Analyses of the Functional Regions of DEAD-Box RNA “Helicases” with Deletion and Chimera Constructs Tested In Vivo and In Vitro

ABSTRACT DEAD box proteins are a family of putative RNA helicases associated with all aspects of cellular metabolism involving the modification of RNA secondary structure. DDX1 is a member of the DEAD box protein family that is overexpressed in a subset of retinoblastoma and neuroblastoma cell lines and tumors. DDX1 is found primarily in the nucleus, where it forms two to four large aggregates called DDX1 bodies. Here, we report a rapid redistribution of DDX1 in cells exposed to ionizing radiation, resulting in the formation of numerous foci that colocalize with γ-H2AX and phosphorylated ATM foci at sites of DNA double-strand breaks (DSBs). The formation of DDX1 ionizing-radiation-induced foci (IRIF) is dependent on ATM, which was shown to phosphorylate DDX1 both in vitro and in vivo. The treatment of cells with RNase H prevented the formation of DDX1 IRIF, suggesting that DDX1 is recruited to sites of DNA damage containing RNA-DNA structures. We have shown that DDX1 has RNase activity toward single-stranded RNA, as well as ADP-dependent RNA-DNA- and RNA-RNA-unwinding activities. We propose that DDX1 plays an RNA clearance role at DSB sites, thereby facilitating the template-guided repair of transcriptionally active regions of the genome.

Download Full-text

Splicing Factor DDX23, Transcriptionally Activated by E2F1, Promotes Ovarian Cancer Progression by Regulating FOXM1

Frontiers in Oncology ◽

10.3389/fonc.2021.749144 ◽

2021 ◽

Vol 11 ◽

Author(s):

Chen Zhao ◽

Yingwei Li ◽

Chunping Qiu ◽

Jingying Chen ◽

Huan Wu ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Progression ◽

Mrna Processing ◽

Splicing Factor ◽

Luciferase Reporter ◽

Ovarian Cancer Cells ◽

Rna Helicases ◽

Dead Box

Ovarian carcinoma remains the most lethal gynecological carcinoma. Abnormal expression of splicing factors is closely related to the occurrence and development of tumors. The DEAD-box RNA helicases are important members of the splicing factor family. However, their role in the occurrence and progression of ovarian cancer is still unclear. In this study, we identified DEAD-box helicase 23 (DDX23) as a key DEAD-box RNA helicase in ovarian cancer using bioinformatics methods. We determined that DDX23 was upregulated in ovarian cancer and its high expression predicted poor prognosis. Functional assays indicated that DDX23 silencing significantly impeded cell proliferation/invasion in vitro and tumor growth in vivo. Mechanistically, transcriptomic analysis showed that DDX23 was involved in mRNA processing in ovarian cancer cells. Specifically, DDX23 regulated the mRNA processing of FOXM1. DDX23 silencing reduced the production of FOXM1C, the major oncogenic transcript of FOXM1 in ovarian cancer, thereby decreasing the FOXM1 protein expression and attenuating the malignant progression of ovarian cancer. Rescue assays indicated that FOXM1 was a key executor in DDX23-induced malignant phenotype of ovarian cancer. Furthermore, we confirmed that DDX23 was transcriptionally activated by the transcription factor (TF) E2F1 in ovarian cancer using luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays. In conclusion, our study demonstrates that high DDX23 expression is involved in malignant behavior of ovarian cancer and DDX23 may become a potential target for precision therapy of ovarian cancer.

Download Full-text

Regulation of RNA helicase activity: principles and examples

Biological Chemistry ◽

10.1515/hsz-2020-0362 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Pascal Donsbach ◽

Dagmar Klostermeier

Keyword(s):

Atp Hydrolysis ◽

Wide Spectrum ◽

Mrna Translation ◽

Rna Degradation ◽

Rna Helicases ◽

Interaction Partners ◽

Rna Duplexes ◽

Self Association

Abstract RNA helicases are a ubiquitous class of enzymes involved in virtually all processes of RNA metabolism, from transcription, mRNA splicing and export, mRNA translation and RNA transport to RNA degradation. Although ATP-dependent unwinding of RNA duplexes is their hallmark reaction, not all helicases catalyze unwinding in vitro, and some in vivo functions do not depend on duplex unwinding. RNA helicases are divided into different families that share a common helicase core with a set of helicase signature motives. The core provides the active site for ATP hydrolysis, a binding site for the non-sequence-specific interactions with RNA, and in many cases a basal unwinding activity. Its activity is often regulated by flanking domains, by interaction partners, or by self-association. In this review, we summarize the regulatory mechanisms that modulate the activities of the helicase core. Case studies on selected helicases with functions in translation, splicing, and RNA sensing illustrate the various modes and layers of regulation in time and space that harness the helicase core for a wide spectrum of cellular tasks.

Download Full-text

Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3463 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3463-3471 ◽

Cited By ~ 62

Author(s):

S R Schmid ◽

P Linder

Keyword(s):

Saccharomyces Cerevisiae ◽

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Clear Correlation ◽

Temperature Sensitive ◽

Rna Helicases ◽

Initiation Of Translation

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

Download Full-text

Functional interplay between DEAD-box RNA helicases Ded1 and Dbp1 in preinitiation complex attachment and scanning on structured mRNAs in vivo

Nucleic Acids Research ◽

10.1093/nar/gkz595 ◽

2019 ◽

Cited By ~ 4

Author(s):

Neelam Dabas Sen ◽

Neha Gupta ◽

Stuart K. Archer ◽

Thomas Preiss ◽

Jon R Lorsch ◽

...

Keyword(s):

Untranslated Region ◽

Double Mutant ◽

Direct Evidence ◽

Translation Efficiency ◽

Rna Structures ◽

Preinitiation Complex ◽

Rna Helicases ◽

Ribosome Binding ◽

Dead Box

Abstract RNA structures that impede ribosome binding or subsequent scanning of the 5′-untranslated region (5′-UTR) for the AUG initiation codon reduce translation efficiency. Yeast DEAD-box RNA helicase Ded1 appears to promote translation by resolving 5′-UTR structures, but whether its paralog, Dbp1, performs similar functions is unknown. Furthermore, direct in vivo evidence was lacking that Ded1 or Dbp1 resolves 5′-UTR structures that impede attachment of the 43S preinitiation complex (PIC) or scanning. Here, profiling of translating 80S ribosomes reveals that the translational efficiencies of many more mRNAs are reduced in a ded1-ts dbp1Δ double mutant versus either single mutant, becoming highly dependent on Dbp1 or Ded1 only when the other helicase is impaired. Such ‘conditionally hyperdependent’ mRNAs contain unusually long 5′-UTRs with heightened propensity for secondary structure and longer transcript lengths. Consistently, overexpressing Dbp1 in ded1 cells improves the translation of many such Ded1-hyperdependent mRNAs. Importantly, Dbp1 mimics Ded1 in conferring greater acceleration of 48S PIC assembly in a purified system on mRNAs harboring structured 5′-UTRs. Profiling 40S initiation complexes in ded1 and dbp1 mutants provides direct evidence that Ded1 and Dbp1 cooperate to stimulate both PIC attachment and scanning on many Ded1/Dbp1-hyperdependent mRNAs in vivo.

Download Full-text

In vivo mapping of the functional regions of the DEAD-box helicase Vasa

Biology Open ◽

10.1242/bio.201410579 ◽

2015 ◽

Vol 4 (4) ◽

pp. 450-462 ◽

Cited By ~ 19

Author(s):

M. Dehghani ◽

P. Lasko

Keyword(s):

Functional Regions ◽

Dead Box ◽

The Dead

Download Full-text

A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

Molecular Biology of the Cell ◽

10.1091/mbc.11.4.1153 ◽

2000 ◽

Vol 11 (4) ◽

pp. 1153-1167 ◽

Cited By ~ 41

Author(s):

Rudolf F. Zirwes ◽

Jens Eilbracht ◽

Sandra Kneissel ◽

Marion S. Schmidt-Zachmann

Keyword(s):

Cultured Cells ◽

Gradient Centrifugation ◽

Intracellular Localization ◽

Ribosomal Subunit ◽

Rnase A ◽

Rna Helicases ◽

Dead Box ◽

Transcription Inhibitor ◽

Calculated Mass

We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is a member of the family of “DEAD-box” proteins, representing a subgroup of putative ATP-dependent RNA helicases. ATPase activity of the recombinant protein is evident and stimulated by a variety of polynucleotides tested. Immunolocalization studies revealed that protein NOH61 (nucleolar helicase of 61 kDa) is highly conserved during evolution and shows a strong accumulation in nucleoli. Biochemical experiments have shown that protein NOH61 synthesized in vitro sediments with ∼11.5 S, i.e., apparently as homo-oligomeric structures. By contrast, sucrose gradient centrifugation analysis of cellular extracts obtained with buffers of elevated ionic strength (600 mM NaCl) revealed that the solubilized native protein sediments with ∼4 S, suggestive of the monomeric form. Interestingly, protein NOH61 has also been identified as a specific constituent of free nucleoplasmic 65S preribosomal particles but is absent from cytoplasmic ribosomes. Treatment of cultured cells with 1) the transcription inhibitor actinomycin D and 2) RNase A results in a complete dissociation of NOH61 from nucleolar structures. The specific intracellular localization and its striking sequence homology to other known RNA helicases lead to the hypothesis that protein NOH61 might be involved in ribosome synthesis, most likely during the assembly process of the large (60S) ribosomal subunit.

Download Full-text

Dhr1p, a Putative DEAH-Box RNA Helicase, Is Associated with the Box C+D snoRNP U3

Molecular and Cellular Biology ◽

10.1128/mcb.20.19.7238-7246.2000 ◽

2000 ◽

Vol 20 (19) ◽

pp. 7238-7246 ◽

Cited By ~ 44

Author(s):

Alan Colley ◽

Jean D. Beggs ◽

David Tollervey ◽

Denis L. J. Lafontaine

Keyword(s):

18S Rrna ◽

Mrna Splicing ◽

Rrna Synthesis ◽

Detectable Effect ◽

Rna Helicases ◽

Structural Reorganization ◽

Rrna Cleavage ◽

Processing Steps

ABSTRACT Putative RNA helicases are involved in most aspects of gene expression. All previously characterized members of the DEAH-box family of putative RNA helicases are involved in pre-mRNA splicing. Here we report the analysis of two novel DEAH-box RNA helicases, Dhr1p and Dhr2p, that were found to be predominantly nucleolar. Both genes are essential for viability, and MET-regulated alleles were therefore created. Depletion of Dhr1p or Dhr2p had no detectable effect on pre-mRNA splicing in vivo or in vitro. Both Dhr1p and Dhr2p were, however, required for 18S rRNA synthesis. Depletion of Dhr2p inhibited pre-rRNA cleavage at sites A0, A1, and A2, while Dhr1p depletion inhibited cleavage at sites A1 and A2. No coprecipitation of snoRNAs was detected with ProtA-Dhr2p, but Dhr1p-ProtA was stably associated with the U3 snoRNA. Depletion of Dhr1p inhibited processing steps that require base pairing of U3 to the 5′ end of the 18S rRNA. We speculate that Dhr1p is targeted to the preribosomal particles by the U3-18S rRNA interaction and is required for the structural reorganization of the rRNA during formation of the central pseudoknot.

Download Full-text

Functional analysis of haplotypes and promoter activity at the 5' region of the DRD2 gene and associations with schizophrenia.

10.21203/rs.3.rs-15812/v1 ◽

2020 ◽

Author(s):

Xicen Zhang ◽

Mei Ding ◽

Yi Liu ◽

Yongping Liu ◽

Jiaxin Xing ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Promoter Region ◽

Regulatory Region ◽

Gene Promoter ◽

Luciferase Reporter ◽

Drd2 Gene ◽

Functional Regions

Abstract Background: In previous studies, we researched the association of the DRD2 gene promoter region SNP loci rs7116768, rs1047479195, rs1799732, rs1799978 and schizophrenia using Sanger sequencing. rs7116768 and rs1799978 were found to be slightly associated with schizophrenia. This study investigated the effects of haplotypes consisted of the four SNPs on protein expression level in vitro and identified the functional sequence in the 5’ regulatory region of DRD2 gene which has a potential link with schizophrenia.Methods: Recombinant plasmids with haplotypes, SNPs and 13 recombinant vectors containing deletion fragments from the DRD2 gene 5' regulatory region were transfected into HEK293 and SK-N-SH cell lines. Relative luciferase activity of the haplotypes, SNPs and different sequences was compared using a dual luciferase reporter assay system.Results: Haplotype H4(G-C-InsC-G) could significantly increase the gene expression in SK-N-SH cell lines. Allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate the gene expression. There were 5~7 functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.Conclusion: We cannot rule out the possibility that different haplotypes may influence DRD2 gene expression in vivo. We observed that allele C of rs7116768, allele A of rs1047479195 and allele del C of rs1799732 could up-regulate gene expression. The truncation results confirmed the existence of functional regions in the promoter region of DRD2 gene that could affect the level of gene expression.

Download Full-text

Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

10.1101/402404 ◽

2018 ◽

Author(s):

Ruchika Sachdev ◽

Maria Hondele ◽

Miriam Linsenmeier ◽

Pascal Vallotton ◽

Christopher F. Mugler ◽

...

Keyword(s):

Phase Separation ◽

Direct Interaction ◽

Liquid Droplets ◽

Processing Bodies ◽

Dead Box ◽

The Dead ◽

Processing Body ◽

Liquid Liquid Phase Separation

AbstractProcessing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. We recently identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo [Mugler et al., 2016]. Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

Download Full-text