DEAD-box RNA Helicase Protein Research Articles

Role and therapeutic potential of DEAD-box RNA helicase family in colorectal cancer.

Colorectal cancer (CRC) is the third most commonly diagnosed and the second cancer-related death worldwide, leading to more than 0.9 million deaths every year. Unfortunately, this disease is changing rapidly to a younger age, and in a more advanced stage when diagnosed. The DEAD-box RNA helicase proteins are the largest family of RNA helicases so far. They regulate almost every aspect of RNA physiological processes, including RNA transcription, editing, splicing and transport. Aberrant expression and critical roles of the DEAD-box RNA helicase proteins have been found in CRC. In this review, we first summarize the protein structure, cellular distribution, and diverse biological functions of DEAD-box RNA helicases. Then, we discuss the distinct roles of DEAD-box RNA helicase family in CRC and describe the cellular mechanism of actions based on recent studies, with an aim to provide future strategies for the treatment of CRC.

Systematic in silico Evaluation of Leishmania spp. Proteomes for Drug Discovery.

Leishmaniasis is a group of neglected infectious diseases, with approximately 1. 3 million new cases each year, for which the available therapies have serious limitations. Therefore, it is extremely important to apply efficient and low-cost methods capable of selecting the best therapeutic targets to speed up the development of new therapies against those diseases. Thus, we propose the use of integrated computational methods capable of evaluating the druggability of the predicted proteomes of Leishmania braziliensis and Leishmania infantum, species responsible for the different clinical manifestations of leishmaniasis in Brazil. The protein members of those proteomes were assessed based on their structural, chemical, and functional contexts applying methods that integrate data on molecular function, biological processes, subcellular localization, drug binding sites, druggability, and gene expression. These data were compared to those extracted from already known drug targets (BindingDB targets), which made it possible to evaluate Leishmania proteomes for their biological relevance and treatability. Through this methodology, we identified more than 100 proteins of each Leishmania species with druggability characteristics, and potential interaction with available drugs. Among those, 31 and 37 proteins of L. braziliensis and L. infantum, respectively, have never been tested as drug targets, and they have shown evidence of gene expression in the evolutionary stage of pharmacological interest. Also, some of those Leishmania targets showed an alignment similarity of <50% when compared to the human proteome, making these proteins pharmacologically attractive, as they present a reduced risk of side effects. The methodology used in this study also allowed the evaluation of opportunities for the repurposing of compounds as anti-leishmaniasis drugs, inferring potential interaction between Leishmania proteins and ~1,000 compounds, of which only 15 have already been tested as a treatment for leishmaniasis. Besides, a list of potential Leishmania targets to be tested using drugs described at BindingDB, such as the potential interaction of the DEAD box RNA helicase, TRYR, and PEPCK proteins with the Staurosporine compound, was made available to the public.

DEAD-box RNA helicase protein DDX21 as a prognosis marker for early stage colorectal cancer with microsatellite instability

DEAD-box RNA helicase DDX21 (also named nucleolar RNA helicase 2) is a nuclear autoantigen with undefined roles in cancer. To explore possible roles of autoimmune recognition in cancer immunity, we examined DDX21 protein expression in colorectal cancer tissue and its association with patient clinical outcomes. Unbiased deep proteomic profiling of two independent colorectal cancer cohorts using mass spectrometry showed that DDX21 protein was significantly upregulated in cancer relative to benign mucosa. We then examined DDX21 protein expression in a validation group of 710 patients, 619 of whom with early stage and 91 with late stage colorectal cancers. DDX21 was detected mostly in the tumor cell nuclei, with high expression in some mitotic cells. High levels of DDX21 protein were found in 28% of stage I, 21% of stage II, 30% of stage III, and 32% of stage IV colorectal cancer cases. DDX21 expression levels correlated with non-mucinous histology in early stage cancers but not with other clinicopathological features such as patient gender, age, tumor location, tumor grade, or mismatch repair status in any cancer stage. Kaplan–Meier analyses revealed that high DDX21 protein levels was associated with longer survival in patients with early stage colorectal cancer, especially longer disease-free survival in patients with microsatellite instability (MSI) cancers, but no such correlations were found for the microsatellite stable subtype or late stage colorectal cancer. Univariate and multivariate analyses also identified high DDX21 protein expression as an independent favorable prognostic marker for early stage MSI colorectal cancer.

Roles of Organellar RNA-Binding Proteins in Plant Growth, Development, and Abiotic Stress Responses.

Organellar gene expression (OGE) in chloroplasts and mitochondria is primarily modulated at post-transcriptional levels, including RNA processing, intron splicing, RNA stability, editing, and translational control. Nucleus-encoded Chloroplast or Mitochondrial RNA-Binding Proteins (nCMRBPs) are key regulatory factors that are crucial for the fine-tuned regulation of post-transcriptional RNA metabolism in organelles. Although the functional roles of nCMRBPs have been studied in plants, their cellular and physiological functions remain largely unknown. Nevertheless, existing studies that have characterized the functions of nCMRBP families, such as chloroplast ribosome maturation and splicing domain (CRM) proteins, pentatricopeptide repeat (PPR) proteins, DEAD-Box RNA helicase (DBRH) proteins, and S1-domain containing proteins (SDPs), have begun to shed light on the role of nCMRBPs in plant growth, development, and stress responses. Here, we review the latest research developments regarding the functional roles of organellar RBPs in RNA metabolism during growth, development, and abiotic stress responses in plants.

A DEAD-box RNA helicase TCD33 that confers chloroplast development in rice at seedling stage under cold stress

Cold stress is one of the most common unfavorable environmental factors affecting the growth, development, and survival of plants. The DEAD-box RNA helicases play important roles in all types of processes of RNA metabolism. However, the function of DEAD-box RNA helicase under cold stress is poorly explored in plants, especially in rice. This study reported the identification of a novel rice thermo-sensitive chlorophyll-deficient mutant, tcd33, which displayed an albino phenotype before the four-leaf stage, then withered and eventually died at 20 °C, while wild-type plants exhibited normal green coloration at 32 °C. The tcd33 seedlings also exhibited less chlorophyll contents and severe defects of chloroplast structure under 20 °C condition. Map-based cloning and complementation experiments suggested that TCD33 encodes a chloroplast-located DEAD-box RNA helicase protein. The transcript expression level of TCD33 indicated that the genes related to chlorophyll (Chl) biosynthesis, photosynthesis, and chloroplast development in tcd33 mutants were down-regulated at 20 °C, while the down-regulated genes were nearly recovered to or slightly higher than the WT level at 32 °C. Together, our results suggest that the cold-inducible TCD33 is essential for early chloroplast development and is important for cold-responsive gene regulation and cold tolerance in rice.

Maheshvara, a Conserved RNA Helicase, Regulates Notch Signaling in Drosophila melanogaster.

Gene expression is regulated at multiple steps after generation of primary RNA transcripts, including mRNA processing, stability, and transport, along with co- and post-transcriptional regulation. These processes are controlled via the involvement of a multitude of RNA binding proteins (RBPs). Innumerable human diseases have been associated with altered expression of RNA binding proteins. In this chapter we have focused on Maheshvara (mahe) which encodes a putative DEAD box RNA helicase protein in Drosophila. We have recently reported that mahe plays an important role in regulation of Notch signaling. Fine tuning of Notch signaling is required at multiple stepsand it's misregulation leads to a variety of human diseases. Additionally, mutation in DDX59, a human homolog of mahe results in broad neurological phenotypesassociated with orofaciodigital syndrome. Drosophila mahe mutants show abnormal peripheral and central nervous system development that resemble neuropathology of patients having mutation in DDX59 gene. This chapter will help in advancing the knowledge as to how mahe regulates Notch signaling and nervous system development.

FLT3-ITD Activates Cytoplasmic Drosha-Dependent Non-Canonical Mechanisms of Mir-155 Biogenesis in Acute Myeloid Leukemia

MiRNAs are small noncoding RNAs that control gene expression by binding to cognate sites in the 3′ untranslated region (3′ UTR) of target messenger RNAs (mRNAs). MiRNAs are transcribed as primary (pri) mRNAs and processed into precursor (pre) miRNAs by nuclear DROSHA before being exported into the cytoplasm via the Exportin 5 (XPO5)/RAN-GTP complex. Once in the cytoplasm, pre-miRNAs are further processed by Dicer into mature miRNAs. The FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common mutations in acute myeloid leukemia (AML) and confers growth and survival advantages to leukemia blasts. Among miRNAs, miR-155 has emerged as one of the most significantly upregulated in the FLT3-ITD+ AML and has been shown to play a pivotal role in uncontrolled blast hyperproliferation and survival. Herein, we report a previously unrecognized activity of FLT3-ITD that leads to deregulation of miR-155 expression by providing the evidence for the existence of FLT3-dependent non-canonical mechanisms of miRNA biogenesis. We initially observed that FLT3-ITD blocked the biogenesis of intronic miRNAs via inhibition of the "gatekeeper" XPO5/RAN-GTP complex that allow nucleus-to-cytoplasm pre-miRNA transport. By using in vitro phosphorylation assay with [32P]-ATP labeling, we demonstrated that FLT3-ITD phosphorylates a Sprouty related EVH1 domain-containing protein 1 (SPRED1), and that phospho-SPRED1 in turn inhibited the XPO5/RAN-GTP complex thereby halting transportation of pre-miRNAs from the nucleus to cytoplasm. This resulted in a decrease of several intronic miRNAs involved in normal hematopoiesis (i.e., miR-29b, miR-181a, miR-146b, miR-126). Accordingly, knocking down of SPRED1 expression in FLT3-ITD+ AML cells resulted in increased production of mature intronic miRNAs. Since the XPO5/RAN-GTP complex is the main gatekeeper for miRNA biogenesis, these results appeared in contradiction with the miR-155 upregulation which has been invariably observed in FLT3-ITD+ AML blasts. Differently from intronic miRNAs, miR-155 is hosted at the genomic site of long non-coding RNA (lnc-RNA) and we therefore hypothesized that it follows a different path of biogenesis. In fact forced expression of FLT3-ITD in lin-Sca1+kit+ (LSK) cells decreased intronic miRNA biogenesis (i.e., miR-126) and increase production of lnc-RNA hosted miRNAs (i.e., miR-155). Accordingly, we demonstrated that upon FLT3-ITD activation, AKT phosphorylated DDX3X, a DEAD-box RNA helicase protein thereby reducing its ability to bind heterogeneous nuclear Ribonucleoprotein U (hnRNP U), a component of the hnRNP complex associated with pre-mRNA splicing. Consequently, in FLT3-ITD cells, BIC-155 splicing is decreased. In fact, we measured significant increased levels of phospho-DDX3X and unspliced BIC-155 in primary FLT3-ITD+ blasts compared with FLT3-ITD- AML blasts. In consistent, DDX3X knock-down (KO) in FLT3-ITD- cells decreased BIC-155 splicing whereas DDX3X re-expression reversed these effects. The excess of unspliced BIC-155 RNA then bound to nuclear RNA export factor 1 (NXF1), a shuttle protein that transports poly A+ RNAs from the nucleus to the cytoplasm. In consistent, NXF1 KO blocked unspliced BIC-155 nucleus-to-cytoplasm transportation. In FLT3-ITD+ blasts, once in the cytoplasm, the unspliced BIC-155 RNA was then processed by cytoplasmic isoforms of DROSHA, as demonstrated using immunostaining, RNA Immunoprecipitation (RIP), and other gain- and loss-of-function experiments. Furthermore, overexpression of cytoplasmic DROSHA but not nuclear DROSHA isoform increased the processing of unspliced BIC-155 to mature miR-155. None of the intronic miRNAs studies interacted with cytoplasmic DROSHA. The bound of BIC-155 RNA with cytoplasmic DROSHA was unique of FLT3-ITD+ blasts, as it was not observed in FLT3-ITD- blasts. Thus, our results indicate a two-fold activity of FLT3-ITD that leads to decreased levels of intronic miRNAs via XPO5/RAN-GTP blockage and upregulation of other lnc-RNA hosted miRNAs via cytoplasmic DROSHA activation. The net result is suppression of intronic miRNAs that participates to the regulation of normal hematopoiesis and upregulation of lnc-RNA-hosted miRNA, especially miR-155 that contribute to aberrant blast hyperproliferation in the FLT3-ITD AML phenotype (Figure 1). Disclosures No relevant conflicts of interest to declare.

Insights into the RNA quadruplex binding specificity of DDX21

Guanine quadruplexes can form in both DNA and RNA and influence many biological processes through various protein interactions. The DEAD-box RNA helicase protein DDX21 has been shown to bind and remodel RNA quadruplexes but little is known about its specificity for different quadruplex species. Previous reports have suggested DDX21 may interact with telomeric repeat containing RNA quadruplex (TERRA), an integral component of the telomere that contributes to telomeric heterochromatin formation and telomere length regulation. Here we report that the C-terminus of DDX21 directly interacts with TERRA. We use, for the first time, 2D saturation transfer difference NMR to map the protein binding site on a ribonucleic acid species and show that the quadruplex binding domain of DDX21 interacts primarily with the phosphoribose backbone of quadruplexes. Furthermore, by mutating the 2′OH of loop nucleotides we can drastically reduce DDX21's affinity for quadruplex, indicating that the recognition of quadruplex and specificity for TERRA is mediated by interactions with the 2′OH of loop nucleotides.

Distinct RNA-unwinding mechanisms of DEAD-box and DEAH-box RNA helicase proteins in remodeling structured RNAs and RNPs.

Structured RNAs and RNA-protein complexes (RNPs) fold through complex pathways that are replete with misfolded traps, and many RNAs and RNPs undergo extensive conformational changes during their functional cycles. These folding steps and conformational transitions are frequently promoted by RNA chaperone proteins, notably by superfamily 2 (SF2) RNA helicase proteins. The two largest families of SF2 helicases, DEAD-box and DEAH-box proteins, share evolutionarily conserved helicase cores, but unwind RNA helices through distinct mechanisms. Recent studies have advanced our understanding of how their distinct mechanisms enable DEAD-box proteins to disrupt RNA base pairs on the surfaces of structured RNAs and RNPs, while some DEAH-box proteins are adept at disrupting base pairs in the interior of RNPs. Proteins from these families use these mechanisms to chaperone folding and promote rearrangements of structured RNAs and RNPs, including the spliceosome, and may use related mechanisms to maintain cellular messenger RNAs in unfolded or partially unfolded conformations.

Comparative proteomic analysis provides insight into the biological role of protein phosphatase inhibitor-2 from Arabidopsis

Protein phosphatase inhibitor-2 (PPI-2) is a conserved eukaryotic effector protein that inhibits type one protein phosphatases (TOPP). A transfer-DNA knockdown of AtPPI-2 resulted in stunted growth in both vegetative and reproductive phases of Arabidopsis development. At the cellular level, AtPPI-2 knockdown had 35 to 40% smaller cells in developing roots and leaves. This developmental phenotype was rescued by transgenic expression of the AtPPI-2 cDNA behind a constitutive promoter. Comparative proteomics of developing leaves of wild type (WT) and AtPPI-2 mutant revealed reduced levels of proteins associated with chloroplast development, ribosome biogenesis, transport, and cell cycle regulation processes. Decreased abundance of several ribosomal proteins, a DEAD box RNA helicase family protein (AtRH3), Clp protease (ClpP3) and proteins associated with cell division suggests a bottleneck in chloroplast ribosomal biogenesis and cell cycle regulation in AtPPI-2 mutant plants. In contrast, eight out of nine Arabidopsis TOPP isoforms were increased at the transcript level in AtPPI-2 leaves compared to WT. A protein-protein interaction network revealed that >75% of the differentially accumulated proteins have at least secondary and/or tertiary connections with AtPPI-2. Collectively, these data reveal a potential basis for the growth defects of AtPPI-2 and support the presumed role of AtPPI-2 as a master regulator for TOPPs, which regulate diverse growth and developmental processes. Biological significanceComparative label-free proteomics was used to characterize an AtPPI-2T-DNA knockdown mutant. The complex, reduced growth phenotype supports the notion that AtPPI-2 is a global regulator of TOPPs, and possibly other proteins. Comparative proteomics revealed a range of differences in protein abundance from various cellular processes such as chloroplast development, ribosome biogenesis, and transporter activity in the AtPPI-2 mutant relative to WT Arabidopsis. Collectively the results of proteomic analysis and the protein-protein network suggest that AtPPI-2 is involved in a wide range of biological processes either directly or indirectly including plastid biogenesis, translational mechanisms, and cell cycle regulation. The proposed protein interaction network comprises a testable model underlying changes in protein abundance in the AtPPI-2 mutant, and provides a better framework for future studies.

Identification of Reference Genes for qRT-PCR Analysis in Yesso Scallop Patinopecten yessoensis

BackgroundBivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and β-actin (ACT) is most frequently used as qRT-PCR reference gene without validation.ResultsIn this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop ( Patinopecten yessoensis ) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ∆Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae.ConclusionsThis work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species.

Toward Understanding Life Under Subzero Conditions: The Significance of Exploring Psychrophilic “Cold-Shock” Proteins

Understanding the behavior of proteins under freezing conditions is vital for detecting and locating extraterrestrial life in cold environments, such as those found on Mars and the icy moons of Jupiter and Saturn. This review highlights the importance of studying psychrophilic "cold-shock" proteins, a topic that has yet to be explored. A strategy for analyzing the psychrophilic RNA helicase protein CsdA (Psyc_1082) from Psychrobacter arcticus 273-4 as a key protein for life under freezing temperatures is proposed. The experimental model presented here was developed based on previous data from investigations of Escherichia coli, P. arcticus 273-4, and RNA helicases. P. arcticus 273-4 is considered a model for life in freezing environments. It is capable of growing in temperatures as cold as -10°C by using physiological strategies to survive not only in freezing temperatures but also under low-water-activity and limited-nutrient-availability conditions. The analyses of its genome, transcriptome, and proteome revealed specific adaptations that allow it to inhabit freezing environments by adopting a slow metabolic strategy rather than a cellular dormancy state. During growth at subzero temperatures, P. arcticus 273-4 genes related to energy metabolism and carbon substrate incorporation are downregulated, and genes for maintenance of membranes, cell walls, and nucleic acid motion are upregulated. At -6°C, P. arcticus 273-4 does not upregulate the expression of either RNA or protein chaperones; however, it upregulates the expression of its cold-shock induced DEAD-box RNA helicase protein A (CsdA - Psyc_1082). CsdA - Psyc_1082 was investigated as a key helper protein for sustaining life in subzero conditions. Proving CsdA - Psyc_1082 to be functional as a key protein for life under freezing temperatures may extend the known minimum growth temperature of a mesophilic cell and provide key information about the mechanisms that underlie cold-induced biological systems in icy worlds.

Mechanism of Mss116 ATPase Reveals Functional Diversity of DEAD-Box Proteins

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein that is essential for efficient in vivo splicing of all group I and group II introns and for activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ∼7-fold by promoting rate-limiting ATP hydrolysis such that inorganic phosphate (Pi) release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-Pi binds RNA more strongly than Mss116 with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA-binding Mss116–ADP-Pi state. Strong RNA binding allows Mss116 to fulfill its biological role in the stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA-binding ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with Pi release dissociates Mss116–RNA complexes, regenerating free Mss116.

Pathway of ATP utilization and duplex rRNA unwinding by the DEAD-box helicase, DbpA

DEAD-box RNA helicase proteins use the energy of ATP hydrolysis to drive the unwinding of duplex RNA. However, the mechanism that couples ATP utilization to duplex RNA unwinding is unknown. We measured ATP utilization and duplex RNA unwinding by DbpA, a non-processive bacterial DEAD-box RNA helicase specifically activated by the peptidyl transferase center (PTC) of 23S rRNA. Consumption of a single ATP molecule is sufficient to unwind and displace an 8 base pair rRNA strand annealed to a 32 base pair PTC-RNA "mother strand" fragment. Strand displacement occurs after ATP binding and hydrolysis but before P(i) product release. P(i) release weakens binding to rRNA, thereby facilitating the release of the unwound rRNA mother strand and the recycling of DbpA for additional rounds of unwinding. This work explains how ATPase activity of DEAD-box helicases is linked to RNA unwinding.

A TCRMimic Monoclonal Antibody Generated from a Directly Discovered Epitope of p68 RNA Helicase Dramatically Debulks Large Established Tumors through Recognition of a Specific MHC/Peptide Complex.

Abstract Debulking large tumors using monoclonal antibodies (mAb) that specifically recognize unique T cell epitopes represents a novel approach to cancer therapy.Using a mAb designated as a TCRm we recently demonstrated the in vivo suppression of tumor growth. The TCRm had specificity for a peptide/MHC class I molecule expressed on the surface of the tumor cells. In this study we use an innovative direct epitope discovery strategy that led to the identification of peptide YLLPAIVHI in the context of HLA-A*0201 (YLL/A2) in human breast carcinoma cell lines. The peptide is derived from the DEAD Box RNA helicase protein p68, a protein overexpressed and highly phosphorylated in malignant cells. A TCRm antibody (RL6A) was generated and characterized for binding specificity, sensitivity and affinity for the YLL/A2 target. The TCRm specifically stained cells that co-expressed HLA-A2 and p68 but not cells lacking expression of p68 protein or HLA-A2 alone. It binds to cognate peptide/HLA-A2 with an affinity constant (KD) of 5.69 x 10-10 M and recognized specifically the YLL/A2 epitope in human breast cancer tissue but not in normal control breast tissue.Mice implanted in the mammary fat pad with human breast tumor cells grew tumors of considerable size, yet when treated with RL6A TCRm there was a marked reduction in tumor size. Examination of frozen tumor tissue sections revealed significantly greater numbers of apoptotic cells in mice treated with RL6A TCRm than with control antibody. Investigation in vitro showed that TCRm engagement of specific YLL/A2 epitope led to the induction of apoptosis providing the first experimental evidence that MHC signaling could directly mediate tumor cell death. Together, these data indicate that TCRm targeting of specific peptide/MHC complexes unique to tumor cells is effective not only in slowing the growth of tumors but also in dramatically reducing the size of established tumors and suggests a possible novel approach to the treatment of cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5072.

Molecular cloning and characterization of the soybean DEAD-box RNA helicase gene induced by low temperature and high salinity stress

A novel gene encoding a DEAD-box RNA helicase designated as GmRH was isolated from soybean. Amino acid sequence alignment and phylogenetic tree analysis revealed a close relationship between GmRH and other orthologous DEAD-box RNA helicases from other plant species. Structural motif analysis revealed that the bipartite lysine rich nuclear localization signal (NLS) is present in the N-terminal variable region of GmRH and that there are ten conserved motifs found in DEAD-box RNA helicase proteins. Southern blot analysis revealed the presence of 2 copies of GmRH in the soybean genome. Northern blot analysis demonstrated that the RNA expression of the GmRH was induced during low temperature or high salinity stress, but not by the exogenous application of abscisic acid or drought stress. Subcellular localization studies showed that GmRH (1–355)-GFP is localized in the nucleus, whereas GmRH (130–355)-GFP is localized both in the cytoplasm and in the nucleus. This provides the evidence that the N-terminal region predicted as NLS is essential for nuclear targeting of the GmRH protein in the plant cell. Purified GST-GmRH recombinant protein was shown to unwind dsRNA independent of ATP in vitro. Here, we propose that GmRH plays an important role in RNA processing during low temperature and high salinity stresses in plants.

OsBIRH1, a DEAD-box RNA helicase with functions in modulating defence responses against pathogen infection and oxidative stress

DEAD-box proteins comprise a large protein family with members from all kingdoms and play important roles in all types of processes in RNA metabolism. In this study, a rice gene OsBIRH1, which encodes a DEAD-box RNA helicase protein, was cloned and characterized. The predicted OsBIRH1 protein contains a DEAD domain and all conserved motifs that are common characteristics of DEAD-box RNA helicases. Recombinant OsBIRH1 protein purified from Escherichia coli was shown to have both RNA-dependent ATPase and ATP-dependent RNA helicase activities in vitro. Expression of OsBIRH1 was activated in rice seedling leaves after treatment with defence-related signal chemicals, for example benzothiadiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid, and jasmonic acid, and was also up-regulated in an incompatible interaction between a resistant rice genotype and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants that overexpress the OsBIRH1 gene were generated. Disease resistance phenotype assays revealed that the OsBIRH1-overexpressing transgenic plants showed an enhanced disease resistance against Alternaria brassicicola and Pseudomonas syringae pv. tomato DC3000. Meanwhile, defence-related genes, for example PR-1, PR-2, PR-5, and PDF1.2, showed an up-regulated expression in the transgenic plants. Moreover, the OsBIRH1 transgenic Arabidopsis plants also showed increased tolerance to oxidative stress and elevated expression levels of oxidative defence genes, AtApx1, AtApx2, and AtFSD1. The results suggest that OsBIRH1 encodes a functional DEAD-box RNA helicase and plays important roles in defence responses against biotic and abiotic stresses.

NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47

Previously, we described a novel nucleolar protein, NOP132, which interacts with the small GTP binding protein RRAG A. To elucidate the function of NOP132 in the nucleolus, we identified proteins that interact with NOP132 using mass spectrometric methods. NOP132 associated mainly with proteins involved in ribosome biogenesis and RNA metabolism, including the DEAD-box RNA helicase protein, DDX47, whose yeast homolog is Rrp3, which has roles in pre-rRNA processing. Immunoprecipitation of FLAG-tagged DDX47 co-precipitated rRNA precursors, as well as a number of proteins that are probably involved in ribosome biogenesis, implying that DDX47 plays a role in pre-rRNA processing. Introduction of NOP132 small interfering RNAs induced a ring-like localization of DDX47 in the nucleolus, suggesting that NOP132 is required for the appropriate localization of DDX47 within the nucleolus. We propose that NOP132 functions in the recruitment of pre-rRNA processing proteins, including DDX47, to the region where rRNA is transcribed within the nucleolus.

Expression of rck/p54, a DEAD‐box RNA helicase, in gametogenesis and early embryogenesis of mice

rck/p54 is a DEAD-box RNA helicase protein with ATP-dependent RNA-unwinding activity. Its ortholog is required for sexual reproduction in yeast and for oocyte survival and sperm fertility in Caenorhabditis elegans. In the current study, we investigated the expression of rck/p54 in mouse gametogenesis and early embryogenesis. Western blot analysis revealed that rck/p54 was highly expressed in both the ovary and testis. In the ovary, maturing oocytes strongly expressed rck/p54 in their cytoplasm. In contrast, in the testis, spermatogonia and primary spermatocytes highly expressed rck/p54 in their cytoplasm, but its expression decreased in the spermatids. Interestingly, rck/p54 was concentrated in the heads of spermatozoa; and then its expression gradually decreased as these cells matured along the epididymal duct. After fertilization, rck/p54 protein and its mRNA remained present in the pronucleus phase; and then their expression levels slightly but definitely decreased in morulae and blastocytes. The injection of a CMV-based rck/p54 expression vector into the pronuclei of fertilized eggs caused a delay in early embryogenesis. In generating RCK transgenic mice, the birth rate of the mice was significantly lower than those of other gene transgenic mice. These findings indicate that rck/p54 may play an important role in gametogenesis and early embryogenesis in mammals.

Human DDX3Y, the Y-encoded isoform of RNA helicase DDX3, rescues a hamster temperature-sensitive ET24 mutant cell line with a DDX3X mutation

We investigated the function of DDX3Y, the Y chromosome AZFa region encoding a putative DEAD-box RNA helicase protein, the loss of which results in oligozoospermia or azoospermia in humans. The human DDX3Y amino acid sequence is similar to that of the X chromosome gene DDX3X (91.7% homology). Here we report that human Y- and X-encoded DEAD box RNA helicase proteins DDX3Y and DDX3X are interchangeable and have an essential function: both proteins rescued a temperature-sensitive mutant hamster cell line (tsET24) that was otherwise incapable of growth at a nonpermissive temperature. Mouse homologues Ddx3y and D1Pas1–PL10 also rescued the mutant cell line at a nonpermissive temperature. In situ hybridization revealed that Ddx3x mRNA was expressed in almost every cell in mouse testis, suggesting that Ddx3x is involved in spermatogenesis. A comparative study of DDX3X and DDX3Y was performed to determine the significance of DDX3Y for cell growth and spermatogenesis. Both DDX3X and DDX3Y promoter DNAs produced a similar degree of transcription in vivo, whereas deletion studies of the promoter DNAs indicated that these genes are differentially regulated. DDX3Y, similar to DDX3X, shuttles between the nucleus and cytoplasm in a crm1-dependent manner.