CCCH-type Zinc Finger Domains Research Articles

Masculinizer gene controls sexual differentiation in Hyphantria cunea.

The Masculinizer gene, Masc, encodes a lepidopteran-specific novel CCCH-type zinc finger protein, which controls sex determination and dosage compensation in Bombyx mori. Considering the potential application of it in pest control, it is necessary to investigate the function of Masc gene in Hyphantria cunea, a globally invasive forest pest. In the present study, we identified and functionally characterized the Masc gene, HcMasc, in H. cunea. Sequence analysis revealed that HcMASC contained the conserved CCCH-type zinc finger domain, nuclear localization signal, and male determining domain, in which the last was confirmed to be required for its masculinization in BmN cell line. However, expression data showed that unlike male-biased expression in B. mori, HcMasc gene expresses in main all developmental stages or tissues in both sexes. Clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9-based disruption of the common exons 1 and 3 of the HcMasc gene resulted in imbalanced sex ratio and abnormal external genitalia of both sexes. Our results suggest that the HcMasc gene is required for both male and female sexual differentiation and dosage compensation in H. cunea and provide a foundation for developing better strategies to control this pest.

OsTZF1, a CCCH-tandem zinc finger protein gene, driven under own promoter produces no pleiotropic effects and confers salt and drought tolerance in rice

ABSTRACT Different abiotic stresses induce OsTZF1, a tandem CCCH-type zinc finger domain gene, in rice. Here, we report that transgenic rice plants overexpressing OsTZF1 under own promoter (POsTZF1:OsTZF1-OX [for overexpression]) transferred to soil showed normal growth similar to vector control plants. The POsTZF1:OsTZF1-OX produced normal leaves without any lesion mimic phenotype and exhibited normal seed setting. The POsTZF1:OsTZF1-OX plants showed significantly increased tolerance to salt and drought stresses and enhanced post stress recovery. Microarray analysis revealed a total of 846 genes up-regulated and 360 genes down-regulated in POsTZF1:OsTZF1-OX salt-treated plants. Microarray analysis of POsTZF1:OsTZF1-OX plants showed the regulation of many abiotic stress tolerance genes. These results suggest that OsTZF1-OX under own promoter show abiotic stress tolerance and produces no pleiotropic effect on phenotype of transgenic rice plant.

Targeting Pathological Tau by Small Molecule Inhibition of the Poly(A):MSUT2 RNA-Protein Interaction.

Neurofibrillary tangles composed of aberrantly aggregating tau protein are a hallmark of Alzheimer's disease and related dementia disorders. Recent work has shown that mammalian suppressor of tauopathy 2 (MSUT2), also named ZC3H14 (Zinc Finger CCCH-Type Containing 14), controls accumulation of pathological tau in cultured human cells and mice. Knocking out MSUT2 protects neurons from neurodegenerative tauopathy and preserves learning and memory. MSUT2 protein functions to bind polyadenosine [poly(A)] tails of mRNA through its C-terminal CCCH type zinc finger domains, and loss of CCCH domain function suppresses tauopathy in Caenorhabditis elegans and mice. Thus, we hypothesized that inhibiting the poly(A):MSUT2 RNA-protein interaction would ameliorate pathological tau accumulation. Here we present a high-throughput screening method for the identification of small molecules inhibiting the poly(A):MSUT2 RNA-protein interaction. We employed a fluorescent polarization assay for initial small molecule discovery with the intention to repurpose hits identified from the NIH Clinical Collection (NIHCC). Our drug repurposing development workflow included validation of hits by dose-response analysis, specificity testing, orthogonal assays of activity, and cytotoxicity. Validated compounds passing through this screening funnel will be evaluated for translational effectiveness in future studies. This preclinical drug development pipeline identified diverse FDA approved drugs duloxetine, saquinavir, and clofazimine as potential repurposing candidates for reducing pathological tau accumulation.

Zinc finger protein ZFP36L1 inhibits influenza A virus through translational repression by targeting HA, M and NS RNA transcripts.

ZFP36L1, a CCCH-type zinc finger protein, is an RNA-binding protein that participates in controlling cellular mRNA abundance and turnover by posttranscriptional regulation. Here, we demonstrated that ZFP36L1 has an important role in host defense against influenza A virus (IAV) infection. Overexpression of ZFP36L1 reduced IAV replication via translational repression of HA, M and NS RNA segment transcripts. IAV infection upregulated cellular ZFP36L1 expression, and endogenous ZFP36L1 knockdown significantly enhanced IAV replication. ZFP36L1 directly binds to IAV NS1 mRNA in the cytoplasm and blocks the expression and function of NS1 protein. Mutation of CCCH-type zinc finger domains of ZFP36L1 lost its antiviral potential and NS1 mRNA binding. Thus, ZFP36L1 can act as a host innate defense by targeting HA, M and NS mRNA transcripts to suppress viral protein translation.

Two CCCH-type zinc finger domains in the Masc protein are dispensable for masculinization and dosage compensation in Bombyx mori

The Masculinizer (Masc) gene encodes a novel lepidopteran-specific protein that controls both masculinization and dosage compensation in the silkworm Bombyx mori. The Masc protein possesses two CCCH-type zinc finger domains (ZFs), a nuclear localization signal, and an 11-amino-acid region that is highly conserved among lepidopteran insects. Using a cell-based assay system, we revealed that two cysteine residues localized in the conserved region, but not ZFs, are required for masculinization. In addition, nuclear localization of the Masc protein is not associated with masculinizing activity. Because dosage compensation is considered to occur in the nucleus, we inferred that the two ZFs play a role in the establishment of dosage compensation. To investigate this hypothesis at the organism level, we utilized the CRISPR/Cas9 system and established three B. mori strains whose Masc is partially deleted at different regions. The strain lacking the 210 C-terminal amino acids of the Masc protein showed male-specific embryonic lethality due to its low abundance and/or instability. The male embryos of this strain expressed the female-type splice variants of B. mori doublesex and did not express the male-type mRNA of B. mori IGF-II mRNA-binding protein. Furthermore, mRNA levels of Z-linked genes were abnormally enhanced only in male embryos. In contrast, the strain lacking both ZFs grew normally and did not show any defective phenotypes including sexual differentiation and the expression of Z-linked genes, demonstrating that the two CCCH-type ZFs, which are conserved in lepidopteran Masc homologs, are dispensable for masculinization and dosage compensation.

The Masc gene product controls masculinization in the black cutworm, Agrotis ipsilon.

Sex determination has been studied in the model lepidopteran species Bombyx mori, but it remains poorly understood in lepidopteran pests. In the present study, we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species, Agrotis ipsilon. Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280). We assessed the masculinizing activity of AiMasc in BmN cells and found that AiMasc induced expression of the male-specific doublesex isoform. Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A. ipsilon caused abnormalities in abdominal segments and external genitalia, resulting in male-specific sterility. These results suggest that Masc participates in the process of sex determination in A. ipsilon. Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control.

PARP13 regulates cellular mRNA post-transcriptionally and functions as a pro-apoptotic factor by destabilizing TRAILR4 transcript.

Poly(ADP-ribose) Polymerase-13 (PARP13/ZAP/ZC3HAV1) is an antiviral factor, active against specific RNA viruses such as MLV, SINV and HIV. During infection, PARP13 binds viral RNA via its four CCCH-type zinc finger domains and targets it for degradation by recruiting cellular mRNA decay factors such as the exosome complex and XRN1. Here we show that PARP13 binds to and regulates cellular mRNAs in the absence of viral infection. Knockdown of PARP13 results in the misregulation of hundreds of transcripts. Among the most upregulated transcripts is TRAILR4 that encodes a decoy receptor for TRAIL - a pro-apoptotic cytokine that is a promising target for the therapeutic inhibition of cancers. PARP13 destabilizes TRAILR4 mRNA posttranscriptionally in an exosome dependent manner by binding to a region in its 3’UTR. As a consequence, PARP13 represses TRAILR4 expression and increases cell sensitivity to TRAIL-mediated apoptosis, acting as a key regulator of the cellular response to TRAIL.

S-1: Regnase-1, a ribonuclease involved in the inflammatory and immune responses

Gene expression is controlled at multiple points, including signal transduction, transcription and mRNA stability. So far, transcriptional regulation has been extensively studied. However, recent studies have revealed that control of gene expression at the mRNA level is as important as transcriptional control in the immune response. The innate immune system is an evolutionally conserved host defense mechanism against pathogens. Innate immune responses are initiated by activation of pattern recognition receptors (PRRs), which recognize microbial components. Among them, Toll-like receptors (TLRs) are capable of sensing organisms ranging from bacteria to fungi, protozoa and viruses, and play a major role in innate immunity. Individual TLRs recognize different microbial components, activate different signaling pathways via selective usage of adaptor molecules, and give rise to different patterns in gene expression. We are now focusing on the role of genes induced in response to TLR stimulation, particularly the genes that are rapidly induced in a MyD88-dependent manner within 30 min after LPS stimulation. Among them, we have recently identified a novel gene named Zc3h12a which has a CCCH-type zinc finger domain. The knockout mice developed spontaneous autoimmune diseases accompanied by splenomegaly and lymphadenopathy. Subsequent studies showed that Zc3h12a is a nuclease involved in destabilization of IL-6 and IL-12mRNA via the stem loop structure present in the 3’UTR of these genes. We renamed it Regulatory RNase-1 (Regnase-1) based on the function. I would like to discuss the role of Regnase-1 in the immune response.

Zinc-finger antiviral protein mediates retinoic acid inducible gene I–like receptor-independent antiviral response to murine leukemia virus

When host cells are infected by an RNA virus, pattern-recognition receptors (PRRs) recognize the viral RNA and induce the antiviral innate immunity. Toll-like receptor 7 (TLR7) detects the genomic RNA of incoming murine leukemia virus (MLV) in endosomes and mediates the antiviral response. However, the RNA-sensing PRR that recognizes the MLV in the cytosol is not fully understood. Here, we definitively demonstrate that zinc-finger antiviral protein (ZAP) acts as a cytosolic RNA sensor, inducing the degradation of the MLV transcripts by the exosome, an RNA degradation system, on RNA granules. Although the retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 detect various RNA viruses in the cytosol and induce the type I IFN-dependent antiviral response, RLR loss does not alter the replication efficiency of MLV. In sharp contrast, the loss of ZAP greatly enhances the replication efficiency of MLV. ZAP localizes to RNA granules, where the processing-body and stress-granule proteins assemble. ZAP induces the recruitment of the MLV transcripts and exosome components to the RNA granules. The CCCH-type zinc-finger domains of ZAP, which are RNA-binding motifs, mediate its localization to RNA granules and MLV transcripts degradation by the exosome. Although ZAP was known as a regulator of RIG-I signaling in a human cell line, ZAP deficiency does not affect the RIG-I-dependent production of type I IFN in mouse cells. Thus, ZAP is a unique member of the cytosolic RNA-sensing PRR family that targets and eliminates intracellular RNA viruses independently of TLR and RLR family members.

Isolation and identification of a wheat gene encoding a zinc finger protein (TaZnFP) responsive to abiotic stresses

Zinc finger protein is one of the proteins with finger-like domains. CCCH-type zinc finger proteins involved in plant stress tolerance are poorly understood. In this paper, a novel CCCH-type zinc finger protein gene TaZnFP was isolated during analysis of an abiotic stress suppression subtractive hybridization expression library from wheat (Triticum aestivum L.). The TaZnFP gene contains a 1,533 bp open reading frame with no intron and encodes a 55.2 kDa protein with 510 amino acids. Its isoelectric point (pI) is 8.94. The TaZnFP protein contains two typical CCCH-type zinc finger domains (CX9CX5CX3H and CX5CX4CX3H). Transient expression of a 35S::TaZnFP-GFP fusion protein in Arabidopsis mesophyll protoplasts showed that TaZnFP was localized in the cytoplasm. Q-RT-PCR results showed that the TaZnFP transcript was induced by cold, salt (NaCl), drought, and ABA. Overexpression of TaZnFP enhanced drought and salt stress tolerance in transgenic Arabidopsis compared to the control. TaZnFP might be an important regulator responsive to abiotic stresses in plants.

Regnase-1, a Ribonuclease Involved in the Regulation of Immune Responses

Zc3h12a is a lipopolysaccharide-inducible gene and has a CCCH-type zinc-finger domain. Zc3h12a knockout mice develop spontaneous autoimmune diseases accompanied by splenomegaly and by lymphadenopathy. Subsequent studies show that Zc3h12a is a nuclease involved in destabilization of interleukin (IL)-6 and IL-12 mRNA via the stem loop structure within the 3' UTR of these genes. Thus, we renamed it regulatory RNase-1 (Regnase-1) based on its function. IκB kinases phosphorylate and degrade Regnase-1 protein in macrophages in response to IL-1R/Toll-like receptor (TLR) stimulation. T-cell-specific deletion of Regnase-1 produces pathogenic T cells with hyperactivated phenotypes as well as autoimmune diseases. Regnase-1 protein is cleaved by Malt1 and inactivated in response to T-cell receptor stimulation. Thus, Regnase-1 is a key molecule in the regulation of immune responses.

Involvement of XZFP36L1, an RNA-binding protein, in <I>Xenopus</I> neural development

Xenopus ZFP36L1 (zinc finger protein 36, C3H type-like 1) belongs to the ZFP36 family of RNA-binding proteins, which contains two characteristic tandem CCCH-type zinc-finger domains. The ZFP36 proteins can bind AU-rich elements in 3' untranslated regions of target mRNAs and promote their turnover. However, the expression and role of ZFP36 genes during neural development in Xenopus embryos remains largely unknown. The present study showed that Xenopus ZFP36L1 was expressed at the dorsal part of the forebrain, forebrain-midbrain boundary, and midbrain-hindbrain boundary from late neurula stages to tadpole stages of embryonic development. Overexpression of XZFP36L1 in Xenopus embryos inhibited neural induction and differentiation, leading to severe neural tube defects. The function of XZP36L1 requires both its zinc finger and C terminal domains, which also affect its subcellular localization. These results suggest that XZFP36L1 is likely involved in neural development in Xenopus and might play an important role in post-transcriptional regulation.

The role of mRNA degradation in immunity and inflammation

The innate immune system is an evolutionally conserved host defense mechanism against pathogens. Innate immune responses are initiated by pattern recognition receptors (PRRs), which recognize specific structures of microorganisms. Among them, Toll-like receptors (TLRs) are capable of sensing organisms ranging from bacteria to fungi, protozoa and viruses, and play a major role in innate immunity. Individual TLRs recognize different microbial components, and give rise to different patterns in gene expression. We are now focusing on the role of genes induced in response to TLR stimulation, particularly the genes that are rapidly induced in a MyD88-dependent manner within 30 min after LPS stimulation. Among them, we have recently identified a novel gene named Zc3h12a which has a CCCH-type zinc finger domain. The knockout mice developed spontaneous autoimmune diseases accompanied by splenomegaly and lymphadenopathy. Subsequent studies showed that Zc3h12a is a nuclease involved in destabilization of IL-6 and IL-12mRNA. We renamed it Regulatory RNase-1 (Regnase-1) based on the function. We recently found that the IKK complex controls Il6 mRNA stability by phosphorylating Regnase-1 in response to IL-1R/TLR stimulation. Phosphorylated Regnase-1 underwent ubiquitination and degradation. Regnase-1 re-expressed in IL-1R/TLR-activated cells exhibited delayed kinetics, and Regnase-1 mRNA was found to be negatively regulated by Regnase-1 itself via a stem-loop region present in the Regnase-1 3' untranslated region. These data demonstrate that the IKK complex phosphorylates not only IkBalpha, activating transcription, but also Regnase-1, releasing the brake on Il6 mRNA expression.

Alternative splicing regulation by Muscleblind proteins: from development to disease

Regulated use of exons in pre-mRNAs, a process known as alternative splicing, strongly contributes to proteome diversity. Alternative splicing is finely regulated by factors that bind specific sequences within the precursor mRNAs. Members of the Muscleblind (Mbl) family of splicing factors control critical exon use changes during the development of specific tissues, particularly heart and skeletal muscle. Muscleblind homologs are only found in metazoans from Nematoda to mammals. Splicing targets and recognition mechanisms are also conserved through evolution. In this recognition, Muscleblind CCCH-type zinc finger domains bind to intronic motifs in pre-mRNA targets in which the protein can either activate or repress splicing of nearby exons, depending on the localization of the binding motifs relative to the regulated alternative exon. In humans, the Muscleblind-like 1 (MBNL1) proteins play a critical role in hereditary diseases caused by microsatellite expansions, particularly myotonic dystrophy type 1 (DM1), in which depletion of MBNL1 activity through sequestration explains most misregulated alternative splicing events, at least in murine models. Because of the involvement of these proteins in human diseases, further understanding of the molecular mechanisms by which MBNL1 regulates splicing will help design therapies to revert pathological splicing alterations. Here we summarize the most relevant findings on this family of proteins in recent years, focusing on recently described functional motifs, transcriptional regulation of Muscleblind, regulatory activity on splicing, and involvement in human diseases.

Von Hippel-Lindau Gene Product Modulates TIS11B Expression in Renal Cell Carcinoma

TIS11B belongs to a group of RNA-binding proteins (including TIS11/tristetraprolin and TIS11D) that share characteristic tandem CCCH-type zinc-finger domains and can be rapidly induced by multiple stimuli. TIS11B has been shown to regulate vascular endothelial growth factor (VEGF) mRNA stability in adrenocorticotropic hormone-stimulated primary adrenocortical cells. TIS11B has also been documented as a negative regulator of VEGF during development, but nothing has yet been reported in the context of human cancers. The Von Hippel-Lindau (VHL) tumor suppressor protein regulates VEGF gene expression at both the transcriptional and post-transcriptional levels in normoxia. However, whether it can do so in hypoxia is still unclear. Here, we report a unique regulatory function of VHL in VEGF expression in hypoxia that is mediated through modulation of TIS11B protein levels in renal cancer cells. In normoxia, we detected increased expression of the microRNA hsa-miR-29b in the VHL-overexpressing renal cancer cell line 786-O. We also show that this increased expression of hsa-miR-29b decreased TIS11B protein expression by post-transcriptional regulation in normoxia. In contrast, in hypoxia, increased TIS11B expression paralleled an increased TIS11B mRNA stability in VHL-overexpressing 786-O cells. This VHL-mediated TIS11B up-regulation in hypoxia may be important for TIS11B-regulated gene expression: we observed a down-regulation of VEGF mRNA in hypoxia in VHL-overexpressing cells compared with parental 786-O cells, and this effect was reversible by silencing TIS11B expression.

O-Linked β-N-Acetylglucosaminyltransferase Substrate Specificity Is Regulated by Myosin Phosphatase Targeting and Other Interacting Proteins

O-GlcNAc-transferase (OGT) substrate specificity is regulated by transiently interacting proteins. To further examine the regulation of OGT, we have identified 27 putative OGT-interacting proteins through a yeast two-hybrid screen. Two of these proteins, Trak1 (OIP106) and O-GlcNAcase, have been shown previously to interact with and regulate OGT. We demonstrate here that MYPT1 and CARM1 also interact with and target OGT. MYPT1 and CARM1 are substrates of OGT in vitro and in vivo. MYPT1 and CARM1 also function to alter OGT substrate specificity in vitro. Furthermore depletion of MYPT1 in Neuro-2a neuroblastoma cells alters GlcNAcylation of several proteins under basal conditions, suggesting that MYPT1 regulates OGT substrate specificity in vivo.

Molecular dissection of mRNA poly(A) tail length control in yeast

In eukaryotic cells, newly synthesized mRNAs acquire a poly(A) tail that plays several fundamental roles in export, translation and mRNA decay. In mammals, PABPN1 controls the processivity of polyadenylation and the length of poly(A) tails during de novo synthesis. This regulation is less well-detailed in yeast. We have recently demonstrated that Nab2p is necessary and sufficient for the regulation of polyadenylation and that the Pab1p/PAN complex may act at a later stage in mRNA metabolism. Here, we show that the presence of both Pab1p and Nab2p in reconstituted pre-mRNA 3′-end processing reactions has no stimulating nor inhibitory effect on poly(A) tail regulation. Importantly, the poly(A)-binding proteins are essential to protect the mature mRNA from being subjected to a second round of processing. We have determined which domains of Nab2p are important to control polyadenylation and found that the RGG-box work in conjunction with the two last essential CCCH-type zinc finger domains. Finally, we have tried to delineate the mechanism by which Nab2p performs its regulation function during polyadenylation: it likely forms a complex with poly(A) tails different from a simple linear deposit of proteins as it has been observed with Pab1p.