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

2019 ◽  
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.

scholarly journals A Role for DEAD Box 1 at DNA Double-Strand Breaks

2008 ◽  
Vol 28 (20) ◽  
pp. 6413-6425 ◽  
Author(s):  
Lei Li ◽  
Elizabeth A. Monckton ◽  
Roseline Godbout
Keyword(s):  
Ionizing Radiation ◽  
Neuroblastoma Cell ◽  
Rnase H ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Rna Helicases ◽  
Strand Breaks ◽  
Dead Box

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.

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

2011 ◽  
Vol 413 (2) ◽  
pp. 451-472 ◽  
Author(s):  
Josette Banroques ◽  
Olivier Cordin ◽  
Monique Doère ◽  
Patrick Linder ◽  
N. Kyle Tanner
Keyword(s):  
Rna Helicases ◽  
Functional Regions ◽  
Dead Box

scholarly journals Distinct interactions of eIF4A and eIF4E with RNA helicase Ded1 stimulate translation in vivo

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Suna Gulay ◽  
Neha Gupta ◽  
Jon R Lorsch ◽  
Alan G Hinnebusch
Keyword(s):  
Amino Acid ◽  
Cell Growth ◽  
Translation Initiation ◽  
Rna Helicase ◽  
Preinitiation Complex ◽  
Terminal Domain ◽  
Dead Box ◽  
Stimulation Of

Yeast DEAD-box helicase Ded1 stimulates translation initiation, particularly of mRNAs with structured 5'UTRs. Interactions of the Ded1 N-terminal domain (NTD) with eIF4A, and Ded1-CTD with eIF4G, subunits of eIF4F, enhance Ded1 unwinding activity and stimulation of preinitiation complex (PIC) assembly in vitro. However, the importance of these interactions, and of Ded1-eIF4E association, in vivo were poorly understood. We identified separate amino acid clusters in the Ded1-NTD required for binding to eIF4A or eIF4E in vitro. Disrupting each cluster selectively impairs native Ded1 association with eIF4A or eIF4E, and reduces cell growth, polysome assembly, and translation of reporter mRNAs with structured 5'UTRs. It also impairs Ded1 stimulation of PIC assembly on a structured mRNA in vitro. Ablating Ded1 interactions with eIF4A/eIF4E unveiled a requirement for the Ded1-CTD for robust initiation. Thus, Ded1 function in vivo is stimulated by independent interactions of its NTD with eIF4E and eIF4A, and its CTD with eIF4G.

scholarly journals Cold-Induced Putative DEAD Box RNA Helicases CshA and CshB Are Essential for Cold Adaptation and Interact with Cold Shock Protein B in Bacillus subtilis

2006 ◽  
Vol 188 (1) ◽  
pp. 240-248 ◽  
Author(s):  
Karen Hunger ◽  
Carsten L. Beckering ◽  
Frank Wiegeshoff ◽  
Peter L. Graumann ◽  
Mohamed A. Marahiel
Keyword(s):  
Bacillus Subtilis ◽  
Cold Shock ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cold Shock Protein ◽  
Nucleic Acid Binding ◽  
Rna Helicases ◽  
Dead Box ◽  
Initiation Of Translation

ABSTRACT The nucleic acid binding cold shock proteins (CSPs) and the cold-induced DEAD box RNA helicases have been proposed separately to act as RNA chaperones, but no experimental evidence has been reported on a direct cooperation. To investigate the possible interaction of the putative RNA helicases CshA and CshB and the CSPs from Bacillus subtilis during cold shock, we performed genetic as well as fluorescence resonance energy transfer (FRET) experiments. Both cshA and cshB genes could be deleted only in the presence of a cshB copy in trans, showing that the presence of one csh gene is essential for viability. The combined gene deletion of cshB and cspD resulted in a cold-sensitive phenotype that was not observed for either helicase or csp single mutants. In addition to the colocalization of the putative helicases CshA and CshB with CspB and the ribosomes in areas surrounding the nucleoid, we detected a strong FRET interaction in vivo between CshB and CspB that depended on active transcription. In contrast, a FRET interaction was not observed for CshB and the ribosomal protein L1. Therefore, we propose a model in which the putative cold-induced helicases and the CSPs work in conjunction to rescue misfolded mRNA molecules and maintain proper initiation of translation at low temperatures in B. subtilis.

scholarly journals RNA G-quadruplex structures exist and function in vivo

2019 ◽  
Author(s):  
Xiaofei Yang ◽  
Jitender Cheema ◽  
Yueying Zhang ◽  
Hongjing Deng ◽  
Susan Duncan ◽  
...  
Keyword(s):  
Direct Evidence ◽  
Rna Structures ◽  
Specific Sequence ◽  
Form Complex ◽  
Folding Potential ◽  
G Quadruplex ◽  
Quadruplex Formation ◽  
And Function

AbstractGuanine-rich sequences are able to form complex RNA structures termed RNA G-quadruplexes in vitro. Because of their high stability, RNA G-quadruplexes are proposed to exist in vivo and are suggested to be associated with important biological relevance. However, there is a lack of direct evidence for RNA G-quadruplex formation in living cells. Therefore, it is unclear whether any purported functions are associated with the specific sequence content or the formation of an RNA G-quadruplex structure. Here, we profiled the landscape of those guanine-rich regions with the in vitro folding potential in the Arabidopsis transcriptome. We found a global enrichment of RNA G-quadruplexes with two G-quartets whereby the folding potential is strongly influenced by RNA secondary structures. Using in vitro and in vivo RNA chemical structure profiling, we determined that hundreds of RNA G-quadruplex structures are strongly folded in both Arabidopsis and rice, providing direct evidence of RNA G-quadruplex formation in living eukaryotic cells. Subsequent genetic and biochemical analysis showed that RNA G-quadruplex folding was sufficient to regulate translation and modulate plant growth. Our study reveals the existence of RNA G-quadruplex in vivo, and indicates that RNA G-quadruplex structures act as important regulators of plant development and growth.

scholarly journals A DEAD-box RNA helicase promotes thermodynamic equilibration of kinetically trapped RNA structures in vivo

RNA ◽  
2016 ◽  
Vol 22 (3) ◽  
pp. 416-427 ◽  
Author(s):  
Dana J. Ruminski ◽  
Peter Y. Watson ◽  
Elisabeth M. Mahen ◽  
Martha J. Fedor
Keyword(s):  
Rna Helicase ◽  
Rna Structures ◽  
Dead Box

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

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.

scholarly journals In Vivo and In Vitro Identification of Structural and Sequence Elements of the Human Parechovirus 5′ Untranslated Region Required for Internal Initiation

2000 ◽  
Vol 74 (14) ◽  
pp. 6269-6277 ◽  
Author(s):  
Abdolrahman S. Nateri ◽  
Pamela J. Hughes ◽  
Glyn Stanway
Keyword(s):  
Sequence Analysis ◽  
Untranslated Region ◽  
Point Mutations ◽  
In Vitro Transcription ◽  
In Vivo Studies ◽  
Translation Efficiency ◽  
Human Parechovirus ◽  
Entry Site

ABSTRACT Sequence analysis of the picornavirus echovirus 22 led to its classification as the first member of a new genus,Parechovirus, and renaming as human parechovirus type 1 (HPeV1). Although distinct from other genera in most of the genome, the 5′ untranslated region (5′UTR) shows similarities to that of cardio/aphthoviruses in some of its structural domains (A to L). The 5′UTR plays an important role in picornavirus translation initiation and in RNA synthesis. To investigate translation in HPeV1, we engineered an extensive range of mutations (including precise deletions and point mutations) into the 5′UTR. Their effects were studied both by in vitro transcription-translation using a bicistronic construct and by in vivo studies using an infectious, full-length HPeV1 cDNA. These approaches allowed the HPeV1 internal ribosome entry site (IRES) to be mapped. Deletions within the first 298 nucleotides had little impact in the in vitro system, while deletions of nucleotides 298 to 538 had a significant effect. Precise removal of domains H and L (nucleotides 287 to 316 and 664 to 682, respectively) did not significantly reduce translation efficiency in vitro, while domains I, J, and K (nucleotides 327 to 545, 551 to 661, and 614 to 645, respectively) appeared to have much more important roles. Mutation of a phylogenetically conserved GNRA motif (positions 421 to 424) within domain I severely reduced translation. We also confirmed the identity of the AUG (positions 710 to 712) which initiates the open reading frame, the positive identification of which has not been possible previously, as the N terminus of the polyprotein is blocked and not amenable to sequence analysis. This is therefore important in understanding parechovirus genome organization. Mutation of the AUG or an upstream polypyrimidine tract leads to aberrant translation, suggesting they both form part of the parechovirus Yn-Xm-AUG motif. In vivo experiments confirmed the importance of domains I, J, and K, the conserved GNRA motif, polypyrimidine sequences, and AUG, as mutations here were lethal. These features are also important in the IRES elements of cardio/aphthoviruses, but other features reported to be part of the IRES of some members of these genera, notably domains H and L, do not appear to be critical in HPeV1. This adds weight to the idea that there may be functional differences between the IRES elements of different picornaviruses, even when they share significant structural similarity.

scholarly journals Methyltransferase like 13 mediates the translation of Snail in head and neck squamous cell carcinoma

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Xiaochen Wang ◽  
Kang Li ◽  
Yuehan Wan ◽  
Fangfang Chen ◽  
Maosheng Cheng ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Head And Neck ◽  
Squamous Cell ◽  
Neck Squamous Cell Carcinoma ◽  
Translation Efficiency ◽  
Mechanism Study ◽  
Clinical Prognosis ◽  
Cellular Phenotypes

AbstractMethyltransferase like 13 (METTL13), a kind of methyltransferase, is implicated in protein binding and synthesis. The upregulation of METTL13 has been reported in a variety of tumors. However, little was known about its potential function in head and neck squamous cell carcinoma (HNSCC) so far. In this study, we found that METTL13 was significantly upregulated in HNSCC at both mRNA and protein level. Increased METTL13 was negatively associated with clinical prognosis. And METTL13 markedly affected HNSCC cellular phenotypes in vivo and vitro. Further mechanism study revealed that METTL13 could regulate EMT signaling pathway by mediating enhancing translation efficiency of Snail, the key transcription factor in EMT, hence regulating the progression of EMT. Furthermore, Snail was verified to mediate METTL13-induced HNSCC cell malignant phenotypes. Altogether, our study had revealed the oncogenic role of METTL13 in HNSCC, and provided a potential therapeutic strategy.

scholarly journals Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically enhance E-cadherin silencing in breast cancer

2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Yawei Wang ◽  
Yingying Sun ◽  
Chao Shang ◽  
Lili Chen ◽  
Hongyu Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Transcription Factors ◽  
Cancer Cells ◽  
Epithelial Mesenchymal Transition ◽  
Epigenetic Mechanism ◽  
Regulation Mechanism ◽  
Rna Helicases ◽  
Mesenchymal Transition ◽  
Dead Box ◽  
E Cadherin

AbstractRing1b is a core subunit of polycomb repressive complex 1 (PRC1) and is essential in several high-risk cancers. However, the epigenetic mechanism of Ring1b underlying breast cancer malignancy is poorly understood. In this study, we showed increased expression of Ring1b promoted metastasis by weakening cell–cell adhesions of breast cancer cells. We confirmed that Ring1b could downregulate E-cadherin and contributed to an epigenetic rewiring via PRC1-dependent function by forming distinct complexes with DEAD-box RNA helicases (DDXs) or epithelial-mesenchymal transition transcription factors (EMT TFs) on site-specific loci of E-cadherin promoter. DDXs-Ring1b complexes moderately inhibited E-cadherin, which resulted in an early hybrid EMT state of epithelial cells, and EMT TFs-Ring1b complexes cooperated with DDXs-Ring1b complexes to further repress E-cadherin in mesenchymal-like cancer cells. Clinically, high expression of Ring1b with DDXs or EMT TFs predicted low levels of E-cadherin, metastatic behavior, and poor prognosis. These findings provide an epigenetic regulation mechanism of Ring1b complexes in E-cadherin expression. Ring1b complexes may be potential therapeutic targets and biomarkers for diagnosis and prognosis in invasion breast cancer.

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