KRAB Zinc Finger protein Znf684 interacts with Nxf1 to regulate mRNA export

AbstractCys2His2 (C2H2) type zinc finger (ZnF) proteins constitute a large class of proteins that are generally considered to be DNA-binding transcription factors. Using affinity purification followed by mass spectrometry, as well as reciprocal co-immunoprecipitation experiments, we determined that the C2H2-ZnF protein Znf684 interacts physically with several proteins involved in mRNA export, including Nxf1 and Alyref. We utilized individual nucleotide resolution cross-linking immunoprecipitation followed by high throughput sequencing (iCLIP-seq) experiments to show that Znf684 binds directly to specific mRNAs in vivo and has an RNA-binding profile similar to those of Nxf1 and Alyref, suggesting a role in mRNA export regulation. Immunofluorescence microscopy (IF) experiments revealed that Znf684 localizes to both the nucleus and cytoplasm. Using cellular fractionation experiments, we demonstrate that overexpression of Znf684 negatively impacts the export of SMAD3 and other target mRNAs. Taken together, our results suggest that Znf684 regulates the export of a subset of transcripts through physical interactions with Nxf1 and specific target mRNAs.

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Multiple nuclear-replicating viruses require the stress-induced protein ZC3H11A for efficient growth

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1722333115 ◽

2018 ◽

Vol 115 (16) ◽

pp. E3808-E3816 ◽

Cited By ~ 11

Author(s):

Shady Younis ◽

Wael Kamel ◽

Tina Falkeborn ◽

Hao Wang ◽

Di Yu ◽

...

Keyword(s):

Zinc Finger ◽

Nuclear Export ◽

High Throughput Sequencing ◽

Rna Binding ◽

Semliki Forest Virus ◽

Zinc Finger Protein ◽

Binding Capacity ◽

Binding Property ◽

Nuclear Regions ◽

Simplex Virus

The zinc finger CCCH-type containing 11A (ZC3H11A) gene encodes a well-conserved zinc finger protein that may function in mRNA export as it has been shown to associate with the transcription export (TREX) complex in proteomic screens. Here, we report that ZC3H11A is a stress-induced nuclear protein with RNA-binding capacity that localizes to nuclear splicing speckles. During an adenovirus infection, the ZC3H11A protein and splicing factor SRSF2 relocalize to nuclear regions where viral DNA replication and transcription take place. Knockout (KO) of ZC3H11A in HeLa cells demonstrated that several nuclear-replicating viruses are dependent on ZC3H11A for efficient growth (HIV, influenza virus, herpes simplex virus, and adenovirus), whereas cytoplasmic replicating viruses are not (vaccinia virus and Semliki Forest virus). High-throughput sequencing of ZC3H11A–cross-linked RNA showed that ZC3H11A binds to short purine-rich ribonucleotide stretches in cellular and adenoviral transcripts. We show that the RNA-binding property of ZC3H11A is crucial for its function and localization. In ZC3H11A KO cells, the adenovirus fiber mRNA accumulates in the cell nucleus. Our results suggest that ZC3H11A is important for maintaining nuclear export of mRNAs during stress and that several nuclear-replicating viruses take advantage of this mechanism to facilitate their replication.

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FLASH: ultra-fast protocol to identify RNA–protein interactions in cells

Nucleic Acids Research ◽

10.1093/nar/gkz1141 ◽

2019 ◽

Vol 48 (3) ◽

pp. e15-e15 ◽

Cited By ~ 1

Author(s):

Ibrahim Avsar Ilik ◽

Tugce Aktas ◽

Daniel Maticzka ◽

Rolf Backofen ◽

Asifa Akhtar

Keyword(s):

High Throughput ◽

Protein Interactions ◽

Binding Sites ◽

Binding Proteins ◽

High Throughput Sequencing ◽

Rna Binding ◽

Rna Binding Proteins ◽

Affinity Purification ◽

Sequencing Library

Abstract Determination of the in vivo binding sites of RNA-binding proteins (RBPs) is paramount to understanding their function and how they affect different aspects of gene regulation. With hundreds of RNA-binding proteins identified in human cells, a flexible, high-resolution, high-throughput, highly multiplexible and radioactivity-free method to determine their binding sites has not been described to date. Here we report FLASH (Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing), which uses a special adapter design and an optimized protocol to determine protein–RNA interactions in living cells. The entire FLASH protocol, starting from cells on plates to a sequencing library, takes 1.5 days. We demonstrate the flexibility, speed and versatility of FLASH by using it to determine RNA targets of both tagged and endogenously expressed proteins under diverse conditions in vivo.

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Zinc finger RNA binding protein Zn72D regulates ADAR-mediated RNA editing in neurons

10.1101/631986 ◽

2019 ◽

Author(s):

Anne L. Sapiro ◽

Emily C. Freund ◽

Lucas Restrepo ◽

Huan-Huan Qiao ◽

Amruta Bhate ◽

...

Keyword(s):

Rna Editing ◽

Zinc Finger ◽

Rna Binding ◽

Zinc Finger Protein ◽

Rna Binding Proteins ◽

Rna Sequences ◽

Protein Levels ◽

Adar Editing ◽

The Brain

AbstractAdenosine-to-inosine RNA editing, catalyzed by ADAR enzymes, alters RNA sequences from those encoded by DNA. These editing events are dynamically regulated, but few trans regulators of ADARs are known in vivo. Here, we screen RNA binding proteins for roles in editing regulation using in vivo knockdown experiments in the Drosophila brain. We identify Zinc-Finger Protein at 72D (Zn72D) as a regulator of editing levels at a majority of editing sites in the brain. Zn72D both regulates ADAR protein levels and interacts with ADAR in an RNA-dependent fashion, and similar to ADAR, Zn72D is necessary to maintain proper neuromuscular junction architecture and motility in the fly. Furthermore, the mammalian homolog of Zn72D, Zfr, regulates editing in mouse primary neurons, demonstrating the conservation of this regulatory role. The broad and conserved regulation of ADAR editing by Zn72D in neurons represents a novel mechanism by which critically important editing events are sustained.

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Understanding RNA Binding by the Nonclassical Zinc Finger Protein CPSF30, a Key Factor in Polyadenylation during Pre-mRNA Processing

Biochemistry ◽

10.1021/acs.biochem.0c00940 ◽

2021 ◽

Author(s):

Jordan D. Pritts ◽

Abdulafeez A. Oluyadi ◽

Weiliang Huang ◽

Geoffrey D. Shimberg ◽

Maureen A. Kane ◽

...

Keyword(s):

Zinc Finger ◽

Rna Binding ◽

Zinc Finger Protein ◽

Mrna Processing ◽

Finger Protein ◽

Key Factor

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Repeated CT elements bound by zinc finger proteins control the absolute and relative activities of the two principal human c-myc promoters

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5710-5724.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5710-5724

Author(s):

E DesJardins ◽

N Hay

Keyword(s):

Zinc Finger ◽

Tandem Repeats ◽

Zinc Finger Protein ◽

Consensus Sequence ◽

Regulatory Region ◽

Maximal Activity ◽

Single Copy ◽

Promoter Usage

Transcription of the human proto-oncogene c-myc is governed by two tandem principal promoters, termed P1 and P2. In general, the downstream promoter, P2, is predominant, which is in contrast to the promoter occlusion phenomenon usually observed in genes containing tandem promoters. A shift in human c-myc promoter usage has been observed in some tumor cells and in certain physiological conditions. However, the mechanisms that regulate promoter usage are not well understood. The present studies identify regulators which are required to promote transcription from both human c-myc promoters, P1 and P2, and have a role in determining their relative activities in vivo. A novel regulatory region located 101 bp upstream of P1 was characterized and contains five tandem repeats of the consensus sequence CCCTCCCC (CT element). The integrity of the region containing all five elements is required to promote transcription from P1 and for maximal activity from P2 in vivo. A single copy of this same element, designated CT-I2, also appears in an inverted orientation 53 bp upstream of the P2 transcription start site. This element has an inhibitory effect on P1 transcription and is required for P2 transcription. The transcription factor Sp1 was identified as the factor that binds specifically to the tandem CT elements upstream of P1 and to the CT-I2 element upstream of P2. In addition, the recently cloned zinc finger protein ZF87, or MAZ, was also able to bind these same elements in vitro. The five tandem CT elements can be functionally replaced by a heterologous enhancer that only in the absence of CT-I2 reverses the promoter usage, similar to what is observed in the translocated c-myc allele of Burkitt's lymphoma cells.

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TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals

10.1101/2020.02.24.962852 ◽

2020 ◽

Cited By ~ 2

Author(s):

Hua Jin ◽

Daxiang Na ◽

Reazur Rahman ◽

Weijin Xu ◽

Allegra Fieldsend ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Growth Control ◽

Ribosome Profiling ◽

Translational Efficiency ◽

Mtor Inhibition ◽

Mrna Targets ◽

Specific Mrnas ◽

Specific Mrna

Abstract4E-BP (eIF4E-BP) represses translation initiation by binding to the 5’cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used hyperTRIBE (Targets of RNA-binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets compared to non-targets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.

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Zinc finger protein ZFP36L1 inhibits flavivirus infection by both 5′-3′ XRN1 and 3′-5′ RNA-exosome RNA decay pathways.

Journal of Virology ◽

10.1128/jvi.01665-21 ◽

2021 ◽

Author(s):

Han Chiu ◽

Hsin-Ping Chiu ◽

Han-Pang Yu ◽

Li-Hsiung Lin ◽

Zih-Ping Chen ◽

...

Keyword(s):

Antiviral Activity ◽

Dengue Virus ◽

Zinc Finger ◽

Rna Binding ◽

Zinc Finger Protein ◽

Viral Rna ◽

Rna Decay ◽

Finger Protein ◽

Zinc Finger Motifs ◽

Rna Exosome

Zinc-finger protein 36, CCCH type-like 1 (ZFP36L1), containing tandem CCCH-type zinc-finger motifs with an RNA-binding property, plays an important role in cellular RNA metabolism mainly via RNA decay pathways. Recently, we demonstrated that human ZFP36L1 has potent antiviral activity against influenza A virus infection. However, its role in the host defense response against flaviviruses has not been addressed. Here, we demonstrate that ZFP36L1 functions as a host innate defender against flaviviruses, including Japanese encephalitis virus (JEV) and dengue virus (DENV). Overexpression of ZFP36L1 reduced JEV and DENV infection, and ZFP36L1 knockdown enhanced viral replication. ZFP36L1 destabilized the JEV genome by targeting and degrading viral RNA mediated by both 5′-3′ XRN1 and 3′-5′ RNA-exosome RNA decay pathways. Mutation in both zinc-finger motifs of ZFP36L1 disrupted RNA-binding and antiviral activity. Furthermore, the viral RNA sequences specifically recognized by ZFP36L1 were mapped to the 3'-untranslated region of the JEV genome with the AU-rich element (AUUUA) motif. We extend the function of ZFP36L1 to host antiviral defense by directly binding and destabilizing the viral genome via recruiting cellular mRNA decay machineries. Importance Cellular RNA-binding proteins are among the first lines of defense against various viruses, particularly RNA viruses. ZFP36L1 belongs to the CCCH-type zinc-finger protein family and has RNA-binding activity; it has been reported to directly bind to the AU-rich elements (AREs) of a subset of cellular mRNAs and then lead to mRNA decay by recruiting mRNA degrading enzymes. However, the antiviral potential of ZFP36L1 against flaviviruses has not yet been fully demonstrated. Here, we reveal the antiviral potential of human ZFP36L1 against Japanese encephalitis virus (JEV) and dengue virus (DENV). ZFP36L1 specifically targeted the ARE motif within viral RNA and triggered the degradation of viral RNA transcripts via cellular degrading enzymes, 5′-3′ XRN1 and 3′-5′ RNA exosome. These findings provide mechanistic insights into how human ZFP36L1 serves as a host antiviral factor to restrict flavivirus replication.

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Residues in the Swi5 Zinc Finger Protein That Mediate Cooperative DNA Binding with the Pho2 Homeodomain Protein

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6436 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6436-6446 ◽

Cited By ~ 19

Author(s):

Leena T. Bhoite ◽

David J. Stillman

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Zinc Finger ◽

Transcriptional Activation ◽

Binding Proteins ◽

Zinc Finger Protein ◽

Interaction Analysis ◽

Dna Binding Proteins ◽

Two Hybrid

ABSTRACT The Swi5 zinc finger and the Pho2 homeodomain DNA-binding proteins bind cooperatively to the HO promoter.Pho2 (also known as Bas2 or Grf10) activates transcription of diverse genes, acting with multiple distinct DNA-binding proteins. We have performed a genetic screen to identify amino acid residues in Swi5 that are required for synergistic transcriptional activation of a reporter construct in vivo. Nine unique amino acid substitutions within a 24-amino-acid region of Swi5, upstream of the DNA-binding domain, reduce expression of promoters that require both Swi5 and Pho2 for activation. In vitro DNA binding experiments show that the mutant Swi5 proteins bind DNA normally, but some mutant Swi5 proteins (resulting from SWI5* mutations) show reduced cooperative DNA binding with Pho2. In vivo experiments show that these SWI5* mutations sharply reduce expression of promoters that require both SWI5 and PHO2, while expression of promoters that require SWI5 but arePHO2 independent is largely unaffected. This suggests that these SWI5* mutations do not affect the ability of Swi5 to bind DNA or activate transcription but specifically affect the region of Swi5 required for interaction with Pho2. Two-hybrid experiments show that amino acids 471 to 513 of Swi5 are necessary and sufficient for interaction with Pho2 and that the SWI5* point mutations cause a severe reduction in this two-hybrid interaction. Analysis of promoter activation by these mutants suggests that this small region of Swi5 has at least two distinct functions, conferring specificity for activation of the HO promoter and for interaction with Pho2.

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A non-canonical monovalent zinc finger stabilizes the integration of Cfp1 into the H3K4 methyltransferase complex COMPASS

Nucleic Acids Research ◽

10.1093/nar/gkz1037 ◽

2019 ◽

Cited By ~ 1

Author(s):

Yidai Yang ◽

Monika Joshi ◽

Yoh-hei Takahashi ◽

Zhibin Ning ◽

Qianhui Qu ◽

...

Keyword(s):

Zinc Finger ◽

Zinc Finger Protein ◽

H3k4 Methylation ◽

Methyltransferase Activity ◽

Vitro Binding ◽

New Type ◽

High Level ◽

In Cells

Abstract COMPlex ASsociating with SET1 (COMPASS) is a histone H3 Lys-4 methyltransferase that typically marks the promoter region of actively transcribed genes. COMPASS is a multi-subunit complex in which the catalytic unit, SET1, is required for H3K4 methylation. An important subunit known to regulate SET1 methyltransferase activity is the CxxC zinc finger protein 1 (Cfp1). Cfp1 binds to COMPASS and is critical to maintain high level of H3K4me3 in cells but the mechanisms underlying its stimulatory activity is poorly understood. In this study, we show that Cfp1 only modestly activates COMPASS methyltransferase activity in vitro. Binding of Cfp1 to COMPASS is in part mediated by a new type of monovalent zinc finger (ZnF). This ZnF interacts with the COMPASS’s subunits RbBP5 and disruption of this interaction blunts its methyltransferase activity in cells and in vivo. Collectively, our studies reveal that a novel form of ZnF on Cfp1 enables its integration into COMPASS and contributes to epigenetic signaling.

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