DEAD-Box RNA Helicases and Genome Stability.

DEAD-box RNA helicases are important regulators of RNA metabolism and have been implicated in the development of cancer. Interestingly, these helicases constitute a major recurring family of RNA-binding proteins important for protecting the genome. Current studies have provided insight into the connection between genomic stability and several DEAD-box RNA helicase family proteins including DDX1, DDX3X, DDX5, DDX19, DDX21, DDX39B, and DDX41. For each helicase, we have reviewed evidence supporting their role in protecting the genome and their suggested mechanisms. Such helicases regulate the expression of factors promoting genomic stability, prevent DNA damage, and can participate directly in the response and repair of DNA damage. Finally, we summarized the pathological and therapeutic relationship between DEAD-box RNA helicases and cancer with respect to their novel role in genome stability.

The DEAD-box RNA helicase RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis.

RNA helicases perform essential housekeeping and regulatory functions in all domains of life by binding and unwinding RNA molecules. The bacterial RhlE-like DEAD-box RNA helicases are among the least well studied of these enzymes. They are widespread especially among Proteobacteria, whose genomes often encode multiple homologs. The significance of the expansion and diversification of RhlE-like proteins for bacterial fitness has not yet been established. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. We show that, in the course of evolution, RhlE1 and RhlE2 have diverged in their biological functions, molecular partners and RNA-dependent enzymatic activities. Whereas RhlE1 is mainly needed for growth in the cold, RhlE2 also acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery. Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.

Sequence- and structure-based analysis of proteins involved in miRNA biogenesis.

miRNA biogenesis is a multistage process for the generation of a mature miRNA and involves several different proteins. In this work, we have carried out both sequence- and structure-based analysis for crucial proteins involved in miRNA biogenesis, namely Dicer, Drosha, Argonaute (Ago), and Exportin-5 to understand evolution of these proteins in animal kingdom and also to identify key sequence and structural features that are determinants of their function. Our analysis reveals that in animals the miRNA biogenesis pathway first originated in molluscs. The phylogeny of Dicer and Ago indicated evolution through gene duplication followed by sequence divergence that resulted in functional divergence. Our detailed structural analysis also revealed that RIIIDb domains of Drosha and Dicer, share significant similarity in sequence, structure, and substrate-binding pocket. On the other hand, PAZ domains of Dicer and Ago show only conservation of the substrate-binding pockets in the catalytic sites despite significant divergence in sequence and overall structure. Based on a comparative structural analysis of all four human Ago proteins (hAgo1-4) and their known biochemical activity, we have also attempted to identify key residues in Ago2 which are responsible for the unique slicer activity of hAgo2 among all isoforms. We have identified six key residues in N domain of hAgo2, which are located far away from the catalytic pocket, but might be playing a major role in slicer activity of hAgo2 protein because of their involvement in mRNA binding.

DExD/H-box RNA helicase genes are differentially expressed between males and females during the critical period of male sex differentiation in channel catfish.

DExD/H-box RNA helicases are motor proteins participating in nearly all aspects of cellular processes, especially in RNA metabolism. In this study, a total of 54 DExD/H-box RNA helicase genes including 37 DDX (DEAD-box) and 17 DHX (DEAH-box) genes were characterized in channel catfish (Ictalurus punctatus), and annotated through phylogenetic and syntenic analyses. All the catfish RNA helicases contained conserved helicase signature motifs, demonstrating that the RNA helicase gene family was highly conserved. Analysis of the relative rates of synonymous (dS) and nonsynonymous (dN) substitutions revealed that the RNA helicase genes were subjected to strong negative (purifying) selection. Meta-analysis was conducted to determine expression of the RNA helicase genes during the critical period (90-110days post-fertilization, dpf) of male gonad differentiation. At 90dpf, 24 RNA helicase genes were highly differentially expressed in the gonad tissues between the males and females; similarly, 24 and 18 RNA helicase genes were found highly differentially expressed in the gonad tissues between the males and females at 100 and 110dpf, respectively (p<0.01). In general, the vast majority of the RNA helicase genes (31) were expressed at higher levels in females than in males. In the male gonad, a set of 8 RNA helicases were expressed at a significantly higher level at 110dpf than at 90dpf. These findings suggested that RNA helicases may play important roles in sex development and differentiation in teleosts.

Integrating multiple evidences in taxonomy: species diversity and phylogeny of mustached bats (Mormoopidae: Pteronotus).

A phylogenetic systematic perspective is instrumental in recovering new species and their evolutionary relationships. The advent of new technologies for molecular and morphological data acquisition and analysis, allied to the integration of knowledge from different areas, such as ecology and population genetics, allows for the emergence of more rigorous, accurate and complete scientific hypothesis on species diversity. Mustached bats (genus Pteronotus) are a good model for the application of this integrative approach. They are a widely distributed and a morphologically homogeneous group, but comprising species with remarkable differences in their echolocation strategy and feeding behavior. The latest systematic review suggested six species with 17 subspecies in Pteronotus. Subsequent studies using discrete morphological characters supported the same arrangement. However, recent papers reported high levels of genetic divergence among conspecific taxa followed by bioacoustic and geographic agreement, suggesting an underestimated diversity in the genus. To date, no study merging genetic evidences and morphometric variation along the entire geographic range of this group has been attempted. Based on a comprehensive sampling including representatives of all current taxonomic units, we attempt to delimit species in Pteronotus through the application of multiple methodologies and hierarchically distinct datasets. The molecular approach includes six molecular markers from three genetic transmission systems; morphological investigations used 41 euclidean distances estimated through three-dimensional landmarks collected from 1628 skulls. The phylogenetic analysis reveals a greater diversity than previously reported, with a high correspondence among the genetic lineages and the currently recognized subspecies in the genus. Discriminant analysis of variables describing size and shape of cranial bones support the rising of the genetic groups to the specific status. Based on multiples evidences, we present an updated taxonomic arrangement composed by 16 extant species and a new and more robust phylogenetic hypothesis for the species included in the genus Pteronotus. Studies developed under such integrative taxonomic approach are timely for a deeper and wider comprehension of Neotropical diversity, representing the first step for answering broader questions on evolutionary and ecological aspects of Neotropical life history.

Comparative Evolution of Duplicated Ddx3 Genes in Teleosts: Insights from Japanese Flounder, Paralichthys olivaceus.

Following the two rounds of whole-genome duplication that occurred during deuterostome evolution, a third genome duplication event occurred in the stem lineage of ray-finned fishes. This teleost-specific genome duplication is thought to be responsible for the biological diversification of ray-finned fishes. DEAD-box polypeptide 3 (DDX3) belongs to the DEAD-box RNA helicase family. Although their functions in humans have been well studied, limited information is available regarding their function in teleosts. In this study, two teleost Ddx3 genes were first identified in the transcriptome of Japanese flounder (Paralichthys olivaceus). We confirmed that the two genes originated from teleost-specific genome duplication through synteny and phylogenetic analysis. Additionally, comparative analysis of genome structure, molecular evolution rate, and expression pattern of the two genes in Japanese flounder revealed evidence of subfunctionalization of the duplicated Ddx3 genes in teleosts. Thus, the results of this study reveal novel insights into the evolution of the teleost Ddx3 genes and constitute important groundwork for further research on this gene family.

DEAD box RNA helicase functions in cancer.

Members of the DEAD box family of RNA helicases are known to be involved in most cellular processes that require manipulation of RNA structure and, in many cases, exhibit other functions in addition to their established ATP-dependent RNA helicase activities. They thus play critical roles in cellular metabolism and in many cases have been implicated in cellular proliferation and/or neoplastic transformation. These proteins generally act as components of multi-protein complexes; therefore their precise role is likely to be influenced by their interacting partners and to be highly context-dependent. This may also provide an explanation for the sometimes conflicting reports suggesting that DEAD box proteins have both pro- and anti-proliferative roles in cancer.

Superfamily 2 helicases.

Superfamily 2 helicases are involved in all aspects of RNA metabolism, and many steps in DNA metabolism. This review focuses on the basic mechanistic, structural and biological properties of each of the families of helicases within superfamily 2. There are ten separate families of helicases within superfamily 2, each playing specific roles in nucleic acid metabolism. The mechanisms of action are diverse, as well as the effect on the nucleic acid. Some families translocate on single-stranded nucleic acid and unwind duplexes, some unwind double-stranded nucleic acids without translocation, and some translocate on double-stranded or single-stranded nucleic acids without unwinding.

Phylogenetic distribution and evolutionary history of bacterial DEAD-Box proteins.

DEAD-box proteins are found in all domains of life and participate in almost all cellular processes that involve RNA. The presence of DEAD and Helicase_C conserved domains distinguish these proteins. DEAD-box proteins exhibit RNA-dependent ATPase activity in vitro, and several also show RNA helicase activity. In this study, we analyzed the distribution and architecture of DEAD-box proteins among bacterial genomes to gain insight into the evolutionary pathways that have shaped their history. We identified 1,848 unique DEAD-box proteins from 563 bacterial genomes. Bacterial genomes can possess a single copy DEAD-box gene, or up to 12 copies of the gene, such as in Shewanella. The alignment of 1,208 sequences allowed us to perform a robust analysis of the hallmark motifs of DEAD-box proteins and determine the residues that occur at high frequency, some of which were previously overlooked. Bacterial DEAD-box proteins do not generally contain a conserved C-terminal domain, with the exception of some members that possess a DbpA RNA-binding domain (RBD). Phylogenetic analysis showed a separation of DbpA-RBD-containing and DbpA-RBD-lacking sequences and revealed a group of DEAD-box protein genes that expanded mainly in the Proteobacteria. Analysis of DEAD-box proteins from Firmicutes and γ-Proteobacteria, was used to deduce orthologous relationships of the well-studied DEAD-box proteins from Escherichia coli and Bacillus subtilis. These analyses suggest that DbpA-RBD is an ancestral domain that most likely emerged as a specialized domain of the RNA-dependent ATPases. Moreover, these data revealed numerous events of gene family expansion and reduction following speciation.

Identification of a vasa homologue gene in grass carp and its expression pattern in tissues and during embryogenesis.

The RNA helicase Vasa is a member of the DEAD box protein family that plays an indispensable role in germ cell determination in eukaryotes such as in Drosophila and Xenopus species. In this study, the grass carp homologue of the Drosophila vasa gene, Civasa (Ctenopharyngodon idella vasa) was obtained using degenerate primers in RT-PCR and 5' and 3' rapid amplification of cDNA end polymerase chain reaction from the grass carp ovary SMART cDNA. This cDNA sequence encodes a 670 amino acid residue protein that contains eight consensus regions for the DEAD box protein family, 9 arginine-glycine repeats and 9 arginine-glycine-glycine repeats, a common character of known Vasa homologues. CiVasa shows high identity to that of zebrafish and other animals, suggesting Vasa is highly conserved through evolution. RT-PCR analysis reveals that in grass carp tissues, both ovaries and testes contain large amounts of vasa gene transcripts whereas no Civasa transcript is detected in somatic tissues examined. The Civasa transcripts are present at a high level from the 2 cell stage to gastrula stages which indicated that Cicasa transcripts are maternally inherited. The predicted protein sequence, localization and conserved pattern of gene expression suggest that Civasa plays an important role in the germ cell determinant and development in grass carp as proposed for other teleost species.

Loss of the plant DEAD-box protein ISE1 leads to defective mitochondria and increased cell-to-cell transport via plasmodesmata.

Plants have intercellular channels, plasmodesmata (PD), that span the cell wall to enable cell-to-cell transport of micro- and macromolecules. We identified an Arabidopsis thaliana embryo lethal mutant increased size exclusion limit 1 (ise1) that results in increased PD-mediated transport of fluorescent tracers. The ise1 mutants have a higher frequency of branched and twinned PD than wild-type embryos. Silencing of ISE1 in mature Nicotiana benthamiana leaves also leads to increased PD transport, as monitored by intercellular movement of a GFP fusion to the tobacco mosaic virus movement protein. ISE1 encodes a putative plant-specific DEAD-box RNA helicase that localizes specifically to mitochondria. The N-terminal 100 aa of ISE1 specify mitochondrial targeting. Mitochondrial metabolism is compromised severely in ise1 mutant embryos, because their mitochondrial proton gradient is disrupted and reactive oxygen species production is increased. Although mitochondria are essential for numerous cell-autonomous functions, the present studies demonstrate that mitochondrial function also regulates the critical cell non-cell-autonomous function of PD.

SpolvlgA is a DDX3/PL10-related DEAD-box RNA helicase expressed in blastomeres and embryonic cells in planarian embryonic development.

Planarian flatworms have an impressive regenerative power. Although their embryonic development is still poorly studied and is highly derived it still displays some simple characteristics. We have identified SpolvlgA, a Schmidtea polychroa homolog of the DDX3/PL10 DEAD-box RNA helicase DjvlgA from the planarian species Dugesia japonica. This gene has been previously described as being expressed in planarian adult stem cells (neoblasts), as well as the germ line. Here we present the expression pattern of SpolvlgA in developing embryos of S. polychroa and show that it is expressed from the first cleavage rounds in blastomere cells and blastomere-derived embryonic cells. These cells are undifferentiated cells that engage in a massive wave of differentiation during stage 5 of development. SpolvlgA expression highlights this wave of differentiation, where nearly all previous structures are substituted by blastomere-derived embryonic cells. In late stages of development SpolvlgA is expressed in most proliferating and differentiating cells. Thus, SpolvlgA is a gene expressed in planarian embryos from the first stages of development and a good marker for the zygote-derived cell lineage in these embryos. Expression in adult worms is also monitored and is found in the planarian germ line, where it is showed to be expressed in spermatogonia, spermatocytes and differentiating spermatids.

Evolution of MDA-5/RIG-I-dependent innate immunity: independent evolution by domain grafting.

Type I Interferons (IFNs) are requisite components in antiviral innate immunity. Classically, a Toll-like receptor-dependent pathway induces type I interferons. However, recent recognition of melanoma differentiation associated gene-5 (MDA-5) and retinoic acid inducible gene-I (RIG-I) as primary sensors of RNA viruses for type I interferon induction highlights a potentially unique pathway for innate immunity. Our present investigation tracing the phylogenetic origin of MDA-5 and RIG-I domain arrangement (CARD1-CARD2-helicase-DEAD/DEAH) indicates that these proteins originated specifically in mammals, firmly linking this family of proteins with interferons in a highly derived evolutionary development of innate immunity. MDA-5, but not RIG-I, orthologs are found in fish, indicating that MDA-5 might have evolved before RIG-I. Our analyses also reveal that the MDA-5 and RIG-I domain arrangement evolved independently by domain grafting and not by a simple gene-duplication event of the entire four-domain arrangement, which may have been initiated by differential sensitivity of these proteins to viral infection.

The DEAD box RNA helicases p68 (Ddx5) and p72 (Ddx17): novel transcriptional co-regulators.

DEAD box [a motif named after its amino acid sequence (Asp-Glu-Ala-Asp)] RNA helicases are known to play key roles in all cellular processes that require modulation of RNA structure. However, in recent years, several of these proteins have been found to function in transcriptional regulation. In the present paper, we shall review the literature demonstrating the action of p68 and, where data are available, p72 as transcriptional co-regulators for a range of transcription factors, namely ERalpha (oestrogen receptor alpha), the tumour suppressor p53, the myogenic regulator MyoD and Runx2, a transcription factor essential for osteoblast development. We shall also discuss evidence indicating that, in some cases at least, p68 and p72 have distinct, non-redundant, roles.

Embryonic expression of p68, a DEAD-box RNA helicase, in the oligochaete annelid Tubifex tubifex.

We have cloned and characterized the expression of a p68 homologue (designated Ttu-p68) from the oligochaete annelid Tubifex tubifex. Ttu-p68 mRNA is distributed broadly throughout the early stages. Ttu-p68 is expressed in all of the early blastomeres, in which Ttu-p68 RNA associates with pole plasms. Ttu-p68 transcripts are concentrated to 4d cell but not to 2d cell. During gastrulation, expression of Ttu-p68 is restricted to elongating germ bands (GBs) and an anteriormost crescent of micromere descendants on both sides of the embryo. During body elongation that follows gastrulation, expression of Ttu-p68 is further restricted to the stomodaeum (derived from the micromere crescent), ventral ganglia, lateral dots (corresponding to dorsal and ventral setal sacs), ventral large cells (that resemble presumptive primordial germ cells) in segments VIII-XII, and a bilateral pair of cell clusters at the caudal end. At the end of embryogenesis, Ttu-p68 expression persists exclusively in the tail and the lining epithelium of the pharynx.

A vasa-like gene in the giant freshwater prawn, Macrobrachium rosenbergii.

A molecular marker for germ cells of the giant freshwater prawn, Macrobrachium rosenbergii, was studied. A vasa-like gene, Mrvlg, from the ovary was isolated and characterized by a reverse transcriptase-polymerase chain reaction (RT-PCR) method. A full-length sequence was obtained by the rapid amplification of cDNA end (RACE) method. Analysis of the nucleotide sequence revealed that Mrvlg comprises 2,686 bps with an open reading frame of 2,130 bps encoding 710 amino acids. The deduced amino acid sequence contains four arginine-glycine-glycine motifs and eight conserved motifs belonging to the DEAD-box protein family. The MrVLG sequence shows high similarity to Vasa homologue of zebrafish (73%). In the adult tissues, the Mrvlg transcripts were specifically detected in the germ cells. In situ hybridization analysis showed that Mrvlg RNA was detected in the cytoplasm of oogonia, previtellogenic, and vitellogenic oocytes and was also detected in the nucleoplasm of mature oocytes. In the testis, the Mrvlg transcript was detected in the cytoplasm of spermatogonia and primary spermatocytes but was detected in the nuclei of secondary spermatocytes and sperm. Sequence similarity and specific localization in the germ cells suggest that Mrvlg is the prawn vasa homologue of the Drosophila gene and can be used as a molecular marker for prawn germ cells.