Expression of the orphan nuclear receptor ERRalpha is under circadian regulation in estrogen-responsive tissues.

Circadian gene expression has been demonstrated in many tissues and involves both positive and negative regulatory loops. The potential interferences of circadian rhythmicity with other well-known biologic rhythms, such as the ovarian cycle, at least in part controlled by estrogens, has not been questioned. The estrogen receptor-related receptor (ERR)alpha is an orphan nuclear receptor that is widely expressed in estrogen-responsive tissues such as liver, uterus and bone. In addition, expression of the ERRalpha gene has been proposed to be transcriptionally controlled by estrogens in the uterus. Here we show that the expression of ERRalpha displays a circadian rhythmicity in liver, bone and uterus. This is in contrast to other uterine estrogen-regulated genes. Analysis of clock/clock mutant mice shows that ERRalpha is an output gene of the circadian clock oscillator. The expression of clock-control genes, such as Bmal1 and Rev-erbalpha, also displays diurnal oscillations in the uterus, but not in bone. In this tissue, however, Per2 displayed a rhythmic expression, altogether suggesting unconventional loops in the regulation of circadian rhythm in bone.

Circadian regulation of diverse gene products revealed by mRNA expression profiling of synchronized fibroblasts.

Genes under a 24-h regulation period may represent drug targets relevant to diseases involving circadian dysfunctions. As a testing model of the circadian clock system, we have used synchronized rat fibroblasts that are known to express at least six genes in a circadian fashion. We have determined the expression patterns of 9957 transcripts every 4 h over a total period of 76 h using high density oligonucleotide microarrays. The spectral analysis of our mRNA profiling data indicated that approximately 2% (85 genes) of all expressed genes followed a robust circadian pattern. We have confirmed the circadian expression of previously known clock or clock-driven genes, and we identified 81 novel circadian genes. The majority of the circadian-regulated gene products are known and are involved in diverse cellular functions. We have classified these circadian genes in seven clusters according to their phase of cycling. Our pathway analysis of the mRNA profiling data strongly suggests a direct link between circadian rhythm and cell cycle.

RNA binding specificity of Unr, a protein with five cold shock domains.

The human unr gene encodes an 85 kDa protein which contains five cold shock domains (CSD). The capacity of Unr to interact in vitro with RNA and its intracellular localization suggest that Unr could be involved in some aspect of cytoplasmic mRNA metabolism. As a step towards identification of Unr mRNA targets, we investigated the RNA-binding specificity of Unr by an in vitro selection approach (SELEX). Purine-rich sequences were selected by Unr, leading to the identification of two related consensus sequences characterized by a conserved core motif AAGUA/G or AACG downstream of a purine stretch. These consensus sequences are 11-14 nt long and appear unstructured. RNAs containing a consensus sequence were bound specifically by Unr with an apparent dissociation constant of 1 x 10(-8) M and both elements, the 5' purine stretch and the core motif, were shown to contribute to the high affinity. When the N-terminal and C-terminal CSD were analyzed individually, they exhibited a lower affinity than Unr for winner sequences (5- and 100-fold, respectively) but with similar binding specificity. Two combinations of CSDs, CSD1-2-3 and CSD1*2-3-4-5 were sufficient to achieve the high affinity of Unr, indicating some redundancy between the CSDs of Unr for RNA recognition. The SELEX-generated consensus motifs for Unr differ from the AACAUC motif selected by the Xenopus Y-box factor FRGY2, indicating that a diversity of RNA sequences could be recognized by CSD-containing proteins.

mRNP3 and mRNP4 are phosphorylatable by casein kinase II in Xenopus oocytes, but phosphorylation does not modify RNA-binding affinity.

mRNP3 and mRNP4 (also called FRGY2) are two mRNA-binding proteins which are major constituents of the maternal RNA storage particles of Xenopus laevis oocytes. The phosphorylation of mRNP3-4 has been implicated in the regulation of mRNA masking. In this study, we have investigated their phosphorylation by casein kinase II and its consequence on their affinity for RNA. Comparison of the phosphopeptide map of mRNP3-4 phosphorylated in vivo with that obtained after phosphorylation in vitro by purified Xenopus laevis casein kinase II strongly suggests that casein kinase II is responsible for the in vivo phosphorylation of mRNP3-4 in oocytes. The phosphorylation occurs on a serine residue in a central domain of the proteins. The affinity of mRNP3-4 for RNA substrates remained unchanged after the treatment with casein kinase II or calf intestine phosphatase in vitro. This suggests that phosphorylation of these proteins does not regulate their interaction with RNA but rather controls their interactions with other proteins.

Transcription of unr (upstream of N-ras) down-modulates N-ras expression in vivo.

The ras proteins (Harvey, Kirsten and N-ras) are key regulators of signal transduction and a perturbation of their GDP/GTP cycle is frequently observed in tumors. In mammals, N-ras constitutes with unr (upstream of N-ras) a tightly linked tandem of ubiquitously expressed genes. Although unr and N-ras appear to be involved in distinct functions, this unusual genetic organization could be important for the regulation of N-ras expression. Specifically, transcription of unr could negatively regulate that of N-ras by transcriptional interference. To investigate this possibility, we have deleted the unr promoter by homologous recombination in murine embryonic stem cells. Analysis of tissues of heterozygous mice revealed an increase in N-ras mRNA accumulation ranging between 20 and 65%, in agreement with the suppression of a transcriptional interference.

Nucleic acid binding and intracellular localization of unr, a protein with five cold shock domains.

The unr gene was identified as a transcription unit located immediately upstream of N-ras in the genome of several mammalian species. While this genetic organization could be important for the transcriptional regulation of unr and N-ras, the function of the protein product of unr is unknown. unr is ubiquitously expressed and codes for an 85 kDa protein which is not closely related to previously characterized proteins. Nevertheless, a search for protein motifs has indicated the presence of five 'cold shock domains' within unr, a motif present in procaryotic cold shock proteins and in the vertebrate Y box factors. As these proteins have been reported to interact with nucleic acids, we investigated whether unr could bind to some classes of nucleic acids. We report here that unr has a high affinity for single-stranded DNA or RNA and a low affinity for double-stranded nucleic acids. Its low affinity for double-stranded DNA clearly distinguishes unr from the Y box factors. The binding of unr to RNA does not appear to depend upon extended sequence motifs but requires some level of sequence complexity as unr has only a low affinity for most simple polymers including polyA stretches. unr is also characterized by its low affinity for double-stranded and structured RNAs. We further determined that unr is mostly localized in the cytoplasm, and is in part associated with the endoplasmic reticulum. These studies indicate that unr is a novel single-stranded nucleic acid binding protein which is likely to be associated with cytoplasmic mRNA in vivo.

Organization of the human N-ras locus: characterization of a gene located immediately upstream of N-ras.

The mammalian ras genes have been implicated in a wide variety of natural and experimental tumors. They code for small GTP binding proteins which are believed to play a central role in the control of cellular proliferation and differentiation. We have investigated the transcriptional organization of the human N-ras locus and characterized a transcription unit located immediately upstream of N-ras, which we designate by NRU for N-ras Upstream. NRU messages contain an open reading frame of 767 amino acids which shows no similarity with the ras proteins and provides no clue to the function of the corresponding protein. Of the order of 150 nucleotides separate the N-ras transcription initiation sites from the last NRU polyadenylation site and the same organization is present in the murine genome. Both genes are simultaneously expressed in all the cell lines and murine tissues we have analysed, and in all cases NRU messages accumulate to a higher level than those of N-ras. The small intergenic distance implies that, during transcription of NRU, RNA polymerases transcribe a substantial part of the N-ras gene, suggesting that this genetic organization participates in the regulation of N-ras expression.