ZC3H12B/MCPIP2, a new active member of the ZC3H12 family

Abstract

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.

Potential limitations of the Sleeping Beauty transposon use in gene expression studies

“…The pSBtet-GP-MCPIP2 construct was prepared by cloning ZC3H12B coding region (gene encoding MCPIP2, NM_001010888.3) into the Sleeping Beauty vector pSBtet-GP (Addgene, #60495). PCR products were amplified from a previously prepared vector pcDNA5/FRT/TO-SF-ZC3H12B, containing ZC3H12B coding region fused with Strep-TagII and FLAG tags using Q5 High Fidelity DNA Polymerase (New England Biolabs, Ipswich) (Wawro et al, 2019). The following primers were used:…”

“…Previously, we have found that MCPIP2 is involved in regulation of inflammation through destabilization of transcripts of proinflammatory cytokines (Wawro et al, 2019) To identify new RNA molecules which are regulated by MCPIP2, we employed the SB system for overexpression of MCPIP2 in a human astrocytoma cell line, U251-MG. Surprisingly, we found that the SB system alone influences expression of some genes.…”

Post‐transcriptional control of immune responses and its potential application

“…Although the findings above provide mechanistic insights into the functions of Regnase family members, the roles of other members, particularly ZC3H12B and ZC3H12C, are not well understood. Very recently, it has been reported that ZC3H12B also regulates IL‐6 and Ier3 in vitro , which are targets of Regnase‐1 . Also, ZC3H12C is associated with an increased risk of psoriasis, although the functional role remains to be clarified.…”

“…Very recently, it has been reported that ZC3H12B also regulates IL-6 and Ier3 in vitro, which are targets of Regnase-1. 31 Also, ZC3H12C is associated with an increased risk of psoriasis, 32 although the functional role remains to be clarified. Given conservation of the PIN-like RNase domain, it is conceivable that these family members may also play a pivotal role in regulating immune responses and this warrants further investigation.…”

RNA binding proteins in the control of autoimmune diseases

“…While the immune regulatory function of Regnase-1 and Regnase-4 has been revealed, the roles of other family members remain to be known. Recently, a study showed that ZC3H12B is also involved in the regulation of IL-6 and Ier3, which are targets of Regnase-1 [72]. Also, another family member, ZC3H12C is associated with an increased risk of psoriasis [73], although the functional role remains to be elucidated.…”