Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M511927200 on May 9, 2006

J. Biol. Chem., Vol. 281, Issue 28, 19100-19106, July 14, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/28/19100    most recent
M511927200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kwak, E.
Right arrow Articles by Kim, K.-T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kwak, E.
Right arrow Articles by Kim, K.-T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Essential Role of 3'-Untranslated Region-mediated mRNA Decay in Circadian Oscillations of Mouse Period3 mRNA*

Eunyee Kwak, Tae-Don Kim, and Kyong-Tai Kim1

From the Systems Bio-dynamics National Core Research Center, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Kyung-Buk 790-784, Republic of Korea

Received for publication, November 4, 2005 , and in revised form, April 27, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Daily oscillations in mRNA levels are a general feature of most clock genes. Although mRNA oscillations largely depend on transcriptional regulation, it has been suggested that post-transcriptional controls also contribute to mRNA oscillations in Drosophila. Currently, however, there is no direct evidence for post-transcriptional regulation of mammalian clock genes. To investigate the roles of post-transcriptional regulations, we focused on the 3'-untranslated region (3'-UTR) of mouse Period3 (mPer3) mRNA, one of the clock genes. Insertion of the entire mPer3 3'-UTR downstream of a reporter gene resulted in a dramatic decrease in mRNA stability. Deletion and point mutation analyses led to the identification of critical sequences responsible for mRNA decay. To explore the effects of the mPer3 3'-UTR-mediated mRNA decay on circadian oscillations, we established NIH3T3 stable cell lines that express luciferase mRNA with wild-type or mutant mPer3 3'-UTR. Interestingly, a stabilizing mutation of 3'-UTR induced a significant alteration in the oscillation profile of luciferase mRNA. Above all, the peak time, during which the mRNAs reached their highest levels, was significantly delayed (for 12 h). In addition, the luciferase mRNA level with mutant 3'-UTR began to increase earlier than that in the presence of wild-type 3'-UTR. Consequently, luciferase mRNA with mutant 3'-UTR displayed oscillation patterns with a prolonged rising phase. Our results indicate that mPer3 3'-UTR-mediated mRNA decay plays an essential role in mRNA cycling and provide direct evidence for post-transcriptional control of circadian mRNA oscillations.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Circadian rhythms, defined as daily behavioral and physiological oscillations, have been observed in a variety of organisms. These oscillations are driven by a self-sustained time-keeping system, the intracellular clock (1, 2). Over the past several years, great effort has been focused on understanding the molecular mechanisms that underlie the circadian clock and indispensable components, called clock genes, have been elucidated (3–8). In addition, it was revealed that the intracellular molecular clock consists of interacting positive and negative transcriptional and translational feedback loops that involve these components (9–12).

Daily oscillations in protein and/or mRNA levels are central features of clock genes (1, 12). As for the underlying mechanism of mRNA cycling, a number of studies have revealed that mRNA oscillations largely depend on transcriptional regulation (10, 13–17). In Drosophila, nevertheless, it has been suggested that post-transcriptional controls also contribute to mRNA oscillations. First, despite the absence of rhythmic transcription, period (per) mRNA showed circadian cycling in transgenic flies carrying promoter-less per gene (18). Second, results of a nuclear run-on assay suggested that post-transcriptional regulation is responsible for mRNA cycling (19).

In mammals, however, much less is known about the relevance of post-transcriptional control to the circadian oscillations of clock gene mRNAs. By computational modeling, it was demonstrated that circadian clocks depend on mRNA stability (20). These authors proposed that the changes in mRNA stability accounted for the time lag between mRNA and protein levels. In the case of the mouse Period1 (mPer1) gene, it was reported that the 3'-UTR2 of mPer1 mRNA repressed its own expression and that this translational repression could be involved in the generation of the same time lag (i.e. the time lag between mRNA and protein levels) (21). Currently, however, there is no experimental evidence for the involvement of post-transcriptional regulation in the circadian mRNA cycling of clock genes.

To investigate the role of post-transcriptional regulation in circadian mRNA cycling, we focused on the 3'-UTR of mouse Period3 (mPer3) mRNA, one of the mammalian clock genes that shows typical mRNA oscillations (22, 23). To obtain direct evidence for the role of the 3'-UTR, we established NIH3T3 stable cell lines that each express luciferase mRNA with wild-type or mutant mPer3 3'-UTR. Previous studies have shown that circadian clocks exist even in cultured fibroblasts and that either dexamethasone or serum shock can synchronize the circadian expression of a variety of clock genes (24–26). Since then, this cell system has been used as an experimental model to examine the molecular mechanisms of the mammalian circadian clock (27–29). In the present study, we demonstrate that the mPer3 3'-UTR regulates mRNA stability and that 3'-UTR-mediated mRNA decay plays an important role in circadian mRNA cycling.


Figure 1
View larger version (102K):
[in this window]
[in a new window]
  		FIGURE 1.
RNA sequence of mouse Period 3 3'-UTR. The first three nucleotides (boxed) represent the translation stop codon. Putative poly(A) additional signals are underlined. The boundaries of the deletion constructs, shown in Fig. 2A, are indicated by solid triangles. The asterisks represent the mutation sites of each of the mutant constructs, shown in Fig. 3B. Numbering begins at the first nucleotide, following the stop codon. The mPer3 3'-UTR sequence was deposited in GeneBankTM with accession number DQ196346.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
3'-Rapid Amplification of cDNA Ends (3'-RACE)—3'-RACE was performed with mouse suprachiasmatic nuclei cDNAs as templates. The sequences of the mPer3 specific forward primers were as follows: F1, 5'-TCATGGAGATGTGTGTGCCCCGG-3' and F2, 5'-CGGAATTCCTTGAAGAGGGGTAAGAGTTTGG-3'. The sequences of the reverse (dT)17-adaptor primer was 5'-GCTCTAGAGGCCTCGAGTTTTTTTTTTTTTTTTT-3'. The nested PCR product was digested with EcoRI and XbaI, and the fragment was ligated into EcoRI/XbaI sites of pBluescript SK(+) vector (Stratagene). Nucleotide sequences were determined by sequencing using ABI PRISM 3100 Genetic Analyzer System.

Plasmids—The pcNAT control vector was constructed using the pcDNA3.1/His C vector (Invitrogen). Hence, the arylalkylamine N-acetyltransferase (AANAT) coding region is followed by bovine growth hormone poly(A) signal without any specific 3'-UTR (30). For the pcNAT/1–2220 construct, full-length 3'-UTR of mPer3 mRNA was amplified with forward primer (5'-CGGAATTCGCAGCTGTAAAGATGTCACACCCC-3') and reverse primer (5'-GCTCTAGATTCCACTTGAACACATATACTGTTT-3'), digested with EcoRI/XbaI, and cloned into EcoRI/XbaI sites of pcNAT control vector.

To generate the deletion constructs, various regions of mPer3 3'-UTR were amplified using pcNAT/1–2220 as a template. The amplified fragments are shown in Fig. 2A. All of the PCR fragments are flanked by an EcoRI site at one end and an XbaI site at the other end. Following EcoRI/XbaI digestion, fragments were introduced into the corresponding restriction sites of pcNAT control vector. For the constructs of point mutations as shown in Fig. 3B, two-step PCR amplification was performed with following primers: for SDM-A former fragment (5'-CGGAATTCGCAGCTGTAAAGATGTCACACCCC-3' (forward) and 5'-AACATAAAAGGGGGAACGAGACGAAGGCCTGCGAC-3' (reverse)), for SDM-A latter fragment (5'-CGTCTCGTTCCCCCTTTTATGTTGTTACAAGATCA-3' (forward) and 5'-GCTCTAGATTCCACTTGAACACATATACTGTTT-3' (reverse)), for SDM-B former fragment (5'-CGGAATTCGCAGCTGTAAAGATGTCACACCCC-3' (forward) and 5'-A-CAACATGGGGGGGCAAACGAGACGAAGGCCTGC-3' (reverse)), for SDM-B latter fragment (5'-CGTTTGCCCCCCCATGTTGTTACAAGATCACTTTT-3' (forward) and 5'-GCTCTAGATTCCACTTGAACACATATACTGTTT-3' (reverse)). The second PCR fragment was digested and cloned into EcoRI/XbaI sites of pcNAT control vector.

To generate D-box-Luc/WT-3'-UTR/Neo vector, pGL3 control vector (Promega) was digested with XbaI and filled up with T4 DNA polymerase (Roche Applied Science). The full-length 3'-UTR of mPer3 mRNA was amplified by PCR, inserted into T4 DNA polymerase treated pGL3 vector, and designated pGL3/3'-UTR. D-box containing sequences of mPer3 promoter (10) as shown in Fig. 4B were inserted into KpnI/XmaI sites of pGL3/3'-UTR vector and named D-box-Luc/3'-UTR vector. The neomycin-resistant gene preceded by the thymidine kinase promoter was amplified from pMC1neo poly(A) vector (Stratagene) with forward primer (5'-GCTCTAGAGCAGTGTGGTTTTGCAAGAGGAA-3') and reverse pri-mer (5'-CAGGTCGACGGATCCGAACAAACG-3'). Following XbaI/SalI digestion, the fragment was cloned into XbaI/SalI sites of D-box-Luc/3'-UTR vector. For D-box-Luc/SDM-3'-UTR/Neo vector, mutant 3'-UTR (SDM-A) was used instead of wild-type mPer3 3'-UTR.

Cell Culture—HEK 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. CHO-K1 cells were cultured in DMEM/nutrient mixture F-12 (DMEM/F-12) supplemented with 10% bovine calf serum and 1% penicillin-streptomycin. NIH3T3 cells were cultured in DMEM supplemented with 10% bovine calf serum and 1% penicillin-streptomycin.

Transient Transfection—HEK 293T cells were seeded in 6-well plates at a density of 2 x 105 cells per well 1 day prior to transfection. Transfection was carried out using Metafectene (Biontex) according to the manufacturer's instructions. After incubation for 17 h, cells were harvested or further incubated with 5 µg of actinomycin D/ml for 6 h before harvest. For reporter mRNA decay kinetics, CHO-K1 cells were transiently co-transfected with 0.5 µg of pcNAT control vector and 2.5 µg of reporter plasmid (pcNAT/1–2220, pcNAT/1–2220(SDM-A), or pcNAT/1–2220(SDM-B)) by the electroporation method (30) at room temperature. After incubation for 5 h, cells were harvested or further incubated for indicated time with 5 µg of actinomycin D/ml before harvest.

Generation of Stable Cell Lines and Dexamethasone Shock Procedure—To generate NIH-Luc-WT 3'-UTR and NIH-Luc-SDM 3'-UTR cell lines, D-box-Luc/WT-3'-UTR/Neo vector, and D-box-Luc/SDM-3'-UTR/Neo vector were introduced to NIH3T3 fibroblasts by calcium phosphate precipitation method, respectively. After 2 days, we started selection with 800 µg of G418 (Invitrogen)/ml. Neomycin-resistant clones were isolated by standard procedure. The resulting cell lines were maintained in DMEM supplemented with 10% bovine calf serum, 1% penicillin-streptomycin, and 200 µg of G418/ml.

The dexamethasone shock was done as described previously, with minor modifications (24). In brief, ~1.5 x 105 cells were seeded in each well of 12-well plate. When the cells reached confluence, the medium was exchanged with 100 nM dexamethasone containing medium. After 2 h, this medium was replaced with complete medium. At the indicated time, cells were harvested and kept at –70 °C until total RNA was extracted from the cells.

Northern Blot Analysis—Northern blot analysis was carried out as described previously (30). In brief, total RNA was isolated by using TRI Reagent (Molecular Research Center) according to the manufacturer's instructions. Five micrograms of total RNA was size-separated by 1% agarose gel electrophoresis containing 0.66 M formaldehyde, transferred to nylon membranes (ICN), and hybridized with a randomly primed probe. For detection of reporter mRNA and loading control, rat AANAT (GeneBankTM accession number NM_012818) and ribosomal protein L32 (GeneBankTM accession number NM_000994) coding regions were used as probes, respectively. Radioactivity was measured by autoradiography.


Figure 2
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 2.
The mPer3 3'-UTR mediates mRNA degradation. A, schematic diagram of the pcNAT control vector and of chimeric reporters containing either the full-length mPer3 3'-UTR or its fragment. B, HEK 293T cells were transiently transfected with the pcNAT control vector (left panel) or the pcNAT/1–2220 reporter (right panel). After incubation for 17 h, cells were harvested (Act.D 0 h) or further incubated with 5 µg of actinomycin D/ml for 6 h before harvest (Act.D 6 h). Reporter mRNA levels were determined by Northern blot analysis (Northern) using the AANAT-coding region as a probe. Both 28 and 18 S rRNAs were used as loading controls and stained with ethidium bromide.

 
Quantitative Real-time RT-PCR—Quantitative real-time RT-PCR was performed as described previously (31). In brief, total RNA was isolated by using TRI Reagent (Molecular Research Center). RNA was reverse transcribed by using Moloney murine leukemia virus reverse transcriptase (Roche Applied Science) according to the manufacturer's instructions. For detection and quantification, MyiQTM real-time PCR detection system (Bio-Rad) was used. The sequences of the forward and reverse primers were as follows: endogenous mPer3, 5'-TTGTCAGGTTGGCCTTCTCT-3' and 5'-GGCATCCTAGCAGAGGTGAG-3'; luciferase, 5'-GAGGTTCCATCTGCCAGGTA-3' and 5'-CACACAGTTCGCCTCTTTGA-3'; TATA-binding protein (TBP), 5'-CAGCCTTCCACCTTATGCTC-3' and 5'-TTGCTGCTGCTGTCTTTGTT-3'.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The mPer3 3'-UTR Regulates mRNA Stability—We cloned the 3'-UTR of mPer3 mRNA by 3'-RACE. Sequence analysis of the cloned cDNA showed that the mPer3 3'-UTR was composed of 2,220 nucleotides, contained a peculiar, U-rich region in the middle of the 3'-UTR, and included three putative poly(A) signals (Fig. 1). When the mPer3 3'-UTR sequence was aligned in the corresponding loci of the genome, the sequence agreed with the mouse genome sequence (data not shown). The mPer3 3'-UTR sequence was deposited in GeneBankTM with accession number DQ196346.


Figure 3
View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 3.
Deletion and mutational analysis of a critical cis-acting element. A, HEK 293T cells were transiently transfected with various deletion constructs. After incubation for 17 h, cells were harvested (Act.D 0 h) or further incubated with 5 µg of actinomycin D/ml for 6 h before harvest (Act.D 6 h). Reporter mRNA levels were determined by Northern blot analysis (Northern). For the detection of reporter mRNAs and loading controls, both AANAT (NAT)- and ribosomal protein L32 (RPL32)-coding regions were used as probes, respectively. Signal intensities were quantified using a PhosphorImager, normalized to RPL32 signal intensity, and indicated as relative values. For each transfection condition, the NAT/RPL32 ratio at time 0 was set to 1.00. B and C, the mRNA decay kinetics of wild-type and mutant reporters are shown. CHO-K1 cells were transiently co-transfected with the pcNAT control vector and pcNAT/1–2220, pcNAT/1–2220(SDM-A), or pcNAT/1–2220(SDM-B) reporter plasmids. Mutated nucleotides of pcNAT/1–2220(SDM-A) and pcNAT/1–2220(SDM-B) reporters are depicted in bold and underlined. Reporter mRNA decay rates were determined by actinomycin D (Act.D) chase experiments and Northern blot analyses using the AANAT-coding region as a probe. Northern blot data are representative of two experiments. Signal intensities were quantified using a PhosphorImager, normalized to NAT control mRNA signal intensity, and plotted as a percentage of the initial value versus time.

 
To examine the function of the mPer3 3'-UTR, a reporter construct (pcNAT/1–2220) was created by inserting the mPer3 3'-UTR downstream from the AANAT-coding sequence of the pcNAT control vector (30) (Fig. 2A). HEK 293T cells were transiently transfected with the pcNAT/1–2220 reporter or the pcNAT control vector and treated with actinomycin D for the indicated times to block new transcription. The effects of the mPer3 3'-UTR were determined by Northern blot analysis. As shown in Fig. 2B, the reporter mRNA with the mPer3 3'-UTR insertion (right panel) was rapidly degraded, whereas control mRNA lacking the mPer3 3'-UTR insertion (left panel) was sufficiently stable to maintain consistent mRNA levels, even after 6 h of actinomycin D treatment. This result suggests that the mPer3 3'-UTR mediates mRNA degradation.

Deletion and Mutational Analyses of a Critical cis-Acting Element—To identify the region critical for mRNA degradation, various reporter constructs containing serially deleted fragments of the mPer3 3'-UTR were generated (Fig. 2A). The effects of these 3'-UTR fragments were analyzed by Northern blot analysis. Compared with the full-length 3'-UTR (pcNAT/1–2220), truncation of the distal region of the 3'-UTR to nucleotides 1164, 741, or 561 had no significant effect on mRNA stability (Fig. 3A, see pcNAT/1–1164, pcNAT/1–741, and pcNAT/1–561, respectively). Interestingly, the additional removal of nucleotides 382–561 caused a dramatic increase in mRNA stability (see pcNAT/1–381). Greater than 80% of the initial mRNA amount was present even after 6 h of actinomycin D treatment. To address whether the fragment 382–561 itself shows mRNA decay activity, we generated pcNAT/382–561 reporter construct and analyzed the effect on mRNA stability (Fig. 3A, right panel). Although the effect was weaker than that of full-length 3'-UTR (1–2220), the fragment 382–561 still showed mRNA decay activity. That is, more than 40% of the initial mRNA was degraded after 6 h of actinomycin D treatment.

We then sought to identify critical sequences within nucleotides 382–561. Since most of the protein bindings against mPer3 3'-UTR were competed out by homopolymeric poly(U) nucleotides (data not shown), we decided to mutate U-rich sequences between nucleotides 382 and 561. We generated two mutant reporter constructs, pcNAT/1–2220(SDM-A) and pcNAT/1–2220(SDM-B), in which four and six nucleotides of the mPer3 3'-UTR were substituted with cytidines, respectively (Fig. 3B).


Figure 4
View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 4.
The mPer3 3'-UTR plays an essential role in circadian mRNA oscillations. A, temporal expression profiles of endogenous mPer3 mRNA are shown from NIH3T3 fibroblasts after dexamethasone (Dex) shock. When dexamethasone (Dex) was treated, the time was set to zero. The mPer3 mRNA level was determined by quantitative real-time RT-PCR and normalized to TBP mRNA levels. Initial levels of mPer3 mRNA were arbitrarily set to 1.0. Each point presented is a mean ± S.E. of three experiments. B, schematic diagram of reporter genes expressed in NIH-Luc-WT 3'-UTR or NIH-Luc-SDM 3'-UTR cell lines is shown. The D-box-containing region of the mPer3 promoter was fused to the SV40 basic promoter (SV40). D-box sequences are underlined. C, temporal expression profiles of luciferase mRNA with wild-type mPer3 3'-UTR are shown. The results were obtained from two independent cell lines. D, temporal expression profiles of luciferase mRNA with mutant mPer3 3'-UTR are shown. The results were obtained from two independent cell lines. C and D, reporter mRNA levels were determined by quantitative real-time RT-PCR and normalized to TBP mRNA levels. Initial levels of each reporter mRNA were arbitrarily set to 1.0. Each point presented is a mean ± S.E. of three experiments.

 
To investigate the effects of these mutations, we examined the kinetics of reporter mRNA degradation by actinomycin D chase experiment and Northern blot analysis (Fig. 3, B and C). Consistent with the results shown in Fig. 2B, wild-type reporter mRNA was rapidly degraded (t1/2 of ~3 h). However, SDM-A mutant mRNA was significantly stable (t1/2 > 8 h), maintaining 58% of its initial levels even after 8 h of actinomycin D treatment. These results demonstrate that the mutated sequences in the SDM-A reporter comprise a cis-acting element important for mRNA decay. Interestingly, despite the existence of overlapping mutation sites, SDM-B mutant mRNA showed completely different degradation kinetics from that of SDM-A mutant mRNA. The SDM-B mutant mRNA was rapidly degraded (t1/2 of ~3.5 h) with a decay rate similar to that of wild-type reporter mRNA.

mPer3 3'-UTR-mediated mRNA Decay Contributes to Circadian mRNA Oscillations—Before analyzing the effects of the mPer3 3'-UTR on circadian mRNA oscillations, we first examined the circadian oscillation profile of endogenous mPer3 mRNA in NIH3T3 mouse fibroblasts. After dexamethasone shock, mPer3 mRNA showed robust circadian rhythms (Fig. 4A). The mRNA levels rapidly decreased immediately after dexamethasone shock and began to increase after 18 h. After reaching their peak level at 30 h, mRNA levels began to decline again.

To explore the roles of mPer3 3'-UTR-mediated mRNA decay in circadian oscillations, we established NIH3T3 stable cell lines that express luciferase mRNA with wild-type or SDM-A mutant mPer3 3'-UTR and designated them NIH-Luc-WT 3'-UTR or NIH-Luc-SDM 3'-UTR cell lines, respectively (Fig. 4B). The expression of the luciferase reporters was regulated by D-box elements of the mPer3 promoter, which were previously verified to be sufficient for the generation of circadian oscillations (10).

As shown in Fig. 4C, luciferase mRNAs with wild-type mPer3 3'-UTR showed similar oscillation profiles to endogenous mPer3 mRNA. The levels of luciferase mRNA began to increase after 18 h of dexamethasone shock. After reaching their peak levels at 30 h, mRNA levels started to decline. We confirmed these oscillation profiles in two different NIH-Luc-WT 3'-UTR cell lines (i.e. #1 and #9). Interestingly, the SDM-A mutation of the mPer3 3'-UTR led to dramatic alterations in these oscillation patterns (Fig. 4D, #3 and #4). Above all, the peak time, during which the mRNAs reached their highest levels, was significantly delayed (for 12 h). In addition, the mRNA levels began to increase earlier than that with wild-type 3'-UTR, and the rising phase continued until 42 h. Consequently, luciferase mRNAs with mutant 3'-UTR showed oscillation patterns with a prolonged rising phase. Based on these results, we concluded that the mPer3 3'-UTR-mediated mRNA decay plays an essential role in mRNA cycling.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Most clock genes show circadian oscillations in protein and/or mRNA levels (1, 12). Although protein oscillations can be generated without mRNA cycling (32), there is no doubt that the precision of the protein rhythm is accomplished by appropriate control of the mRNA levels (33).

In this study, we sought to obtain direct evidence for the relevance of post-transcriptional regulation to the mRNA oscillations of mammalian clock genes. To study rhythmic regulation of mPer3 mRNA levels, we focused on the function of the 3'-UTR. The mPer3 3'-UTR induced dramatic mRNA decay when inserted immediately downstream from a reporter gene. We cannot exclude the possibility that the mPer3 3'-UTR plays additional roles in other post-transcriptional events, such as splicing or translation. Nevertheless, it is obvious that the 3'-UTR modulates mRNA stability since, despite similar initial levels, reporter mRNA that is fused to the mPer3 3'-UTR (WT) is more rapidly degraded than is NAT control mRNA (Fig. 3B, upper panel).

Although it is clear that the mutated sequences in the SDM-A mutant are important for mRNA decay, it should be noted that the mPer3 3'-UTR could have additional cis-acting elements for the regulation of mRNA stability, given that SDM-A mutant mRNA is still more unstable than NAT control mRNA (Fig. 3B, middle panel, and C). Furthermore, even when nucleotides 1–1164 were deleted, the remaining 3'-UTR exhibited mRNA decay activity, suggesting that nucleotides 1165–2220 contain additional cis-acting elements involved in mRNA stability (data not shown).

The relevance of the 3'-UTR to circadian behavior was previously studied in the Drosophila per gene (33). Replacement of the per 3'-UTR with the tubulin 3'-UTR altered the accumulation profile of per mRNA. Interestingly, most of the per 3'-UTR could be deleted without severely altering the circadian rhythm; the authors suggested that the regulatory elements for post-transcriptional control may be located in a different region of the per mRNA. Supporting this idea, Stanewsky et al. (34) revealed that the per 5'-UTR contains a regulatory element required for post-transcriptional control, although they did not demonstrate which post-transcriptional events are regulated by the per 5'-UTR. Our results suggest that, in contrast to the Drosophila per gene, mPer3 3'-UTR plays an essential role in circadian mRNA oscillations. In view of the finding that a stabilizing mutation of 3'-UTR induced dramatic alterations in mRNA cycling, we propose that precise regulation of mRNA stability is essential for the control of mPer3 mRNA oscillations.

Although the oscillation profile of the luciferase mRNA with wild-type 3'-UTR was similar to that of endogenous mPer3 mRNA, it should be noted that the two profiles were not identical. First, this discrepancy could be the result of a different promoter structure. In our study, transcription of luciferase mRNA is driven by a D-box fused SV40 promoter. While the D-box is sufficient for circadian oscillations (10), we cannot exclude the possibility that additional regulatory sequences are required for precise control of transcription. Second, it is possible that the 5'-UTR or coding region of mPer3 mRNA contributes to mRNA cycling. As mentioned above, 5'-UTR-mediated post-transcriptional regulation is involved in circadian mRNA oscillations of the Drosophila per gene. In this respect, it will be interesting to explore the contribution of the mPer3 5'-UTR to post-transcriptional regulation as well as to circadian mRNA oscillations.

In conclusion, our study demonstrated that the mPer3 3'-UTR mediated mRNA degradation and that the stabilizing mutation of the mPer3 3'-UTR caused a dramatic change in the oscillations of reporter mRNA. Based on these results, we propose that 3'-UTR-mediated mRNA decay plays an essential role in the circadian oscillations of mPer3 mRNA.


   FOOTNOTES
 
* This work was supported by the Brain Neurobiology Research Program (M10412000023-02310) and National Core Research Center for Systems Bio-Dynamics (R15-2004-033) sponsored by the Korean Ministry of Science and Technology. This work was also supported by the basic research grant from the Korea Science & Engineering Foundation (KOSEF) and Brain Korea 21 Program of the Koran Ministry of Education. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) DQ196346. Back

1 To whom correspondence should be addressed: Dept. of Life Science, Pohang University of Science and Technology, San 31, Hyoja Dong, Pohang, Kyung-Buk 790-784, Republic of Korea. Tel.: 82-54-279-2297; Fax: 82-54-279-2199; E-mail: ktk{at}postech.ac.kr.

2 The abbreviations used are: UTR, untranslated region; mPer3, mouse Period3; SDM, site-directed mutagenesis; AANAT, arylalkylamine N-acetyltransferase; RACE, rapid amplification of cDNA ends; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcription; TBP, TATA-binding protein; WT, wild-type. Back


   ACKNOWLEDGMENTS
 
We thank Dae-Cheong Ha and Kyung-Chul Woo for critical discussions and experimental assistance, respectively.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bell-Pedersen, D., Cassone, V. M., Earnest, D. J., Golden, S. S., Hardin, P. E., Thomas, T. L., and Zoran, M. J. (2005) Nat. Rev. Genet. 6, 544–556[CrossRef][Medline] [Order article via Infotrieve]
  2. Reppert, S. M., and Weaver, D. R. (2002) Nature 418, 935–941[CrossRef][Medline] [Order article via Infotrieve]
  3. Vitaterna, M. H., King, D. P., Chang, A. M., Kornhauser, J. M., Lowrey, P. L., McDonald, J. D., Dove, W. F., Pinto, L. H., Turek, F. W., and Takahashi, J. S. (1994) Science 264, 719–725[Abstract/Free Full Text]
  4. Bunger, M. K., Wilsbacher, L. D., Moran, S. M., Clendenin, C., Radcliffe, L. A., Hogenesch, J. B., Simon, M. C., Takahashi, J. S., and Bradfield, C. A. (2000) Cell 103, 1009–1017[CrossRef][Medline] [Order article via Infotrieve]
  5. Albrecht, U., Zheng, B., Larkin, D., Sun, Z. S., and Lee, C. C. (2001) J. Biol. Rhythms 16, 100–104[Abstract/Free Full Text]
  6. Kume, K., Zylka, M. J., Sriram, S., Shearman, L. P., Weaver, D. R., Jin, X., Maywood, E. S., Hastings, M. H., and Reppert, S. M. (1999) Cell 98, 193–205[CrossRef][Medline] [Order article via Infotrieve]
  7. Preitner, N., Damiola, F., Lopez-Molina, L., Zakany, J., Duboule, D., Albrecht, U., and Schibler, U. (2002) Cell 110, 251–260[CrossRef][Medline] [Order article via Infotrieve]
  8. Sato, T. K., Panda, S., Miraglia, L. J., Reyes, T. M., Rudic, R. D., McNamara, P., Naik, K. A., FitzGerald, G. A., Kay, S. A., and Hogenesch, J. B. (2004) Neuron 43, 527–537[CrossRef][Medline] [Order article via Infotrieve]
  9. Reppert, S. M., and Weaver, D. R. (2001) Annu. Rev. Physiol. 63, 647–676[CrossRef][Medline] [Order article via Infotrieve]
  10. Ueda, H. R., Hayashi, S., Chen, W., Sano, M., Machida, M., Shigeyoshi, Y., Iino, M., and Hashimoto, S. (2005) Nat. Genet. 37, 187–192[CrossRef][Medline] [Order article via Infotrieve]
  11. Hardin, P. E. (2004) J. Biol. Rhythms 19, 348–360[Abstract/Free Full Text]
  12. Reppert, S. M., and Weaver, D. R. (2000) J. Biol. Rhythms 15, 357–364[Free Full Text]
  13. Hardin, P. E., Hall, J. C., and Rosbash, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11711–11715[Abstract/Free Full Text]
  14. Darlington, T. K., Lyons, L. C., Hardin, P. E., and Kay, S. A. (2000) J. Biol. Rhythms 15, 462–471[Abstract/Free Full Text]
  15. Wang, G. K., Ousley, A., Darlington, T. K., Chen, D., Chen, Y., Fu, W., Hickman, L. J., Kay, S. A., and Sehgal, A. (2001) J. Neurobiol. 47, 161–175[CrossRef][Medline] [Order article via Infotrieve]
  16. Yamamoto, T., Nakahata, Y., Soma, H., Akashi, M., Mamine, T., and Takumi, T. (2004) BMC Mol. Biol. 5, 18[CrossRef][Medline] [Order article via Infotrieve]
  17. Yoo, S. H., Ko, C. H., Lowrey, P. L., Buhr, E. D., Song, E. J., Chang, S., Yoo, O. J., Yamazaki, S., Lee, C., and Takahashi, J. S. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 2608–2613[Abstract/Free Full Text]
  18. Frisch, B., Hardin, P. E., Hamblen-Coyle, M. J., Rosbash, M., and Hall, J. C. (1994) Neuron 12, 555–570[CrossRef][Medline] [Order article via Infotrieve]
  19. So, W. V., and Rosbash, M. (1997) EMBO J. 16, 7146–7155[CrossRef][Medline] [Order article via Infotrieve]
  20. Forger, D. B., and Peskin, C. S. (2004) J. Theor. Biol. 230, 533–539[CrossRef][Medline] [Order article via Infotrieve]
  21. Kojima, S., Hirose, M., Tokunaga, K., Sakaki, Y., and Tei, H. (2003) Biochem. Biophys. Res. Commun. 301, 1–7[CrossRef][Medline] [Order article via Infotrieve]
  22. Zylka, M. J., Shearman, L. P., Weaver, D. R., and Reppert, S. M. (1998) Neuron 20, 1103–1110[CrossRef][Medline] [Order article via Infotrieve]
  23. Takumi, T., Taguchi, K., Miyake, S., Sakakida, Y., Takashima, N., Matsubara, C., Maebayashi, Y., Okumura, K., Takekida, S., Yamamoto, S., Yagita, K., Yan, L., Young, M. W., and Okamura, H. (1998) EMBO J. 17, 4753–4759[CrossRef][Medline] [Order article via Infotrieve]
  24. Balsalobre, A., Damiola, F., and Schibler, U. (1998) Cell 93, 929–937[CrossRef][Medline] [Order article via Infotrieve]
  25. Ueda, H. R., Chen, W., Adachi, A., Wakamatsu, H., Hayashi, S., Takasugi, T., Nagano, M., Nakahama, K., Suzuki, Y., Sugano, S., Iino, M., Shigeyoshi, Y., and Hashimoto, S. (2002) Nature 418, 534–539[CrossRef][Medline] [Order article via Infotrieve]
  26. Akashi, M., and Nishida, E. (2000) Genes Dev. 14, 645–649[Abstract/Free Full Text]
  27. Nagoshi, E., Saini, C., Bauer, C., Laroche, T., Naef, F., and Schibler, U. (2004) Cell 119, 693–705[Medline] [Order article via Infotrieve]
  28. Yagita, K., Tamanini, F., van Der Horst, G. T., and Okamura, H. (2001) Science 292, 278–281[Abstract/Free Full Text]
  29. Yamamoto, Y., Yagita, K., and Okamura, H. (2005) Mol. Cell. Biol. 25, 1912–1921[Abstract/Free Full Text]
  30. Kim, T. D., Kim, J. S., Kim, J. H., Myung, J., Chae, H. D., Woo, K. C., Jang, S. K., Koh, D. S., and Kim, K. T. (2005) Mol. Cell. Biol. 25, 3232–3246[Abstract/Free Full Text]
  31. Lee, J. H., and Kim, K. T. (2004) J. Neurochem. 91, 634–647[CrossRef][Medline] [Order article via Infotrieve]
  32. Cheng, Y., and Hardin, P. E. (1998) J. Neurosci. 18, 741–750[Abstract/Free Full Text]
  33. Chen, Y., Hunter-Ensor, M., Schotland, P., and Sehgal, A. (1998) J. Biol. Rhythms 13, 364–379[Abstract/Free Full Text]
  34. Stanewsky, R., Lynch, K. S., Brandes, C., and Hall, J. C. (2002) J. Biol. Rhythms 17, 293–306[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
K.-C. Woo, T.-D. Kim, K.-H. Lee, D.-Y. Kim, W. Kim, K.-Y. Lee, and K.-T. Kim
Mouse period 2 mRNA circadian oscillation is modulated by PTB-mediated rhythmic mRNA degradation
Nucleic Acids Res., January 1, 2009; 37(1): 26 - 37.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
E. Yakir, D. Hilman, M. Hassidim, and R. M. Green
CIRCADIAN CLOCK ASSOCIATED1 Transcript Stability and the Entrainment of the Circadian Clock in Arabidopsis
Plant Physiology, November 1, 2007; 145(3): 925 - 932.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
T.-D. Kim, K.-C. Woo, S. Cho, D.-C. Ha, S. K. Jang, and K.-T. Kim
Rhythmic control of AANAT translation by hnRNP Q in circadian melatonin production
Genes & Dev., April 1, 2007; 21(7): 797 - 810.
[Abstract] [Full Text] [PDF]

Home page
 Cold Spring Harb Symp Quant BiolHome page
E. Garbarino-Pico and C. B. Green
Posttranscriptional Regulation of Mammalian Circadian Clock Output
Cold Spring Harb Symp Quant Biol, January 1, 2007; 72(0): 145 - 156.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/28/19100    most recent
M511927200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kwak, E.
Right arrow Articles by Kim, K.-T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kwak, E.
Right arrow Articles by Kim, K.-T.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement