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.

The members of the ZC3H12/MCPIP/Regnase family of RNases have emerged as important regulators of inflammation. In contrast to Regnase-1, -2 and -4, a thorough characterization of Regnase-3 (Reg-3) has not yet been explored. Here we demonstrate that Reg-3 differs from other family members in terms of NYN/PIN domain features, cellular localization pattern and substrate specificity. Together with Reg-1, the most comprehensively characterized family member, Reg-3 shared IL-6, IER-3 and Reg-1 mRNAs, but not IL-1β mRNA, as substrates. In addition, Reg-3 was found to be the only family member which regulates transcript levels of TNF, a cytokine implicated in chronic inflammatory diseases including psoriasis. Previous meta-analysis of genome-wide association studies revealed Reg-3 to be among new psoriasis susceptibility loci. Here we demonstrate that Reg-3 transcript levels are increased in psoriasis patient skin tissue and in an experimental model of psoriasis, supporting the immunomodulatory role of Reg-3 in psoriasis, possibly through degradation of mRNA for TNF and other factors such as Reg-1. On the other hand, Reg-1 was found to destabilize Reg-3 transcripts, suggesting reciprocal regulation between Reg-3 and Reg-1 in the skin. We found that either Reg-1 or Reg-3 were expressed in human keratinocytes in vitro. However, in contrast to robustly upregulated Reg-1 mRNA levels, Reg-3 expression was not affected in the epidermis of psoriasis patients. Taken together, these data suggest that epidermal levels of Reg-3 are negatively regulated by Reg-1 in psoriasis, and that Reg-1 and Reg-3 are both involved in psoriasis pathophysiology through controlling, at least in part different transcripts.

MCPIP2 is the least known member of the MCPIP family of proteins. Recently we have found that it is a new RNase involved in transcript turnover. However, the full spectrum of its cellular targets is still unidentified. To discover transcripts which are regulated by this protein we have employed Sleeping Beauty transposons. This tool allows for rapid generation of a stable transgenic cell line with inducible expression of the desired gene. In this study, we analysed how the Sleeping Beauty system itself influences expression of chosen genes, namely IL-6, Regnase-1 and VEGF. We found that the system alone may influence expression of IL-6. Our results indicate that Sleeping Beauty transposons should be used with caution in studies that are focused on changes in the transcript level.

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