate. These data suggest the presence of both A2B and A3 adenosine receptors in mouse pineal tumour cells and that the A2B receptor is positively coupled to adenylyl cyclase whereas the A3 receptor is negatively coupled to adenylyl cyclase and also coupled to phospholipase C.

Vitamin D3 Upregulated Protein 1 Suppresses TNF-α–Induced NF-κB Activation in Hepatocarcinogenesis

Vitamin D3 upregulated protein 1 (VDUP1) is a candidate tumor suppressor, the expression of which is dramatically reduced in various tumor tissues. In this study, we found that VDUP1 expression is suppressed during human hepatic carcinogenesis, and mice lacking VDUP1 are much more susceptible to diethylnitrosamine-induced hepatocarcinogenesis compared with wild type mice. VDUP1-deficient tumors proliferated significantly more than wild type tumors and had corresponding changes in the expression of key cell cycle regulatory proteins. In addition, the hepatomitogen-induced response was associated with a considerable increase in the release of TNF-α and subsequent enhancement of NF-κB activation in VDUP1-deficient mice. When cells were treated with TNF-α, the VDUP1 level was markedly reduced, concomitant with elevated NF-κB activation. Furthermore, the overexpression of VDUP1 resulted in the robust suppression of TNF-α–activated NF-κB activity via association with HDAC1 and HDAC3. These results indicate that VDUP1 negatively regulates hepatocarcinogenesis by suppressing TNF-α–induced NF-κB activation.

Circadian Amplitude of Cryptochrome 1 Is Modulated by mRNA Stability Regulation via Cytoplasmic hnRNP D Oscillation

ABSTRACT The mammalian circadian rhythm is observed not only at the suprachiasmatic nucleus, a master pacemaker, but also throughout the peripheral tissues. Its conserved molecular basis has been thought to consist of intracellular transcriptional feedback loops of key clock genes. However, little is known about posttranscriptional regulation of these genes. In the present study, we investigated the role of the 3′-untranslated region (3′UTR) of the mouse cryptochrome 1 (mcry1) gene at the posttranscriptional level. Mature mcry1 mRNA has a 610-nucleotide 3′UTR and mediates its own degradation. The middle part of the 3′UTR contains a destabilizing cis-acting element. The deletion of this element led to a dramatic increase in mRNA stability, and heterogeneous nuclear ribonucleoprotein D (hnRNP D) was identified as an RNA binding protein responsible for this effect. Cytoplasmic hnRNP D levels displayed a pattern that was reciprocal to the mcry1 oscillation. Knockdown of hnRNP D stabilized mcry1 mRNA and resulted in enhancement of the oscillation amplitude and a slight delay of the phase. Our results suggest that hnRNP D plays a role as a fine regulator contributing to the mcry1 mRNA turnover rate and the modulation of circadian rhythm.

TXNIP Maintains the Hematopoietic Cell Pool by Switching the Function of p53 under Oxidative Stress

Reactive oxygen species (ROS) are critical determinants of the fate of hematopoietic stem cells (HSCs) and hematopoiesis. Thioredoxin-interacting protein (TXNIP), which is induced by oxidative stress, is a known regulator of intracellular ROS. Txnip(-/-) old mice exhibited elevated ROS levels in hematopoietic cells and showed a reduction in hematopoietic cell population. Loss of TXNIP led to a dramatic reduction of mouse survival under oxidative stress. TXNIP directly regulated p53 protein by interfering with p53- mouse double minute 2 (MDM2) interactions and increasing p53 transcriptional activity. Txnip(-/-) mice showed downregulation of the antioxidant genes induced by p53. Introduction of TXNIP or p53 into Txnip(-/-) bone marrow cells rescued the HSC frequency and greatly increased survival in mice following oxidative stress. Overall, these data indicate that TXNIP is a regulator of p53 and plays a pivotal role in the maintenance of the hematopoietic cells by regulating intracellular ROS during oxidative stress.

Rhythmic interaction between Period1 mRNA and hnRNP Q leads to circadian time-dependent translation.

ABSTRACT The mouse PERIOD1 (mPER1) protein, along with other clock proteins, plays a crucial role in the maintenance of circadian rhythms. 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 was controlled by an internal ribosomal entry site (IRES) element in the 5′ untranslated region (5′-UTR) of mPer1 mRNA along with the trans-acting factor mouse heterogeneous nuclear ribonucleoprotein Q (mhnRNP Q). Knockdown of mhnRNP Q 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 interactions between mPer1 mRNA and mhnRNP Q. Here, we demonstrate 5′-UTR-mediated rhythmic mPer1 translation and provide evidence for posttranscriptional regulation of the circadian rhythmicity of core clock genes.

hnRNP Q and PTB modulate the circadian oscillation of mouse Rev-erb α via IRES-mediated translation

The physiological and behavioral circadian rhythms of most creatures are controlled by a harmony of functional relationships between clock genes. In mammals, several core clock genes show rhythmic profiles of their mRNA and protein expression. Among them, Rev-erb α functions as a transcriptional repressor, affecting expression patterns of other clock genes. For the continuous and robust oscillation of the molecular clock system, the levels of Rev-erb α protein are expected to be tightly regulated with the correct timing. Here, we demonstrate that Rev-erb α has an internal ribosomal entry site (IRES) in its 5′ untranslated region. Furthermore, we demonstrate that heterogeneous nuclear ribonucleoprotein Q and polypyrimidine tract-binding protein (PTB) modulate the IRES-mediated translation of Rev-erb α. We suggest that the rhythmic binding affinity of hnRNP Q to the Rev-erb α IRES and the change in PTB cytosolic levels lead to maintenance of the oscillation profile of the Rev-erb α protein.

Thioredoxin-Interacting Protein Regulates Hematopoietic Stem Cell Quiescence and Mobilization under Stress Conditions

Hematopoietic stem cells (HSCs) are maintained in a quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signals. The mechanisms regulating the quiescence and mobilization of HSCs, however, remain unclear. In this study, we report that the expression of thioredoxin-interacting protein (TXNIP) is decreased during HSC activation. In Txnip−/− mice, the long-term reconstituting HSC population is decreased and exhausted, and its capacity to repopulate is rapidly lost. These effects are associated with hyperactive Wnt signaling, an active cell cycle, and reduced p21 expression under conditions of stress. TXNIP deficiency reduced the CXCL12- and osteopontin-mediated interaction between HSCs and the bone marrow, and impaired homing and retention in the osteoblastic niche, resulting in mobilized HSCs. Therefore, we propose that TXNIP is essential for maintaining HSC quiescence and the interaction between HSCs and the BM niche.