Alternative titles; symbols
HGNC Approved Gene Symbol: CNOT6L
Cytogenetic location: 4q21.1 Genomic coordinates (GRCh38) : 4:77,713,387-77,820,269 (from NCBI)
CNOT6L is a subunit of the CCR4-NOT complex and functions as a cytoplasmic deadenylase with 3-prime-to-5-prime exoribonuclease activity (Morita et al., 2007).
By searching databases for orthologs of yeast Ccr4, Morita et al. (2007) identified human CNOT6L, which they called CCR4B. The deduced 555-amino acid protein has an N-terminal leucine-rich repeat (LRR) domain and a C-terminal nuclease domain. Human CNOT6L shares significant homology with human CCR4A (CNOT6; 608951) and with orthologs in mouse, fly, and yeast. Northern blot analysis detected wide expression of 2 CNOT6L transcripts in human tissues, with highest levels in placenta, skeletal muscle, pancreas, and testis. CNOT6L protein localized to cytoplasm in transfected NIH-3T3 cells.
Using mass spectrometry and coimmunoprecipitation experiments, Morita et al. (2007) demonstrated that CNOT6L interacted with human orthologs of the yeast CCR4-NOT complex. In an in vitro deadenylase assay with single-stranded poly(A) RNA as substrate, human CNOT6L displayed 3-prime-to-5-prime poly(A) exoribonuclease activity. Suppression of CNOT6L in NIH-3T3 cells severely reduced proliferation and inhibited cell cycle progression from G0/G1 to S phase. Immunoblot analysis showed that G0/G1 cell cycle arrest in CNOT6L-depleted cells was caused by increased expression of p27(KIP1) (CDKN1B; 600778). Expression of p27(KIP1) increased in CNOT6L-depleted cells due to enhanced stability of its mRNA resulting from the absence of deadenylation and degradation by the 3-prime-to-5-prime poly(A) exoribonuclease activity of CNOT6L. The authors also demonstrated that the deadenylase activity of CNOT6L played an important part in cell growth regulation.
Mittal et al. (2011) found that knockdown of the CCR4 deadenylase paralogs CNOT6 and CNOT6L in MCF7 breast cancer cells resulted in reduced cell proliferation, cell cycle arrest, and decreased cell survival. They also found that the LRR domain of human CNOT6L impacted its subcellular localization. CNOT6L was predominantly localized to cytoplasm of HEK293 cells, with some found in nucleus, but deletion of the LRR domain resulted in an almost exclusive cytoplasmic localization. The LRR domain of CNOT6L was also required for its interaction with CAF1A (601245), another subunit of the CCR4-NOT complex, through which CNOT6L interacts with other subunits in the complex. Ectopic expression in HEK293 cells and overexpression in MCF7 cells showed that, unlike in yeast, deletion of the LRR domain of CNOT6L affected cell proliferation but had not effect on deadenylase activity or cell viability. Knockdown experiments in MCF7 cells revealed that CNOT6/CNOT6L regulated distinct gene sets with limited overlap with other subunits in the complex. One of the genes that showed the most enhanced expression upon CNOT6/CNOT6L knockdown was insulin-like growth factor-binding protein-5 (IGFBP5; 146734), which mediates cell cycle arrest and senescence via a p53 (TP53; 191170)-dependent pathway.
Polesskaya et al. (2016) found that CNOT6L had an inhibitor role in human muscle cell differentiation, independently of a modification of the myoblast growth rate. Transcriptomic analysis of CNOT6L-knockdown immortalized human LHCN myoblasts identified target mRNAs of CNOT6L, with the chemokine IL8 (146930) as the most upregulated gene in knockdown cells. Analyses showed that IL8 modulated myoblast differentiation and was a direct functional target of CNOT6L deadenylase. CNOT6L bound to IL8 mRNA in differentiating skeletal myoblasts and destabilized the mRNA, most likely by initially shortening its poly(A) tail, resulting in modulation of IL8 protein expression and thereby influencing differentiation of LHCN myoblasts.
Wang et al. (2010) determined the crystal structure of the catalytic nuclease domain of human CNOT6L, covering amino acids 169 to 539, to 1.94-angstrom resolution. The overall structure consisted of 11 alpha helices and 17 beta strands and featured a 2-layered alpha-beta sandwich with approximately 2-fold internal symmetry. The 2 mostly antiparallel beta sheets sit on the interior, flanked by 2 alpha-helical layers on the outside, typical of hydrolases with highly conserved active core residues, similar to APE1 (107748). The active site of CNOT6L catalytic domain contains 2 bound Mg(2+) ions and 5 essential amino acids (asn195, glu240, asp410, asp489, and his529) that form a negatively charged pocket around the 2 Mg(2+). The crystal structure combined with mutational and biochemical data indicated that CNOT6L displays full Mg(2+)-dependent deadenylase activity with strict substrate recognition and preference for poly(A) RNA. The authors proposed a deadenylase mechanism for CNOT6L involving a pentacovalent phosphate transition.
Zhang et al. (2016) reported the crystal structures of the human CNOT6L nuclease domain in complex with representatives from 2 classes of deadenylase inhibitors, natural nucleotides and aminoglycosides. Structural analysis confirmed that both classes of deadenylase inhibitors occupied the substrate- and magnesium-binding sites in CNOT6L, suggesting that they compete with both substrate and divalent magnesium ions for overlapping binding sites.
Gross (2018) mapped the CNOT6L gene to chromosome 4q21.1 based on an alignment of the CNOT6L sequence (GenBank AK304397) with the genomic sequence (GRCh38).
Gross, M. B. Personal Communication. Baltimore, Md. 7/26/2018.
Mittal, S., Aslam, A., Doidge, R., Medica, R., Winkler, G. S. The Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the human Ccr4-Not complex contribute to the prevention of cell death and senescence. Molec. Biol. Cell 22: 748-758, 2011. [PubMed: 21233283] [Full Text: https://doi.org/10.1091/mbc.E10-11-0898]
Morita, M., Suzuki, T., Nakamura, T., Yokoyama, K., Miyasaka, T., Yamamoto, T. Depletion of mammalian CCR4b deadenylase triggers elevation of the p27(Kip1) mRNA level and impairs cell growth. Molec. Cell. Biol. 27: 4980-4990, 2007. [PubMed: 17452450] [Full Text: https://doi.org/10.1128/MCB.02304-06]
Polesskaya, A., Pinna, G., Sassi, Y., Vandamme, M., Bigot, A., Mouly, V., Morozova, N., Harel-Bellan, A., Degerny, C. Post-transcriptional modulation of interleukin 8 by CNOT6L regulates skeletal muscle differentiation. Biochim. Biophys. Acta 1863: 263-270, 2016. [PubMed: 26608607] [Full Text: https://doi.org/10.1016/j.bbamcr.2015.11.018]
Wang, H., Morita, M., Yang, X., Suzuki, T., Yang, W., Wang, J., Ito, K., Wang, Q., Zhao, C., Bartlam, M., Yamamoto, T., Rao, Z. Crystal structure of human CNOT6L nuclease domain reveals strict poly(A) substrate specificity. EMBO J. 29: 2566-2576, 2010. [PubMed: 20628353] [Full Text: https://doi.org/10.1038/emboj.2010.152]
Zhang, Q., Yan, D., Guo, E., Ding, B., Yang, W., Liu, R., Yamamoto, T., Bartlam, M. Structural basis for inhibition of the deadenylase activity of human CNOT6L. FEBS Lett. 590: 1270-1279, 2016. [PubMed: 27013054] [Full Text: https://doi.org/10.1002/1873-3468.12160]