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. 2010 Jan;30(1):197-205.
doi: 10.1128/MCB.01154-09.

Circadian amplitude of cryptochrome 1 is modulated by mRNA stability regulation via cytoplasmic hnRNP D oscillation

Kyung-Chul Woo  1 , Dae-Cheong Ha, Kyung-Ha Lee, Do-Yeon Kim, Tae-Don Kim, Kyong-Tai Kim
Affiliations

Circadian amplitude of cryptochrome 1 is modulated by mRNA stability regulation via cytoplasmic hnRNP D oscillation

Kyung-Chul Woo et al. Mol Cell Biol. 2010 Jan.

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.

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Figures

FIG. 1.
FIG. 1.
mcry1 3′UTR contains a destabilizing element. (A) Oscillation of endogenous mcry1 mRNA in NIH 3T3 cells was measured by quantitative real-time PCR. NIH 3T3 cells were treated with 100 nM dexamethasone (Dex.) twice for 2 h, at −24 h and 0 h, to synchronize the circadian oscillation. Total RNA (1 μg) from each time point was subjected to quantitative real-time RT-PCR with specific oligonucleotides for mcry1 and mtbp (TATA box binding protein, used as a control for normalization). Declining and rising phases are indicated. All results are expressed as the mean ± standard error of the mean of the results of three independent experiments. (B) mRNA degradation kinetics were measured during both declining (closed squares) and rising (open squares) phases within a 1-day cycle after dexamethasone treatment. Cells were treated with 5 μM actinomycin D (Act. D) at 6 h during the declining phase and at 18 h during the rising phase and then harvested at 3-h intervals. Total RNA (1 μg) was subjected to quantitative real-time RT-PCR with specific primer pairs against mcry1 or mrpl32, used as a control for normalization. All results are expressed as the mean ± standard error of the mean of the results of three independent experiments. First-order decay constants and mRNA half-lives were calculated from the formula for each fitted line. (C) RNA sequence of the mcry1 3′UTR (immediately after the stop codon). Nucleotides 201 to 400 of the 3′UTR are shown in bold font. Underlining indicates the sequences of competitive oligonucleotides used in later experiments. Gray font shows poly(U) tracts. Italicized bold font shows a putative poly(A) signal. (D) Change of reporter mRNA levels after actinomycin D (Act. D) treatment. HEK 293T cells were transiently transfected with the pcNAT-wt610 and pcNAT reporters and incubated with actinomycin D for the times indicated. The reporter mRNA levels were analyzed by Northern blot analysis, using a specific probe for Aanat mRNAs. Ethidium bromide-stained 18S rRNA was used as a loading control.
FIG. 2.
FIG. 2.
U tracts within the mcry1 3′UTR affect mRNA stability. (A) Diagram of reporter constructs containing the full-length or serially deleted mcry1 3′UTR, with numbers indicating the positions of the starting nucleotide within the 3′UTR. CMV, cytomegalovirus promoter; NAT, AANAT open reading frame. (B) NIH 3T3 cells were transiently transfected by electroporation with the NAT reporter or pGL3 control or the NAT reporter fused to serially deleted mcry1 3′UTR constructs (shown in the schematic in panel A) or pGL3 control. Transfected cells were incubated for 12 h before treatment with 5 μM actinomycin D (Act. D). Total RNA (1 μg) from the time points indicated (0 h or 6 h after treatment with 5 μM actinomycin D) was subjected to quantitative real-time RT-PCR with specific oligonucleotides for Aanat, fluc (a control for transfection efficiency), and mrpl32 (a control used for normalization). All results are expressed as the mean ± standard error of the mean of the results of three independent experiments. First-order decay constants (const.) and mRNA half-lives were calculated from the formula for each fitted line. (C) Each 3′UTR, transcribed in vitro with [α-32P]-rUTP, was subjected to an in vitro binding and UV cross-linking assay with HEK 293T cytoplasmic extract (HEK_cyto). The arrows indicate the proteins that show differential binding. Molecular sizes in kDa are shown on the left. +, present. (D) The radiolabeled 3′UTR was subjected to an in vitro binding and UV cross-linking assay with HEK 293T cytoplasmic extract (HEK_cyto) with or without the deoxyoligonucleotide competitors (1 μM), indicated above the gel by the positions of the first nucleotide of the target sequences (target sequences are shown in Fig. 1C). The arrows indicate the proteins that show differential binding. Molecular sizes in kDa are shown on the left. +, present.
FIG. 3.
FIG. 3.
hnRNP D binds to the mcry1 3′UTR. (A) In vitro binding/UV cross-linking/immunoprecipitation were performed with the full-length 3′UTR as described in Materials and Methods. In the experiment whose results are shown in lane 1, the reaction mixture was coincubated with 1 μM c. oligo-252, which binds specifically to the changed bands. Lanes 4 and 5 show immunoprecipitation (IP) with monoclonal anti-hnRNP D antibody or rabbit immunoglobulin G (IgG), as a control, respectively. The arrows indicate hnRNP D, and the asterisk indicates unidentified proteins binding to the hnRNP D complex. Molecular sizes in kDa are shown on the left. +, present. (B) The in vitro-transcribed 3′UTR construct was labeled with biotin-UTP and incubated with HEK 293T cell cytoplasmic extract (HEK_cyto). Streptavidin-affinity-purified samples were separated by SDS-PAGE and subjected to Western blotting with anti-hnRNP D antibody or anti-PTB, as a negative control. hnRNP D was detected with biotin-labeled mRNA (lane 3). hnRNP D binding decreased in the presence of a threefold excess of unlabeled wild-type 3′UTR mRNA (wt610; lane 4) or when 1 μM of the competitor deoxyoligonucleotide c. oligo-252 was added (lane 5), but PTB was not detected. Molecular sizes in kDa are shown on the right. +, present.
FIG. 4.
FIG. 4.
Cytoplasmic hnRNP D oscillates and binds to mcry1 3′UTR with a rhythmic profile. (A) NIH 3T3 cells were treated with 100 nM dexamethasone for 2 h and then harvested at the indicated times, and cytoplasmic extract (cyto) was prepared for Western blot analysis. Cytoplasmic hnRNP D was detected with monoclonal hnRNP D antibody, and 14-3-3ζ was included as an internal control. RNA polymerase II (RNA Pol. II), used for a nuclear marker, was not detected in cytoplasmic extract but appeared in the nuclear fraction (Nuc.). The results shown are representative of three independent experiments. (B) The same cytoplasmic extracts used for the experiments whose results are shown in panel A were subjected to an in vitro binding assay (IVBA) with radiolabeled 3′UTR mRNA. The arrow indicates cytoplasmic hnRNP D binding to 3′UTR mRNA (upper gel). 14-3-3ζ was used as an internal control to demonstrate that the same amount of extract was used for all time points (lower gel). hnRNP D binding intensities were quantified by normalization (black bars) to 14-3-3ζ levels in the same extracts (gray and white bars in lower graph). The error bars represent the standard errors of the means of the results. The results shown are representative of three independent experiments. sync., synchronization. IB, immunoblot; A.U., arbitrary unit. (C) Relative levels of cytoplasmic (cyto.) hnRNP D, normalized to 14-3-3ζ levels, were calculated and plotted (closed ovals/dotted line) for the second day after treatment. Relative levels of endogenous mcry1 mRNA (closed squares/solid line) were also quantified, using mtbp as an internal control for each time point. Relative degradation rates of mcry1 mRNA were calculated for each time point for 4 h after actinomycin D treatment; mgapdh was used as an internal control for each value (open diamonds/dashed line). The results shown are representative of three independent experiments. Error bars represent the standard errors of the means of the results. sync., synchronization.
FIG. 5.
FIG. 5.
hnRNP D modulates mcry1 stability and circadian rhythm. (A) NIH 3T3 cells were transiently transfected with a pcNAT reporter containing the mcry1 3′UTR, β-galactosidase as a control used for normalization, and siRNA for knockdown of endogenous hnRNP D (sihnD) or control siRNA (con-si) and then incubated for 24 h, followed by treatment with 5 μM actinomycin D (Act. D). Total RNA (1 μg) was reverse transcribed using oligo(dT) primer and then quantified by real-time PCR (graph). Each value was normalized to the level of mrpl32. Open or closed squares indicate control siRNA or siRNA against hnRNP D, respectively. The error bars represent the standard errors of the means of the results of three independent experiments. Reduction of the hnRNP D protein level in total cell lysate (TCL) by siRNA compared to the level of 14-3-3ζ (used as an internal control) was validated by Western blotting (gel). +, present. (B) Effect of hnRNP D depletion on endogenous mcry1 mRNA oscillation. NIH 3T3 cells were transfected with control siRNA (con-si; open squares) or siRNA against hnRNP D (sinhD; closed squares) and grown to confluence prior to treatment with 100 nM dexamethasone (Dex.) to synchronize the circadian oscillation (time zero). Total RNA (1 μg) from each time point was subjected to quantitative real-time RT-PCR with primers specific to mcry1 or to mtbp as a normalizing control (graph). Each value was relative to that for control siRNA at time zero. Results shown are representative of four independent experiments. The error bars represent the standard errors of the means of the results. Reduction of the hnRNP D protein level in total cell lysate (TCL) by siRNA compared to the level of 14-3-3ζ (used as an internal control) was validated by Western blotting (gel).
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