Interleukin-1-inducible MCPIP protein has structural and functional properties of RNase and participates in degradation of IL-1b mRNA Danuta Mizgalska1, Paulina Wegrzyn1, Krzysztof Murzyn2, Aneta Kasza1, Aleksander Koj1, ˛ Jacek Jura3, Barbara Jarzab4 and Jolanta Jura1 ˛ Department of Cell Biochemistry, Jagiellonian University, Krakow, Poland Department of Biophysics, Jagiellonian University, Krakow, Poland National Research Institute of Animal Production, Balice, Poland M Sklodowska-Curie Memorial Institute, Gliwice, Poland Keywords IL-1b transcript degradation; inflammation; MCPIP; PIN domain; RNase Correspondence J Jura, Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Gronostajowa Street, 30-387 Krakow, Poland Fax: +48 12 664 6902 Tel: +48 12 664 6359 E-mail: jolanta.jura@uj.edu.pl (Received 28 April 2009, revised 23 September 2009, accepted 20 October 2009) doi:10.1111/j.1742-4658.2009.07452.x In human monocyte-derived macrophages, the MCPIP gene (monocyte chemoattractant protein-induced protein) is strongly activated by interleukin-1b (IL-1b) Using bioinformatics, a PIN domain was identified, spanning amino acids 130-280; such domains are known to possess structural features of RNases Recently, RNase properties of MCPIP were confirmed on transcripts coding for interleukins IL-6 and IL-12p40 Here we present evidence that siRNA-mediated inhibition of the MCPIP gene expression increases the level of the IL-1b transcript in cells stimulated with LPS, whereas overexpression of MCPIP exerts opposite effects Cells with an increased level of wild-type MCPIP showed lower levels of IL-1b mRNA However, this was not observed when mutant forms of MCPIP, either entirely lacking the PIN domain or with point mutations in this domain, were used The results of experiments with actinomycin D indicate that lower levels of IL-1b mRNA are due to shortening of the IL-1b transcript half-life, and are not related to the presence of AU-rich elements in the 3¢ UTR The interaction of the MCPIP with transcripts of both IL-1b and MCPIP observed in an RNA immunoprecipitation assay suggests that this novel RNase may be involved in the regulation of expression of several genes Introduction Macrophages and hepatic cells are important players in the inflammatory processes initiated in response to a variety of agents, including viral or bacterial infections, thermal and mechanical trauma, or malignant growth Recently, we have used human monocyte-derived macrophages exposed to interleukins IL-1b and IL-6, and studied changes of gene expression using microarrays [1] Among the cytokine-modulated genes, one was highly activated by IL-1b, but not by IL-6 This tran- script was found to correspond to the ZC3H12A gene (also called the MCPIP gene), originally described as a gene activated by monocyte chemoattractant protein-1 (MCP-1) and encoding MCPIP [2] This protein consists of 599 amino acids, and has a putative nuclear localization signal and two proline-rich potential activation domains, one between residues 100 and 126 and the other between residues 458 and 536 [2] Moreover, amino acid sequence analysis revealed a single zinc- Abbreviations ARE, AU-rich element; dsRBD, double-stranded RNA-binding domain; GM-CSF, granulocyte ⁄ macrophage colony-stimulating factor; iNOS, inducible nitric oxide synthase; IL-1b, interleukin-1b; IL-6, interleukin-6; KH domain, K homology domain; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein-1; MCPIP, MCP-1 induced protein; PAZ, Piwi Argonaut and Zwille; PDB, protein data bank; PMA, phorbol 12-myristate-13-acetate; RRM, RNA-recognition motif; TNF, tumor necrosis factor 7386 FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS D Mizgalska et al finger motif (CCCH) in MCPIP, which prompted Zhou et al to propose a hypothetical function for this molecule as a novel transcription factor [2] They also showed that MCPIP is a negative regulator of macrophage activation, affecting LPS-induced tumor necrosis factor (TNF) and inducible nitric oxide synthase (iNOS) promoters [3] Recently, Matsushita et al [4] reported that MCPIP has an essential role in preventing immune disorders Mice with MCPIP deficiency (Zc3h12a) ⁄ )) showed growth retardation, severe splenomegaly and lymphoadenopathy Matsushita et al [4] also observed infiltration of plasma cells in the lung, the para-epithelium of the bile duct, the pancreas, lymph nodes and spleen Moreover, Zc3h12a-deficient mice suffered from severe anemia, had increased serum immunoglobulin levels and showed auto-antibody production Transcriptome comparison of LPS-treated macrophages from wild-type and Zc3h12a-deficient mice revealed that a particular set of genes, namely IL-6, Ifng, Calcr and Sprr2d, was highly expressed in knockout mice [4] Further experiments showed that MCPIP has RNase properties and regulates IL-6 and IL12p40 mRNA stability [4] Regulation of the half-life of the IL-6 transcript involved its 3¢ UTR but was independent of AU-rich elements (AREs) [4] In our studies, we have observed that, in addition to IL-1b, the MCPIP gene is also strongly activated by LPS, TNFa and phorbol 12-myristate-13-acetate (PMA) Moreover, we found that MCPIP has RNase properties and regulates the half-life of IL-1b mRNA and its own transcript Overexpression of MCPIP in cells activated with LPS results in downregulation of the IL-1b transcript and silencing of MCPIP gene expression has an opposite effect on the IL-1b mRNA level RNase properties of MCPIP are exerted by the PIN domain, as shown by experiments with mutated MCPIP (either lacking the PIN domain, or with two conservative amino acid mutations within PIN domain) Interaction of MCPIP with the IL-1b transcript and its own transcript was confirmed by an RNA immunoprecipitation assay Our findings are in agreement with those of Matsushita et al [4] The data show that MCPIP has more inflammatory targets than described so far, and that this protein is an important regulator of inflammatory processes Results Influence of proinflammatory cytokines on MCPIP transcript level Real-time PCR was used to determine the modulation of expression of the MCPIP gene in two types of cells: MCPIP protein as an RNase HepG2 cells stimulated with IL-1b or IL-6, and monocyte lymphoma cells stimulated with IL-1b, TNFa, PMA or LPS As shown in Fig 1A, the MCPIP transcript level in HepG2 cells stimulated with IL-1b for A B C Fig Regulation of MCPIP gene expression Changes in MCPIP gene expression in HepG2 and U937 cells measured by real-time PCR The results are means ± SD of three independent experiments (*P < 0.05; **P < 0.01 versus control) (A) Changes in the expression of the MCPIP gene after stimulation of HepG2 cells with IL-1b (15 ngỈmL)1) Cells were stimulated for 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h Unstimulated cells collected at each time point served as controls (B) Expression of the MCPIP gene after stimulation of HepG2 cells with IL-6 (15 ngỈmL)1) Cells were stimulated for 4, 8, 12 and 24 h Unstimulated cells served as a control (C) Changes in expression of the MCPIP gene after stimulation of U937 cells with IL-1b (15 ngỈmL)1), TNFa (10 ngỈmL)1), LPS (100 ngỈmL)1) or PMA (100 nM) Cells were stimulated for 1, and h with the above factors Unstimulated cells served as a control FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS 7387 MCPIP protein as an RNase D Mizgalska et al periods ranging from 0.25–4 h increases up to approximately 35 times compared to unstimulated controls The maximum expression is observed at h after stimulation, followed by a slow decrease In contrast to IL-1b, another cytokine important for hepatic cells, IL-6, had no effect on MCPIP gene expression (Fig 1B) In U937 cells, TNFa appears to be the strongest stimulant, increasing the MCPIP transcript level more than 7-fold in comparison with unstimulated cells, but IL-1b, LPS and PMA also activated the MCPIP gene (Fig 1C) These experiments showed that MCPIP belongs to the group of early-response genes Bioinformatic analysis of MCPIP amino acid sequence Application of fold recognition methods allowed to identify potential structural templates for the 130-290 region of the MCPIP1 sequence [5] Ten top scoring Protein Data Bank (PDB) records included 1O4W (the PIN domain from Archaeoglobus fulgidus AF0591 protein; six hits from the various fold recognition servers), 1A76 (FLAP endonuclease-1 from Methanococcus jannaschii) and 1W8I (AF1683 protein of unknown function from Archeoglobus Fulgidus) For greater confidence that the structural alignments created by the GeneSilico.pl server [6] are meaningful, we compared the reported AF0591 PIN ⁄ MCPIP PIN alignment with previously published alignments of human SMG6 (PDB ID: 2HWW) PIN and AF0591 PIN sequences [7,8] The resulting alignment is shown in Fig 2B Four acidic residues (D141, E185, D226, D244) in PIN domains involved in binding Mg2+ ions are conserved in all three sequences Other conserved residues are R263, V139 and N144 Determination of the MCPIP domain architecture was completed using the DISOPRED [9] and SPRITZ servers [10], which consistently indicated that regions 1-50, 90-130 and 290-540 of MCPIP are disordered, with as yet unidentified function (Fig 2A) Distribution of MCPIP transcript in human tissues and cellular localization of MCPIP protein A blot loaded with mRNAs from several human tissues was subjected to Northern blot analysis using a molecular probe spanning the entire coding sequence of the MCPIP gene or the b-actin gene (control) In comparison with the b-actin transcript, the level of MCPIP mRNA was very low From the analyzed tissues, highest expression of the MCPIP gene was observed in leukocytes, with intermediate levels in heart and placenta, and the lowest levels in spleen, kidney, liver and lung The transcript was not detectable in brain, thymus, muscles, small intestine and colon (Fig 3A) The high expression of the MCPIP gene in leucocytes suggests the functional importance of the protein encoded by this transcript for the immunological system Originally, Zhou et al [2] described localization of MCPIP in the nucleus Recently Matsushita et al [4] showed cytoplasmic localization of MCPIP We analyzed the cellular localization of MCPIP using two approaches First, subcellular fractions were isolated A B Fig The domain architecture of MCPIP (A) The disordered regions (D.R.) are shown as grey rectangles (positions 1-50, 90-130 and 340540), the PIN domain is shown as a striped rectangle (positions 130-280), and the C3H type zinc finger (Z.F.) as a white rectangle (positions 306-322) Unannotated protein regions are shown as black bars (B) Alignment of the SMG6 PIN domain (PDB: 2hww), the bacterial hypothetical protein PIN domain (PDB: 1o4w) and the MCPIP PIN domain Identical residues are indicated by asterisks, conserved substitutions as semi-colons and semi-conserved substitution as dots The PIN domain of MCPIP has conserved four acidic residues (D141, E185, D226 and D244) involved in binding Mg2+ ions during RNA degradation 7388 FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS Cytoskeleton Leukocytes Nucleus Small intestine Placenta Lungs Cytosol B Membrane MCPIP protein as an RNase Spleen Kidney Liver Colon Thyroid Brain Heart A Skeletal muscle D Mizgalska et al C Fig Tissue distribution of transcript and cellular localization of MCPIP (A) A human tissue Northern blot (Clontech) was hybridized with a radioactively labelled MCPIP cDNA probe and visualized by autoradiography A b-actin probe served as a control for equal loading A single 2.0 kb band was seen in all lanes A 1.8 kb actin isoform is visible for heart and skeletal muscle The duration of exposure of the Northern blot with MCPIP probe was 20 times longer compared to the blot with the b-actin probe (B) A Qproteome kit (Qiagen) was used to isolate cytosolic, membrane, nuclear and cytoskeletal proteins Protein concentrations were determined using a BCA protein assay (Sigma) Aliquots of 10 lg from each fraction were used in western blot analysis Polyclonal antibodies against MCPIP were used at concentration of lgỈmL)1 As markers specific for cytoskeleton, nucleus, membrane and cytosolic fraction, respectively, antibodies against actin (mouse monoclonal, 1:4000, Sigma), lamin C (rabbit polyclonal, 1:1000, Abcam), MnSOD (rabbit polyclonal, 1:2000, Sigma) and GAPDH (rabbit polyclonal, 1:1000, Abcam) were used (C) A genetic construct overexpressing recombinant MCPIP with fluorescent red protein in the pmaxFP-Red-N vector (Amaxa) was generated for determination of the cellular localization of MCPIP HepG2 cells were transfected with a vector overexpressing MCPIP and with an empty vector (control) using Lipofectamine 2000, according to the manufacturer’s protocol 4¢-6-diamidino-2-phenylindole (DAPI) staining was performed to visualize nuclei Localization of MCPIP in cytoplasm was determined by use of a fluorescent microscope from HepG2 cells overexpressing MCPIP The results of western blot analysis indicate that MCPIP is localized in the cytoskeleton fraction (Fig 3B) In the second approach, HepG2 cells were transfected either with an empty vector, or with a vector expressing a fusion of MCPIP with red fluorescent protein (RFP) Using various concentrations of the plasmid vector overexpressing the fusion protein MCPIP–RFP, we observed that the protein was localized in the cytoplasm, in the form of granules (Fig 3C) It is possible that formation of granules is the result of stress (e.g related to overexpression) Such accumulation of protein has been described previously for other factors engaged in mRNA degradation: for example, tristetraproline and T-cell-restricted intracellular antigen both accumulate in stress granules during environmental stress, which are regarded as dynamic cytoplasmic foci that contain untranslated mRNAs [11,12] Furthermore, the results of western blots show that MCPIP is detected in cytoskeleton These data are in agreement with results obtained by Henics et al [13], who showed that cytoskeleton proteins have binding capacity for proteins involved in mRNA metabolism Moreover, cytoskeleton proteins, such as actin or tubulin, are thought to be crucial for compartmentalization and translation of various mRNAs, including those containing AU-rich instability motifs in the 3¢ UTR [14,15] We performed co-immunoprecipitation and mass spectrometry analyses to identify putative proteins interacting with MCPIP (data not shown) Of the identified proteins, 73% are proteins involved in mRNA stability, but there are also proteins involved in other cellular processes, such as actin Further studies characterizing proteins interacting with MCPIP will clarify the dynamics and features of this protein Role of MCPIP in regulation of the endogenous IL-1b transcript level After identification of a PIN domain in the MCPIP by use of bioinformatics, we decided to check whether MCPIP has any effect on IL-1b transcript level We hypothesized that if IL-1b strongly regulates MCPIP gene expression, MCPIP may be involved in regulation of the IL-1b mRNA level To elucidate the effect of MCPIP on the endogenous IL-1b transcript level, we used primary cultures of human fibroblasts These cells are a good model to study inflammatory processes, because, in contrast to HepG2 cells, expression of IL-1b in fibroblasts is significant and these cells are also important in inflammatory defence Human fibroblasts were transfected with either MCPIP-specific siRNA or a vector overexpressing MCPIP and then FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS 7389 MCPIP protein as an RNase D Mizgalska et al Fig MCPIP regulates IL-1b transcript in LPS-treated human fibroblasts (A) Human fibroblasts were transfected with siRNA specific either for MCPIP (50 nM) or GAPDH (50 nM), or with nonspecific siRNA (negative control, 50 nM) The transfection efficiency observed for carboxyfluorescein (FAM)-labeled scrambled siRNA was estimated as 25% Cells treated only with the transfection reagent siPORT NeoFX were used as a control At 48 h post-transfection, cells were treated with LPS (100 ngỈmL)1) for h RNA was isolated and real-time PCR was performed using primers specific for MCPIP cDNA (control of silencing, right panel) or IL-1b (left panel), using GAPDH as a positive control (data not shown) For GAPDH, 50% inhibition of gene expression was obtained (data not shown) and for MCPIP approximately 40% inhibition of gene expression was obtained The IL-1b transcript level after LPS treatment was expressed as a percentage of the basal transcript level in untreated cells The IL-1b transcript level was compared to that in cells transfected with scrambled siRNA (assumed to be 100%) The values represent the means of three independent experiments (**P < 0.01, versus control) (B) Human fibroblasts were transfected with an empty vector (pcDNA3) or a vector overexpressing wildtype MCPIP The obtained transfection efficiency was 30%, as estimated by use of GFP coding vector Twenty-four hours post-transfection, cells were treated with LPS (100 ngỈmL)1) for h RNA was isolated and real-time PCR was performed using primers specific for MCPIP cDNA (control of overexpression, right panel) or for IL-1b cDNA (left panel) In each case, the IL-1b transcript level after LPS treatment was expressed as a percentage of the basal transcript level in untreated cells The IL-1b transcript level was compared to that in cells transfected with pcDNA3 (assumed to be 100%) The values represent the means of three independent experiments (**P < 0.01, versus control) A B stimulated with LPS (Fig 4A,B) Inhibition of MCPIP gene expression by siRNA (by approximately 40%) led to an increase in the endogenous IL-1b transcript level (by approximately 60%) in comparison to control cells transfected with scrambled siRNA In contrast, cells overexpressing MCPIP had lower IL-1b transcript level (by approximately 80%) in comparison to control cells transfected with an empty vector Despite the moderate transfection efficiency (30% for overexpression and 25% for siRNA), the overall conclusion from the experiment with fibroblasts is that MCPIP regulates the amount of IL-1b mRNA Involvement of PIN domain of MCPIP in the stability of IL-1b mRNA In order to find out whether the PIN domain in MCPIP is responsible for IL-1b mRNA regulation, we 7390 performed an experiment with HepG2 cells (Fig 5A) These cells were transfected with a genetic construct encoding IL-1b cDNA and either wild-type MCPIP, a mutant form of MCPIP (D141A and D226A; D ⁄ AMCPIP), or an empty vector A drastic decrease in the IL-1b transcript level was observed when cells were co-transfected with a vector coding for wild-type MCPIP in comparison to cells transfected with an empty vector or one expressing D ⁄ A-MCPIP (Fig 5, lanes 2,3 and 4, lower band) The levels of mutant and wild-type MCPIP mRNAs were higher when these vectors were co-transfected with a vector overexpressing IL-1b (Fig 5, lanes and 4, upper bands) The absence of a plasmid encoding IL-1b prevented the increase in the MCPIP mRNA level for both forms (Fig 5A, lanes and 6, upper bands) Moreover, in a co-transfection study with the IL-1b-expressing vector, the level of mRNA for the mutant form of MCPIP was higher (Fig 5A, lane 4, upper band) than when wildtype MCPIP was co-transfected (Fig 5A, lane 3, upper band) At this stage of the study, the following hypothesis may be presented: some overexpressed IL-1b is secreted and stimulates the endogenous form of MCPIP, resulting in the observed higher level of MCPIP when FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS D Mizgalska et al MCPIP protein as an RNase A B Fig Involvement of MCPIP in IL-1b mRNA stability (A) For determination of the stability of exogenous IL-1b mRNA, HepG2 cells were transfected with an empty vector pcDNA3, a vector overexpressing IL-1b mRNA, and a vector overexpressing either wild-type MCPIP or its mutant form containing the D141A and D226A mutations (D ⁄ A-MCPIP) The level of transcripts coding for MCPIP and IL-1b was determined by Northern blot Lane 1, pcDNA3; lane 2, pcDNA3 ⁄ IL-1b; lane 3, wild-type MCPIP ⁄ IL-1b; lane 4, D ⁄ A-MCPIP ⁄ IL1b; lane 5, pcDNA3 ⁄ MCPIP; lane 6, pcDNA3 ⁄ D ⁄ A-MCPIP The figure is representative of three independent experiments, and the level of analyzed transcripts was compared to that for 28S rRNA (B) To determine the half life of the IL-1b transcript, HepG2 cells stably overexpressing IL-1b mRNA were transfected either with an empty vector or with vector overexpressing wild-type MCPIP or its mutant forms: mutMCPIP without the PIN domain or D ⁄ A-MCPIP with two mutated amino acids located within the PIN domain (D141A and D226A) Transcription was inhibited by addition of actinomycin D, and RNA was collected after 3, and h The mRNA level for IL-1b (top panel) and MCPIP (bottom panel) was determined by Northern blot The middle panel shows inverted images of ethidium bromide-stained 18S and 28S rRNA on the membrane The graphs below the Northern blot present data from densitometry of the IL-1b transcript level normalized to the rRNA amount, calculated from four independent experiments (*P < 0.05 compared with control) plasmids expressing IL-1b are co-transfected Evidence supporting this idea includes the presence of MCPIP in HepG2 cells overexpressing IL-1b and its absence in cells transfected with an empty vector (data not shown) Wild-type MCPIP (endogenous and exogenous) starts degradation of IL-1b mRNA and also of its own transcript For this reason, the level of mRNA for the wildtype form of MCPIP and the level of mRNA for IL-1b (Fig 5A, lane 3) were lower in comparison to the level of mRNA for MCPIP with a mutation within the PIN domain (Fig 5A, lane 4) The same tendency is seen at the protein level (data not shown) In order to determine whether the observed IL-1b mRNA changes are due to a decrease in transcription or an increased degradation rate, we performed experiments with actinomycin D HepG2 cells stably overexpressing IL-1b mRNA were co-transfected with an empty vector or a vector overexpressing wild-type MCPIP or its mutant forms [mutMCPIP (without the PIN domain) and D ⁄ A-MCPIP (with two mutated residues within PIN domain)] The cells were harvested 0, 3, and h after addition of actinomycin D, and the level of exogenous IL-1b mRNA was analyzed by Northern blot A significant reduction in the IL-1b transcript level was observed only when the wild-type MCPIP was overexpressed (Fig 5B) Importance of AREs in regulation of IL-1b mRNA by MCPIP In order to determine whether AREs are important in mRNA degradation triggered by MCPIP, we used FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS 7391 MCPIP protein as an RNase D Mizgalska et al A B C Fig Importance of AREs in MCPIP-dependent mRNA degradation (A) The 3¢ UTR sequence of the transcript coding for IL-1b (NCBI accession number NM_000576) The genetic construct used in the transfection experiment encoding IL-1b cDNA without AREs (IL-1)ARE) contained 343 bp of the 3¢ UTR region (nucleotides 898–1227 of the 3¢ UTR region) The construct encoding IL-1b cDNA with AREs (IL1+ARE) contained 423 bp of the 3¢ UTR (nucleotides 898–1318 of the 3¢ UTR region) The stop codon (positions 895-897) and AU-rich region (positions 1242-1258) are indicated by open boxes Capital letters indicate the sequence of reverse primers used in generation of the PCR products used for construction of vectors expressing transcripts for IL-1b with ⁄ without AREs (IL-1)ARE and IL-1+ARE) Primers used for amplification of AREs used in the generation of a genetic construct containing a minimal promoter and cDNA for luciferase with AREs (p-lucARE) are indicated by shaded boxes (B) Three genetic constructs were generated in order to determine whether MCPIP is involved in IL-1b mRNA degradation: IL-1b cDNA without AREs [IL-1)ARE; detailed description in (A)], IL-1b cDNA with AREs [IL-1+ARE; detailed description in (A)] and MCPIP cDNA (MCPIP) HepG2 cells were transfected with the following combination of vectors: pcDNA3, pcDNA3 ⁄ IL-1+ARE, pcDNA3 ⁄ IL-1)ARE, pcDNA3 ⁄ MCPIP, MCPIP ⁄ IL-1+ARE, MCPIP ⁄ IL-1)ARE and pcDNA3 ⁄ MCPIP The pcDNA3 vector served as a control for the experiment, and to equalize the amount of introduced genetic material The level of IL-1b and MCPIP transcripts was measured by Northern blot 24 h after transfection In lanes and 6, the endogenous IL-1b transcript is visible The left panel is representative of three independent experiments The panel on the right shows densitometric measurements of the IL-1b transcript level normalized to the 18S rRNA band Student’s t test was used to determine significant differences from the control (*P < 0.05, **P < 0.01) (C) Two genetic constructs consisting of a minimal promoter and cDNA for luciferase with (p-luc-ARE) or without AREs (p-luc) from the IL-1b transcript were generated HepG2 cells were co-transfected with combination of p-luc-ARE and pcDNA3 empty vector (control), or wild-type MCPIP or a mutant form of MCPIP lacking the domain with RNase activity The same combinations were used for the plasmid expressing luciferase transcript without ARE sequences in the 3¢ UTR (p-luc) As an internal transfection control, a pEF1 ⁄ Myc-His ⁄ Gal vector encoding b-galactosidase was used The bars represent the luciferase activity calculated from three independent experiments, normalized to that for b-galactosidase Overexpression of MCPIP was confirmed by western blotting (right upper panel) Semi-quantitative RT-PCR was performed for the luciferase transcript to show the reporter transcript level (lower panel) The GAPDH transcript served as a housekeeping gene 7392 FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS D Mizgalska et al three genetic constructs: (a) a construct overexpressing IL-1b transcript with AREs (long 3¢ UTR, Fig 6A), (b) a construct overexpressing IL-1b transcript without AREs (short 3¢ UTR, Fig 6A), and (c) a construct overexpressing the MCPIP transcript HepG2 cells were co-transfected with vector containing IL-1b cDNA with or without AREs or with the vector containing MCPIP cDNA The levels of IL-1b transcript with ⁄ without AREs and the level of MCPIP transcript were determined using Northern blotting 24 h after transfection The endogenous MCPIP transcript is not detectable due to the short exposure time The level of IL-1b transcripts containing AREs was lower than the level of IL-1b mRNA without AREs (Fig 6B, lanes 2–5) These results are in agreement with data indicating that transcripts with AREs are more susceptible to degradation [16] In both cases, overexpression of MCPIP (IL-1b + ARE and IL - 1b )ARE) triggers a significant decrease in the level of IL-1b mRNAs Thus, the MCPIP level correlates with the fast degradation of IL-1b transcript independently of the presence of AREs (Fig 6B, lanes and 5) It is possible that in the region located before the AREs, consisting of 343 bp of the 3¢ UTR of the IL-1b transcript, there is a putative RNA stem–loop structure sequence similar to that present in the 3¢ UTR of IL-6 mRNA [4,17] The presence of such a sequence may account for the degra- MCPIP protein as an RNase dation of both types of IL-1b transcripts, with and without AREs The influence of MCPIP on the IL-1b transcript level was also noted for the endogenous IL-1b transcript (Fig.6B, lane versus lane 6) To confirm that AREs are not involved in the observed IL-1b transcript turnover, we prepared two genetic constructs consisting of a minimal promoter and the cDNA for luciferase with (p-luc)ARE) or without AREs (p-luc) from the IL-1b transcript (Fig 6A) The plasmid p-luc)ARE was transfected into HepG2 cells in combination with a plasmid coding for wild-type MCPIP or its mutant form lacking the PIN domain The same combination was used for the plasmid expressing the luciferase transcript without ARE sequences in the 3¢ UTR (p-luc) The luciferase activity was measured after 22 h As shown in Fig 6C, the presence of ARE sequence has no influence on the MCPIP-dependent turnover of luciferase transcript by MCPIP In both cases, when the constructs p-luc)ARE or p-luc were used, differences in luciferase transcript level ⁄ activity were not detectable between the control and samples overexpressing wild-type MCPIP or its mutant form lacking the PIN domain Nevertheless, the levels of luciferase transcript (Fig 6C, right panel) and luciferase activity (Fig 6C, left panel) were significantly lower in all samples (including control) when AREs were present This A B Fig RNA immunoprecipitation (A) Control of the experimental model HepG2 cells were transfected with a vector overexpressing wildtype MCPIP RNA was isolated and cDNA synthesis was performed Expression of genes encoding exogenous MCPIP (cells with a vector overexpressing MCPIP) or endogenous transcripts encoding IL-1b and GADPH was determined by semi-quantitative PCR (with EF-2 as a reference transcript) A total RNA sample (0.1 lg RNA) served as a negative control (cDNA-) (B) To bind RNA templates to proteins, HepG2 cells were subjected to crosslinking with formaldehyde, lysed and incubated with antibodies specific for MCPIP or with IgG as a negative control Then the samples were digested with DNase to remove traces of genomic DNA Reverse transcription was performed using RNA isolated from immunoprecipitates, followed by PCR with MCPIP-, IL-1b- and GADPH-specific primers (35 cycles) The same RNA but not reverse-transcribed served as a negative control for the PCR reaction Specific transcripts coding for MCPIP and IL-1b were detected only in immunoprecipitates obtained with antibodies specific for MCPIP, but not with IgG antibodies Specificity of the PCR product was determined by restriction analysis (data not shown) PCR product was not observed for the negative control, i.e with primers specific for the GAPDH gene FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS 7393 MCPIP protein as an RNase D Mizgalska et al means that IL-1b AREs are important in luciferase mRNA degradation but this process is performed in an MCPIP-independent manner Interaction of MCPIP protein with the IL-1b transcript An RT-PCR reaction was performed to confirm that HepG2 cells synthesize endogenous IL-1b mRNA As shown in Fig 7A, there is expression of the gene encoding IL-1b, although the transcript level is low in comparison to that of the GAPDH transcript To investigate interactions of MCPIP with the IL-1b transcript, immunoprecipitation of RNA-binding protein was carried out In this experiment, HepG2 cells were transfected with vector overexpressing MCPIP Then HepG2 cells were subjected to crosslinking with formaldehyde in order to bind RNA to protein complexes, lysed and incubated with antibodies specific for MCPIP, or with IgG as a negative control After extraction of RNA from immunoprecipitates, reverse transcription was performed, followed by PCR with primers specific for MCPIP, IL-1b and GADPH (Fig 7B) The amount of IL-1b product was significantly lower than that of MCPIP because endogenous expression of the IL-1b gene in HepG2 cells is quite low (Fig 7A) Amplification of the MCPIP transcript in the immunoprecipitates indicates that this protein interacts with its own transcript and probably regulates its stability We did not observe any product in PCR reactions with primers specific for GAPDH This indicates that IL-1b and MCPIP mRNAs, but not GAPDH mRNA, interact with MCPIP The obtained result confirms the involvement of MCPIP in mRNA processing Discussion It is well known that gene expression of proinflammatory mediators is tightly controlled, both at the transcriptional and post-transcriptional levels The majority of transcripts coding for cytokines and chemokines (IL-1, IL-2, IL-6, TNFa, GM-CSF, interferons b and c) contain regulatory elements in the 3¢ UTR that are important for the half life of cytokine mRNAs For this reason, transcription of genes encoding cytokines is not sufficient to guarantee the appearance of functional proteins As reported by Kaspar and Gehrke [18] and Dinarello [19], stimulation of macrophages by mild stimulants (such as C5a complement or some modified proteins) leads to accumulation of specific mRNAs without their translation This is due to the formation of translationally inactive 7394 mRNA–protein complexes These complexes may either result in mRNA degradation or its conversion into translationally active forms by the action of strong stimulants, such as endotoxin (LPS) One of the regulatory sequences involved in determining mRNA half life are the AREs, which can stimulate deadenylation of mRNA [20] and its degradation by the exosome in the 3¢ fi 5¢ direction [21] In addition to mRNA degradation mediated by the exosome, RNA can be degraded through removal of the 7-methyl guanosine cap, followed by 5¢ fi 3¢ mRNA degradation in cytoplasmic processing bodies (P-bodies) [22] One of the best known examples of AREs binding proteins involved in inflammatory processes is tristetraprolin [23] It was shown that tristetraprolin enhances the deadenylation and degradation of mRNA for granulocyte ⁄ macrophage colony-stimulating factor, TNFa and IL-2 Using human monocyte-derived macrophages exposed to IL-1b, we found that the MCPIP gene is strongly regulated by this cytokine [1] Our study on the function of the novel protein started with the use of bioinformatic tools to analyse MCPIP structure It was already known that MCPIP has a single CCCH zinc-binding domain [2], which, similarly to K homology (KH), RNA-recognition motif (RRM), double-stranded RNA-binding domain (dsRBD) and Piwi Argonaut and Zwille (PAZ), represents an RNA-binding domain These domains are usually linked to larger globular enzymatic domains, such as nucleases and nucleotide modifying enzymes that are important in RNA metabolism [24,25] Our bioinformatic analyses of the MCPIP sequence showed that MCPIP contains a centrally located PIN-like domain, presumably with endo ⁄ exonuclease activity, together with relatively large fragments that show disordered structure The PIN domain spans amino acids 130-280 Despite only marginal sequence similarity, the various PIN domains that are found in all kingdoms of life have a highly conserved structure with 5¢ fi 3¢ exonuclease activity [26] We found that the PIN domain of MCPIP, similar to another protein with confirmed RNase activity – SMG6 (2hww) [7] – has the canonical four conserved acidic residues (D141, E185, D226 and D244; Fig 2B) that are involved in binding Mg2+ ions during RNA degradation By analogy to SMG6, we hypothesized that MCPIP may be directly involved in mRNA degradation, with IL-1b mRNA being one of the targets To confirm our hypothesis, we used genetic constructs overexpressing full-length cDNA for IL-1b, wild-type MCPIP and its mutant forms The level of IL-1b mRNA was decreased in cells overexpressing FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS D Mizgalska et al wild-type MCPIP in comparison with a mutant form Inhibition of MCPIP expression by siRNA leads to the increase in IL-1b transcript level These data are in agreement with the results obtained by Liang et al [3] MCPIP-mediated downregulation of the half life of the IL-1b transcript was confirmed in the presence of actinomycin D in HepG2 cells stably overexpressing IL-1b mRNA We found that the stability of IL-1b transcript is dependent on the type of MCPIP expressed by the vector introduced into these cells In control cells (empty vector) and cells overexpressing a mutant form of MCPIP (without the PIN domain, or MCPIP with two mutated amino acid residues), the transcript coding for IL-1b was stable However, when wild-type MCPIP was present, a decrease in the transcript level for IL-1b was observed We have also shown that IL-1b transcript degradation is ARE-independent We used genetic constructs with cDNA coding for interleukin-1 but either containing or lacking the region with ARE sequences Co-transfection of constructs with or without ARE sequences with the vector overexpressing MCPIP resulted in efficient disappearance of the IL-1b transcript The degradation was independent of the presence of AREs, as confirmed in the experiment with luciferase vector Recently, Matsushita et al showed that MCPIP is engaged in the regulation of mRNA stability in inflammatory processes These authors found that MCPIP has RNase properties and regulates the stability of IL-6 and IL-12p40 mRNA [4] MCPIP-dependent degradation of IL-6 mRNA is performed via its 3¢ UTR, but the mechanism is independent of ARE sequences [4] Data obtained in knockout mice for the MCPIP gene showed that MCPIP is essential for inhibition of the development of severe autoimmune responses that culminate in lethality [4] Knockout mice showed increased production of IL-6 and IL-12p40 but not TNF alpha in macrophages, due to failure of mRNA degradation Our data showing that MCPIP is involved in mRNA degradation are in agreement with the results of Matsushita et al [4] As for IL-6 mRNA [4], cis-acting elements that are important in MCPIP-mediated IL-1b mRNA degradation are localized in the 3¢ UTR of this transcript Moreover, as for the IL-6 transcript [4], IL-1b ARE sequences not participate in degradation performed by MCPIP In addition to regulation of transcripts encoding proinflammatory cytokines (IL-1b and IL-6), it appears that MCPIP regulates its own transcript This was demonstrated by the RNA immunoprecipitation experiment Self-regulation has already been described for proteins engaged in the control of RNA stability, such as tristetraprolin [27] MCPIP protein as an RNase Our results confirm the data of Matsushita et al [4] showing that MCPIP has RNase properties performed by the PIN domain We found that MCPIP is activated by many stimulants, and regulates the level of IL-1b mRNA and of its own transcript Further studies are necessary to clarify the biological role of MCPIP as a nuclease and to identify the cis-acting sequences in the 3¢ UTR of the IL-1b and MCPIP transcripts that directly interact with MCPIP However, as MCPIP regulates the stability of transcripts encoding the two major proinflammatory cytokines IL-1b and IL-6, as well as of its own transcript, it may be regarded as a very important potential regulator of inflammatory processes Experimental procedures Cell culture and stimulation with cytokines All cell lines were grown under standard conditions ()37 °C and 5% CO2) HepG2 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium with 1000 mgỈL)1 d-glucose (Gibco, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum Cells were grown in plastic Petri dishes (6 cm in diameter, BD Biosciences, Franklin Lakes, NJ, USA) to approximately 70% confluence At 15 h before stimulation with cytokine, cells were washed twice in NaCl ⁄ Pi and placed in Dulbecco’s modified Eagle’s medium containing 0.5% fetal bovine serum U937 cells (American Type Culture Collection) were cultured and stimulated in RPMI-1640 medium (Gibco ⁄ BRL) supplemented with fetal bovine serum to a final concentration of 10% Human skin fibroblasts were grown in modified Eagle’s medium (MEM) with 1000 mgỈL)1 d-glucose supplemented with 10% fetal bovine serum The final concentrations of stimulants were optimized experimentally, and were as follows: human IL-1b, 15 ngỈmL)1 (BiolMol, Plymouth Meeting, PA, USA); human recombinant IL-6, 15 ngỈmL)1 (Strathmann Biotec AG, Hamburg, Germany); human TNFa, 10 ngỈmL)1 (Suntory Pharmaceuticals, Cambridge, MA, USA); LPS, 100 ngỈmL)1 (Sigma, St Louis, MO, USA); PMA, 100 nm (Calbiochem, Darmstadt, Germany) Real-time PCR Total RNA was isolated from unstimulated cells (control) and cytokine-stimulated cells, as described previously [28] The first strand of cDNA was synthesized from lg of total RNA using MMLV reverse transcriptase (Promega, Madison, WI, USA) and oligo(dT) primer Real-time PCR was performed using SYBR Green PCR master mix (DyNAmoÔ HS SYBR Green qPCR; Finnzymes, Espoo, Finland), ll cDNA and 20 ng suitable forward and reverse primers For FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS 7395 MCPIP protein as an RNase D Mizgalska et al amplification, the following primers were used: IL-1b cDNA, 5¢-GATGTCTGGTCCATATGAACTG-3¢ (forward) and 5¢-TGGGATCTACACTCTCCAGC-3¢ (reverse); MCPIP cDNA, 5¢-GGAAGCAGCCGTGTCCCTATG-3¢ (forward) and 5¢-TCCAGGCTGCACTGCTCACTC-3¢ (reverse) Each sample was normalized to reference genes: elongation factor (EF-2), 5¢-GACATCACCAAGGGTGTGCA-3¢ (forward) and 5¢-TTCAGCACACTGGCATAGAGGC-3¢ (reverse); GAPDH, 5¢-CCGAGCCACATCGCTCAGAC-3¢ (forward) and 5¢-GTTGAGGTCAATGAAGGGGTC-3¢ (reverse) The relative level of transcripts was quantified by the DDCT method Northern blot analysis For determination of MCPIP and IL-1b transcript levels, 10 lg of total RNA were separated in a 1% formaldehyde agarose gel and blotted to a nitrocellulose membrane Prehybridization and hybridization were performed at 65 °C in 1% SDS, m NaCl, 10% dextran sulfate solution Probes were generated by PCR amplification of the entire coding region of studied genes A random primer labelling kit (Promega) was used to label 30 ng of PCR product with [a-32P]dCTP After washing (20 at room temperature in 2· SSC, 20 at 65 °C in 2· SSC, and twice for 20 at 65 °C in 1· SSC ⁄ 1% SDS), RNA blots were scanned using a Molecular Imager FX (Bio-Rad, Hercules, CA, USA) All densitometry measurements were performed using Quantity One software, and transcripts amounts were normalized to the rRNA level In order to analyse the tissue distribution of the MCPIP transcript, a blot loaded with lg of each of the poly(A)+ RNAs from several human tissues (Clontech, Mountain View, CA, USA) was used The blot was later stripped and reprobed using the b-actin probe provided with the kit to ensure equal loading The probes for MCPIP and actin transcript were labelled with the same specific activity Hybridization was performed as described above Bioinformatic analysis of the MCPIP amino acid sequence The Genesilico.pl server [5] was used to predict the domain architecture of MCPIP The disordered regions of MCPIP were predicted using the DISOPRED [9] and SPRITZ [10] servers The PCONS method [6] was used for consensus fold recognition prediction for the MCPIP PIN domain Multiple sequence alignment of target ⁄ template PIN domain sequences was performed using clustalx [8] Generation of genetic constructs The coding sequences of the wild-type and mutant forms of MCPIP lacking the PIN domain were obtained by two-step 7396 PCR For the MCPIP gene, a first round of PCR was carried out using forward primer 5¢-CCGCTGGCGCATG GCGGGTAGG-3¢ and reverse primer 5¢-GGGGTGGGCC TCAGGGCTGGG-3¢ Then nested primers 5¢-GTCTGA GCTATGAGTGGCCC-3¢ (forward) with a restriction site for KpnI and 5¢-CAGCTTACTCACTGGGGTGC-3¢ (reverse) with a restriction site for EcoRI were used to obtain a region of 1813 bp, corresponding to positions -9 to 1804 relative to the start codon (ATG) of the sequence NM_025079 The obtained fragment was cloned into a pcDNA3 vector (Invitrogen, Carlsbad, CA) The same sequence was used to design primers for generation of a mutant form of MCPIP, without a PIN domain (mutMCPIP) The fragment from 412-888 bp, corresponding to the PIN domain, was removed from the entire coding sequence of the MCPIP gene The coding sequence lacking the PIN domain was obtained in two steps First, the fragment of 423 bp situated in front of the PIN domain was amplified by PCR using forward primer 5¢CGGGATCCCGGAGTCTGAGCTATGAGTG-3¢ with a restriction site for BamHI and reverse primer 5¢-CACT GGTCTCAGGTCGCTG-3¢ with a restriction site for BsaI, and cloned into pcDNA3 at the same restriction sites Then a second fragment situated after the PIN domain was generated by PCR using primers 5¢-AGCGACCTGAGAC CACTCACTTTGGAGCAC-3¢ (BsaI) and 5¢-GGAATTC CCTCACTGGGGTGCTGG-3¢ (EcoRI) This fragment was cloned into pcDNA3 containing the first fragment at the BsaI ⁄ EcoRI restriction sites A genetic construct with mutation of two conserved amino acids residues within the PIN domain (D141A and D226A) was generated by sitedirected mutagenesis (Stratagene, Cedar Creek, TX, USA) A genetic construct overexpressing recombinant MCPIP with fluorescent red protein (pmaxFN-MCPIP-RFP) was generated by PCR using forward primer 5¢-CGCTAG CTATGGGCCCCTGTGGAGA-3¢ with a restriction site for NheI and reverse primer 5¢-GAGATCTAACTCACTG GGGTGCTGGG-3¢ with a restriction site for BglII The PCR product was cloned into the pmaxFP-Red-N vector (Amaxa, Cologne, Germany) using the same restriction sites to produce pmaxFP-MCPIP-RFP The coding sequences of IL-1b with AREs (IL-1+ARE) and IL-1b without AREs (IL-1)ARE) were obtained by two-step PCR For the IL1b transcript with ⁄ without AREs, the first round of PCR was performed using forward primer 5¢-CAAGGCACAA CAGGCTGCTC-3¢ and reverse primer 5¢-GAGAGCA CACCAGTCCAAATTG-3¢ Then a second round of PCR was performed with nested primers 5¢-TGAAGCA GCCATGGCAG-3¢ (forward) with a restriction site for KpnI and 5¢-GGAAGCGGTTGCTCATCAG-3¢ (reverse) with a restriction site for XbaI for IL-1)ARE or 5¢-CAGA CACTGCTACTTCTTG-3¢ (reverse), also with an XbaI restriction site, for IL-1+ARE (Fig 6A) Primers were designed using the sequence accession number NM_000576 FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS D Mizgalska et al All PCR products were cloned into the pcDNA3 vector using appropriate restriction sites All genetic constructs were sequenced before the transfection experiment To prepare a reporter vector consisting of a minimal promoter and cDNA for luciferase with or without AREs from IL-1b cDNA (p-luc)ARE and p-luc), the region flanking the AREs in IL-1b cDNA was amplified using forward primer 5¢-ATTCGCTCCCACATTCTGATG-3¢ and reverse primer 5¢-ACACTGCTACTTCTTGCC-3¢ Both primers had restriction sites for XbaI The PCR product was cloned into pGL3-Promoter (Promega) at the XbaI site, and orientation of the insert was confirmed by PCR Transient transfection For transient transfection experiments, HepG2 cells and human fibroblasts were cultured in six-well plates to approximately 80% confluency The next day, cells were transfected using Lipofectamine 2000 (Invitrogen) in serumfree Opti-MEM (Invitrogen), according to the manufacturer’s protocol Empty pcDNA3 vector was used as a control for the experiment or to equalize the amount of genetic material introduced MCPIP protein as an RNase by adding lgỈmL)1 actinomycin D The cells were harvested, and RNA was isolated at various time points as indicated Northern blot analyses were performed to determine the level of exogenous IL-1b mRNA Reporter gene assay Transient transfection experiments were performed using Lipofectamine 2000 reagent (Invitrogen) in a 12-well plate In each well, the pGL3 reporter gene with ARE from IL-1b 3¢ UTR (p-luc)ARE) or without AREs (p-luc) was co-transfected with an empty vector (pcDNA3), and vector expressing either wild-type or mutant MCPIP lacking the PIN domain (mutMCPIP) Luciferase assays were performed 22 h after transfection using the Dual-Light System (Tropix, Foster City, CA, USA) reporter gene as described by the manufacturer The luciferase activity of each construct was normalized to b-galactosidase activity as an integral transfection control Total RNA was isolated as described above Semi-quantitative RT-PCR was performed using primers specific for luciferase (forward 5¢-AGA GATACGCCCTGGTTCCT-3¢; reverse 5¢-AATCTGACG CAGGCAGTTCT-3¢) and GAPDH transcripts (sequences given above) siRNA transfection The pre-designed siRNAs obtained from Ambion (Austin, TX, USA) included GAPDH siRNA as a positive control and siRNA with a scrambled sequence as a negative control We tested six different siRNA specific for the MCPIP gene The most potent inhibitor appeared to be the sequence 5¢CCCUGUUGAUACACAUUGUTT-3¢ Human fibroblasts were cultured in 12-well plates The siRNA concentration and amount of transfection reagent required were optimized experimentally Before transfection, cells were trypsinized, centrifuged at 1000 g at °C for min, and resuspended in fresh medium The lipid-based transfection reagent siPORT NeoFX (4 ll per well) from Ambion and siRNAs (50 nm final concentration) were separately diluted in 50 lL Opti-MEM and then mixed together Transfection complexes were allowed to form for 10 min, and overlaid with · 104 cells per well As an additional control, some wells were treated with transfection reagent only Silencing of MCPIP was confirmed by real-time PCR in three independent experiments Stability assay for IL-b1 mRNA HepG2 cells stably transfected with a vector overexpressing IL-1b mRNA were transfected with an empty vector or one overexpressing either wild-type MCPIP or its mutant forms – mutMCPIP without the PIN domain and D ⁄ A-MCPIP with two conservative amino acid mutations within the PIN domain Transcription was stopped 24 h after transfection Generation of rabbit antibodies specific for MCPIP, and western blot analysis E coli BL21 cells were transfected with pQE-31 vector (Qiagen, Dusseldorf, Germany) containing the entire coding ă sequence of the MCPIP transcript The recombinant protein was purified from bacterial culture using BD Talon metal affinity resins with cobalt ions (BD Biosciences) and used for immunization of a New Zealand White rabbit (three times 300 lg of antigen) Antibodies were purified from rabbit sera by protein A–Sepharose chromatography and then checked for specificity Generated antibodies were used for western blot analysis of protein extracts from HepG2 cells transfected with a vector overexpressing MCPIP Several independent experiments showed that there was no cross-reactivity, and only a specific product of the expected size was observed on membranes For subcellular fractionation of HepG2 cells overexpressing MCPIP, the Qproteome kit (Qiagen) was used, and all steps were performed as described by the manufacturer Consecutive centrifugation and extraction with suitable buffers allowed isolation of cytosolic, membrane, nuclear and cytoskeletal proteins Protein concentrations were determined using a BCA protein assay (Sigma) Aliquots of 10 lg from each fraction were used in western blot analysis To detect markers of particular fractions, the following antibodies were used: polyclonal antibodies against lamin C (Abcam, Cambridge, MA, USA) for the nuclear fraction, polyclonal antibodies against GAPDH FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS 7397 MCPIP protein as an RNase D Mizgalska et al (Abcam) for the cytosolic fraction, polyclonal antibodies against MnSOD (Sigma) for the membrane fraction and monoclonal antibodies against b-actin (Sigma) for the cytoskeleton fraction Secondary antibodies used: goat anti-mouse (BD Biosciences), goat anti-rabbit (Sigma) RNA immunoprecipitation HepG2 cells were transfected with plasmid coding for MCPIP After 24 h, cells were crosslinked with 1% formaldehyde, and subsequently washed with NaCl ⁄ Pi containing mm phenylmethanesulfonyl fluoride and 125 mm glycine RNAsin (100 mL)1, Promega) was added to all buffers Cells were lysed with buffer containing 1% SDS, 10 mm EDTA, 50 mm Tris ⁄ HCl, pH Lysates were centrifuged at 14 000 g for 10 at °C, and the supernatant obtained was diluted 10-fold using IP buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm NaCl, protease inhibitor complex (Roche, Basel, Switzerland), 16.7 mm Tris ⁄ HCl, pH 8] Protein A–agarose (Roche) used for pre-cleaning of the lysate, was washed with IP buffer, blocked with mgỈmL)1 BSA, and 20 lgỈmL)1 salmon DNA (Sigma) Then the sample was divided into two parts and antibodies were added: anti-MCPIP to the first and rabbit polyIgG as a negative control to the second Samples were incubated overnight at °C with constant mixing The next day, blocked protein A–agarose beads were added After h of incubation at °C, the immunoprecipitates were centrifuged (at 14 000 g, 10 min, °C) and washed twice each with four buffers (B-I: 0.1% SDS, 1% Triton X-100, mm EDTA, 150 mm NaCl, 20 mm Tris ⁄ HCl, pH 8; B-II: 0.1% SDS, 1% Triton X-100, mm EDTA, 500 mm NaCl, 20 mm Tris ⁄ HCl, pH 8; B-III: 0.25 m LiCl, 1% Nonidet P-40, 1% sodium deoxycholate, mm EDTA, 10 mm Tris ⁄ HCl, pH 8; B-IV: mm EDTA,10 mm Tris ⁄ HCl, pH 8) RNA was eluted from beads with buffer containing 1% SDS and 0.1 m NaHCO3 After addition of NaCl to 0.2 m concentration, samples were reverse crosslinked by incubation at 65 °C for h, with subsequent protein digestion by proteinase K RNA was isolated from the immunoprecipitates using the phenol:chlorophorm method To facilitate precipitation with isopropanol, yeast tRNA (Sigma) was added to the samples to final concentration of mgỈmL)1 Residual DNA removal was performed by DNase digestion (Ambion) The RNA obtained was reverse-transcribed, and PCR reactions were performed using IL-1b-, MCPIP- and GAPDH-specific primers (sequences given above) Statistical analysis The data from real-time PCR, Northern blotting and measurements of luciferase activity were examined for statistical significance using Student’s t test All values are means ± SD (n = 3) 7398 Acknowledgments The study was supported by the European Union’s FP6 project Savebeta (LSHM-CT-2006-036903, MTKD-CT-2006-042586 and COST Action BM0602) and by the Polish Ministry of Scientific Research and Information Technology (2 P05A01127, 63 ⁄ 6PRUE ⁄ 2007 ⁄ and 339 ⁄ 6PR-UE ⁄ 2007 ⁄ 7) We would like to thank Professor Sigurd Lenzen (Institute of Clinical Biochemistry, Hannover Medical School, Hannover ⁄ Germany) and Dr Lindsay Ramage (Center of Inflammation, University of Edinburgh, UK) for their constructive criticism References ´ Jura J, Wegrzyn P, Korostynski M, Guzik K, OczkoWojciechowska M, Jarzab M, Kowalska M, Piechota ˛ M, Przewocki P & Koj A (2008) Identification of interleukin-1 and interleukin-6-responsive genes in human monocyte-derived macrophages using microarrays Biochim Biophys Acta 1779, 383–389 Zhou L, Azfer A, Niu J, Graham S, Choudhury M, Adamski FM, Younce C, Binkley PF & Kolattukudy PE (2006) Monocyte chemoattractant protein-1 induces a novel transcription factor that causes cardiac myocyte apoptosis and ventricular dysfunction Circ Res 98, 1177–1185 Liang J, Wang J, Azfer A, Song W, Tromp G, Kolattukudy PE & Fu M (2008) A novel CCCH-zinc finger protein family regulates proinflammatory activation of macrophages J Biol Chem 283, 6337–6346 Matsushita K, Takeuchi O, Standley DM, Kumagai Y, Kawagoe T, Miyake T, Satoh T, Kato H, Tsujimura T, Nakamura H et al (2009) Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay Nature 458, 1185–1190 Wallner B & Elofsson A (2006) Identification of correct regions in protein models using structural, alignment, and consensus information Protein Sci 15, 900–913 Kurowski MA & Bujnicki JM (2003) GeneSilico protein structure prediction meta-server Nucleic Acids Res 31, 3305–3307 Glavan F, Behm-Ansmant I, Izaurralde E & Conti E (2006) Structures of the PIN domains of SMG6 and SMG5 reveal a nuclease within the mRNA surveillance complex EMBO J 25, 5117–5125 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F & Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 25, 76–82 Ward JJ, Sodhi JS, McGuffin LJ, Buxton BF & Jones DT (2004) Prediction and functional analysis of native FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS D Mizgalska et al 10 11 12 13 14 15 16 17 18 disorder in proteins from the three kingdoms of life J Mol Biol 337, 635–645 Vullo A, Bortolami O, Pollastri G & Tosatto S (2006) Spritz: a server for the prediction of intrinsically disordered regions in protein sequences using kernel machines Nucleic Acids Res 34, W164–W168 Stoecklin G, Stubbs T, Kedersha N, Wax S, Rigby WF, Blackwell TK & Anderson P (2004) MK2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and ARE-mRNA decay EMBO J 23, 1313–1324 Kedersha NL, Gupta M, Li W, Miller I & Anderson P (1999) RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2a to the assembly of mammalian stress granules J Cell Biol 147, 1431–1442 Henics T (1999) Microfilament-dependent modulation of cytoplasmic protein binding to TNFa mRNA AU-rich instability element in human lymphoid cells Cell Biol Int 23, 561–570 Sirenko O, Bocker U, Morris JS, Haskill JS & Watson ă JM (2002) IL-1b transcript stability in monocytes is linked to cytoskeletal reorganization and the availability of mRNA degradation factors Immunol Cell Biol 80, 328–339 Sweet TJ, Boyer B, Hu W, Baker KE & Coller J (2007) Microtubule disruption stimulates P-body formation RNA 13, 493–502 Roca FJ, Cayuela ML, Secombes CJ, Meseguer J & Mulero V (2007) Post-transcriptional regulation of cytokine genes in fish: a role for conserved AU-rich elements located in the 3¢-untranslated region of their mRNAs Mol Immunol 44, 472–478 Paschoud S, Dgar AM, Kuntz C, Grisoni-Neupert B, Richman L & Kuhn LC (2006) Destabilization of interleukin-6 mRNA requires a putative RNA stem–loop structure, an AU-rich element, and the RNA-binding protein AUF1 Mol Cell Biol 26, 8228–8241 Kaspar RL & Gehrke L (1994) Peripheral blood mononuclear cells stimulated with C5a or lipopolysaccharide to synthesize equivalent levels of IL-1beta MCPIP protein as an RNase 19 20 21 22 23 24 25 26 27 28 mRNA show unequal IL-1beta protein accumulation but similar polyribosome profile J Immunol 153, 277– 286 Dinarello C (1996) Biologic basis for interleukin-1 in disease Blood 87, 2095–2147 Lai WS, Carballo E, Strum JR, Kennington EA, Phillips RS & Blackshear PJ (1999) Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA Mol Cell Biol 19, 4311–4323 Haile S, Estevez AM & Clayton C (2003) A role for the exosome in the in vivo degradation of unstable mRNAs RNA 9, 1491–1501 Sheth U & Parker R (2003) Decapping and decay of messenger RNA occur in cytoplasmic processing bodies Science, 300, 805–808 Hau HH, Walsh RJ, Ogilvie RL, Williams DA, Reilly CS & Bohjanen PR (2007) Tristetraprolin recruits functional mRNA decay complexes to ARE sequences J Cell Biochem 100, 1477–1492 Cerutti L, Mian N & Bateman A (2000) Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain Trends Biochem Sci 25, 481–482 Anantharaman V & Aravind L (2006) The NYN domains: novel predicted RNAses with a PIN domainlike fold RNA Biol 3, 18–27 Clissold PM & Ponting CP (2000) PIN domains in nonsense-mediated mRNA decay and RNAi Curr Biol 10, R888–R890 Brooks SA, Connolly JE & Rigby WF (2004) The role of mRNA turnover in the regulation of tristetraprolin expression: evidence for an extracellular signal-regulated kinase-specific, AU-rich element-dependent, autoregulatory pathway J Immunol 172, 7263–7271 Jura J, Wegrzyn P, Zarebski A, Wladyka B & Koj A (2004) Identification of changes in the transcriptome profile of human hepatoma HepG2 cells stimulated with interleukin-1b Biochim Biophys Acta 1689, 120–133 FEBS Journal 276 (2009) 7386–7399 ª 2009 The Authors Journal compilation ª 2009 FEBS 7399 ... that MCPIP regulates the amount of IL-1b mRNA Involvement of PIN domain of MCPIP in the stability of IL-1b mRNA In order to find out whether the PIN domain in MCPIP is responsible for IL-1b mRNA. .. interacting with MCPIP will clarify the dynamics and features of this protein Role of MCPIP in regulation of the endogenous IL-1b transcript level After identification of a PIN domain in the MCPIP. .. shown) Of the identified proteins, 73% are proteins involved in mRNA stability, but there are also proteins involved in other cellular processes, such as actin Further studies characterizing proteins