HnRNP Q Has a Suppressive Role in the Translation of Mouse Cryptochrome1

Abstract

Precise regulation of gene expression is especially important for circadian timekeeping which is maintained by the proper oscillation of the mRNA and protein of clock genes and clock-controlled genes. As a main component of the core negative arm feedback loops in the circadian clock, the Cry1 gene contributes to the maintenance of behavioral and molecular rhythmicity. Despite the central role of Cry1, the molecular mechanisms regulating expression levels of Cry1 mRNA and protein are not well defined. In particular, the post-transcriptional regulation of Cry1 mRNA fate decisions is unclear. Here, we demonstrate that hnRNP Q binds to mCry1 mRNA via the 5'UTR. Furthermore, hnRNP Q inhibits the translation of mCry1 mRNA, leading to altered rhythmicity in the mCRY1 protein profile.

Fig 1. hnRNP Q binds to the 5′UTR of mCry1 and suppresses its translation.

(A) RNAi screening in NIH3T3 cells. Five different siRNAs targeting different hnRNPs, including hnRNP Q, were transfected into cells. Control siRNA was used as a negative control. The level of mCRY1 protein was analyzed by Western blotting. On the right side, the circadian phases of cell populations were synchronized by temporal treatment with 100nM dexamethasone, to clearly check the collective effect of hnRNP downregulation on each cell. At 12 hours after synchronization, samples were analyzed. GAPDH and 14-3-3ζ were used as a loading control. The arrow indicates weakly detected mCRY1 protein. (B) mRNA stability of endogenous mCry1 under hnRNP Q downregulation. mRNA degradation kinetics of mTBP was also evaluated as a control. Error bars represent the SEM of three independent experiments. *P<0.05. (C) Identification of the interaction between the mCry1 5′UTR and hnRNP Q by RNA affinity purification followed by immunoblotting. (D) The translation enhancement mediated by the 5′UTR of mCry1 after reduction of hnRNP Q is shown. The 5′UTR of mCry1 was inserted at the upstream of the Fluc coding sequence. Fluc activity was normalized with β-Gal activity. Error bars represent the SEM of seven independent experiments. *P<0.05. (E) Knockdown of hnRNP Q was confirmed by immunoblotting.

Fig 2. The cis-acting region for hnRNP Q resides in the forepart of the mCry1 5′UTR.

(A) Schematic description of serially deleted mCry1 5′UTRs. (B) Cellular proteins that bound to the full-length or truncated forms of the mCry1 5′UTR were analyzed by in vitro binding followed by UV-crosslinking assays. The arrow indicates the bands corresponding to hnRNP Q. (C) The translation efficiency of the full-length or deleted forms of the mCry1 5′UTR was determined. Transfection consistency was compensated with β-Gal activity, and translation efficiency was further calculated with mRNA amount derived from each construct. Error bars represent the SEM of four independent experiments. P-value of mCry1 1–583 vs mCry1 480–583 = 0.032. P-value of mCry1 100–583 vs mCry1 480–583 = 0.048. P-value of mCry1 202–583 vs mCry1 480–583 = 0.026. P-value of mCry1 283–583 vs mCry1 480–583 = 0.036. (D) The enhancement of translation mediated by the full-length or deleted forms of the mCry1 5′UTR under hnRNP Q silencing is shown. Error bars represent the SEM of three independent experiments. (E) Downregulation of hnRNP Q was confirmed by immunoblotting.

Fig 3. mCry1 protein oscillation is less evident under hnRNP Q silencing.

(A) qRT-PCR analysis for endogenous mCry1 mRNA levels after circadian phase synchronization. Error bars represent the SEM of five independent experiments. The initial amount of mCry1 mRNA with siCon was arbitrarily set as 1. *P<0.05. (B) Western blot analysis of mCRY1 in control and hnRNP Q-downregulated cells. (C) The normalized relative expression profile of mCRY1 protein in (B) was plotted. The intensities at 0 hour of siCon group were arbitrarily set as 1. (D) Downregulation of hnRNP Q was confirmed by immunoblotting.