The target of rapamycin (TOR) kinase regulates cell growth and division. Rapamycin only inhibits a subset of TOR activities. Here we show that in contrast to the mild impact of rapamycin on cell division, blocking the catalytic site of TOR with the Torin1 inhibitor completely arrests growth without cell death in Schizosaccharomyces pombe. A mutation of the Tor2 glycine residue (G2040D) that lies adjacent to the key Torin-interacting tryptophan provides Torin1 resistance, confirming the specificity of Torin1 for TOR. Using this mutation, we show that Torin1 advanced mitotic onset before inducing growth arrest. In contrast to TOR inhibition with rapamycin, regulation by either Wee1 or Cdc25 was sufficient for this Torin1-induced advanced mitosis. Torin1 promoted a Polo and Cdr2 kinase-controlled drop in Wee1 levels. Experiments in human cell lines recapitulated these yeast observations: mammalian TOR (mTOR) was inhibited by Torin1, Wee1 levels declined and mitotic commitment was advanced in HeLa cells. Thus, the regulation of the mitotic inhibitor Wee1 by TOR signalling is a conserved mechanism that helps to couple cell cycle and growth controls.

ZC3H12B is the most enigmatic member of the ZC3H12 protein family. The founding member of this family, Regnase-1/ MCPIP1/ZC3H12A, is a well-known modulator of inflammation and is involved in the degradation of inflammatory mRNAs. In this study, for the first time, we characterized the properties of the ZC3H12B protein. We show that the biological role of ZC3H12B depends on an intact NYN/PIN RNase domain. Using RNA immunoprecipitation, experiments utilizing actinomycin D and ELISA, we show that ZC3H12B binds interleukin-6 (IL-6) mRNA in vivo, regulates its turnover, and results in reduced production of IL-6 protein upon stimulation with IL-1β. We verified that regulation of IL-6 mRNA stability occurs via interaction of ZC3H12B with the stem-loop structure present in the IL-6 3 ′ ′ ′ ′ ′ UTR. The IL-6 transcript is not the only target of ZC3H12B. ZC3H12B also interacts with other known substrates of Regnase-1 and ZC3H12D, such as the 3 ′ ′ ′ ′ ′ UTRs of IER3 and Regnase-1, and binds IER3 mRNA in vivo. Using immunofluorescence, we examined the localization of ZC3H12B within the cell. ZC3H12B forms small, granule-like structures in the cytoplasm that are characteristic of proteins involved in mRNA turnover. The overexpression of ZC3H12B inhibits proliferation by stalling the cell cycle in the G2 phase. This effect of ZC3H12B is also NYN/PIN dependent. The analysis of the ZC3H12B mRNA level reveals its highest expression in the human brain and the neuroblastoma cell line SH-SY5Y, although the factors regulating its expression remain elusive. Down-regulation of ZC3H12B in SH-SY5Y cells by specific shRNAs results in up-regulation of ZC3H12B-target mRNAs.

We used RNA sequencing (RNA-Seq) technology to investigate changes in the transcriptome profile in the Caki-1 clear cell renal cell carcinoma (ccRCC) cells, which overexpress monocyte chemoattractant protein-induced protein 1 (MCPIP1). RNA-Seq data showed changes in 11.6% and 41.8% of the global transcriptome of Caki-1 cells overexpressing wild-type MCPIP1 or its D141N mutant, respectively. Gene ontology and KEGG pathway functional analyses showed that these transcripts encoded proteins involved in cell cycle progression, protein folding in the endoplasmic reticulum, hypoxia response and cell signalling. We identified 219 downregulated transcripts in MCPIP1-expressing cells that were either unchanged or upregulated in D141N-expressing cells. We validated downregulation of 15 transcripts belonging to different functional pathways by qRT-PCR. The growth and viability of MCPIP1-expressing cells was reduced because of elevated p21Cip1 levels. MCPIP1-expressing cells also showed reduced levels of DDB1 transcript that encodes component of the E3 ubiquitin ligase that degrades p21Cip1. These results demonstrate that MCPIP1 influences the growth and viability of ccRCC cells by increasing or decreasing the transcript levels for proteins involved in cell cycle progression, protein folding, hypoxia response, and cell signaling.

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Adipogenesis is a process of preadipocyte differentiation that requires action of numerous factors. Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) possesses the N-terminus of the PilT protein (PilT N-terminus or PIN domain) that has RNase properties. This protein degrades transcripts coding for inflammation and differentiation - related proteins. Moreover, MCPIP1 is a broad suppressor of the miRNA biogenesis. We previously found that MCPIP1 degrades transcript encoding CCAAT/Enhancer Binding Protein Beta (C/EBPβ) and influences adipogenesis. Subsequently, we aimed to determine adipocyte miRNA expression profile in differentiating mouse preadipocytes, 3T3-L1, by overexpressing MCPIP1. Using Next-Generation Sequencing (NSG) we showed that MCPIP1 overexpression results in modulated levels of 58 miRNAs in adipocytes on day 2 of differentiation. Among them, 30 miRNAs showed significantly reduced levels and 28 showed increased levels in comparison to control. Approximately one third of the modulated miRNAs were not previously reported to be involved in adipocytes differentiation. Our analysis revealed that 24 down-regulated and 23 up-regulated miRNAs (at least 1.5-fold) influence 19 signaling pathways that are important for adipogenesis. Furthermore, reduced miRNA levels result in the up-regulation of their targets. By using luciferase reporter assay, we demonstrated that miR-32-5p and miR-9-3p directly target the 3'UTR region of Mapk8 and Tiam1, respectively. In addition, activation of MAP kinases pathway (JNK and p38), proposed as being regulated by down-regulated miRNAs, was higher in MCPIP1 than inMCPIP1 or control 3T3-L1 adipocytes. Our results indicate a considerable impact of MCPIP1 on miRNAs levels and its significance in adipogenesis.

The monocyte chemoattractant protein-induced protein (MCPIP) family consists of 4 members (MCPIP1–4) encoded by the ZC3h12A-D genes, which are located at different loci. The common features of MCPIP proteins are the zinc finger domain, consisting of three cysteines and one histidine (CCCH), and the N-terminal domain of the PilT protein (PilT-N-terminal domain (PIN domain)). All family members act as endonucleases controlling the half-life of mRNA and microRNA (miRNA). The best-studied member of this family is MCPIP1 (also known as Regnase-1).In this review, we discuss the current knowledge on the role of MCPIP1 in cancer-related processes. Because the characteristics of MCPIP1 as a fundamental negative regulator of immune processes have been comprehensively described in numerous studies, we focus on the function of MCPIP1 in modulating apoptosis, angiogenesis and metastasis.

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