Identification of four families of yCCR4- and Mg2+-dependent endonuclease-related proteins in higher eukaryotes, and characterization of orthologs of yCCR4 with a conserved leucine-rich repeat essential for hCAF1/hPOP2 binding.

BACKGROUND: The yeast yCCR4 factor belongs to the CCR4-NOT transcriptional regulatory complex, in which it interacts, through its leucine-rich repeat (LRR) motif with yPOP2. Recently, yCCR4 was shown to be a component of the major cytoplasmic mRNA deadenylase complex, and to contain a fold related to the Mg2+-dependent endonuclease core. RESULTS: Here, we report the identification of nineteen yCCR4-related proteins in eukaryotes (including yeast, plants and animals), which all contain the yCCR4 endonuclease-like fold, with highly conserved CCR4-specific residues. Phylogenetic and genomic analyses show that they form four distinct families, one of which contains the yCCR4 orthologs. The orthologs in animals possess a leucine-rich repeat domain. We show, using two-hybrid and far-Western assays, that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2, in a LRR-dependent manner. CONCLUSIONS: We have identified the mammalian orthologs of yCCR4 and have shown that the human member binds to the human yPOP2 homologs, thus strongly suggesting conservation of the CCR4-NOT complex from yeast to human. All members of the four identified yCCR4-related protein families show stricking conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal domain and therefore constitute a new family of potential deadenylases in mammals.

Characterization of a mammalian gene related to the yeast CCR4 general transcription factor and revealed by transposon insertion.

Murine intracisternal A-particles (IAPs) are reiterated retrovirus-like transposable elements that can act as insertional mutagens. Accordingly, we previously identified a chimeric transcript initiated at an IAP promoter and extending through a 3'-located open reading frame with significant similarity to the C-terminal domain of the yeast CCR4 general transcription factor. In this report, we characterize the corresponding murine gene, mCCR4, and its human homologue, thus providing the first description of CCR4-like factors in mammals. cDNA cloning revealed two mCCR4 mRNAs of 2.7 and 3.1 kilobases, differing by their transcription start sites within the native mCCR4 gene promoter, and encoding a putative 430-amino acid protein. The mCCR4 gene contains three exons and two introns spanning almost 27 kilobases. The IAP insertion, detected only in some laboratory mouse strains, is recent and lies within the first intron. The 5'-region of the gene has features of housekeeping gene promoters. It lacks a TATA box but contains a CpG island and Sp1 sites. This region discloses strong promoter activity in transient transfection assays and also stimulates transcription in the reverse orientation, a feature common to other CpG island-containing promoters. Transcripts were detected in all the organs tested, although at a variable level, and displayed no strain-dependent differences relative to the IAP insertion, suggesting the existence of mechanisms preserving mCCR4 transcription from the usually deleterious effects of intronic transposition. The strong amino acid conservation between the human, murine, and the previously identified Xenopus CCR4-like proteins, is consistent with an important and conserved role for this protein in vertebrates.