A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs

In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures

DEAD-Box Protein Ddx46 Is Required for the Development of the Digestive Organs and Brain in Zebrafish

Spatially and temporally controlled gene expression, including transcription, several mRNA processing steps, and the export of mature mRNA to the cytoplasm, is essential for developmental processes. It is well known that RNA helicases of the DExD/H-box protein family are involved in these gene expression processes, including transcription, pre-mRNA splicing, and rRNA biogenesis. Although one DExD/H-box protein, Prp5, a homologue of vertebrate Ddx46, has been shown to play important roles in pre-mRNA splicing in yeast, the in vivo function of Ddx46 remains to be fully elucidated in metazoans. In this study, we isolated zebrafish morendo (mor), a mutant that shows developmental defects in the digestive organs and brain, and found that it encodes Ddx46. The Ddx46 transcript is maternally supplied, and as development proceeds in zebrafish larvae, its ubiquitous expression gradually becomes restricted to those organs. The results of whole-mount in situ hybridization showed that the expression of various molecular markers in these organs is considerably reduced in the Ddx46 mutant. Furthermore, splicing status analysis with RT-PCR revealed unspliced forms of mRNAs in the digestive organ and brain tissues of the Ddx46 mutant, suggesting that Ddx46 may be required for pre-mRNA splicing during zebrafish development. Therefore, our results suggest a model in which zebrafish Ddx46 is required for the development of the digestive organs and brain, possibly through the control of pre-mRNA splicing

Role of the amino terminal RHAU-specific motif in the recognition and resolution of guanine quadruplex-RNA by the DEAH-box RNA helicase RHAU

Under physiological conditions, guanine-rich sequences of DNA and RNA can adopt stable and atypical four-stranded helical structures called G-quadruplexes (G4). Such G4 structures have been shown to occur in vivo and to play a role in various processes such as transcription, translation and telomere maintenance. Owing to their high-thermodynamic stability, resolution of G4 structures in vivo requires specialized enzymes. RHAU is a human RNA helicase of the DEAH-box family that exhibits a unique ATP-dependent G4-resolvase activity with a high affinity and specificity for its substrate in vitro. How RHAU recognizes G4-RNAs has not yet been established. Here, we show that the amino-terminal region of RHAU is essential for RHAU to bind G4 structures and further identify within this region the evolutionary conserved RSM (RHAU-specific motif) domain as a major affinity and specificity determinant. G4-resolvase activity and strict RSM dependency are also observed with CG9323, the Drosophila orthologue of RHAU, in the amino terminal region of which the RSM is the only conserved motif. Thus, these results reveal a novel motif in RHAU protein that plays an important role in recognizing and resolving G4-RNA structures, properties unique to RHAU among many known RNA helicases

ATP-Dependent Unwinding of U4/U6 snRNAs by the Brr2 Helicase Requires the C Terminus of Prp8

The spliceosome is a highly dynamic machine requiring multiple RNA-dependent ATPases of the DExD/H-box family. A fundamental unanswered question is how their activities are regulated. Brr2 function is necessary for unwinding the U4/U6 duplex, a step essential for catalytic activation of the spliceosome. Here we show that Brr2-dependent dissociation of U4/U6 snRNAs in vitro is activated by a fragment from the C terminus of the U5 snRNP protein Prp8. In contrast to its helicase-stimulating activity, this fragment inhibits Brr2 U4/U6-dependent ATPase activity. Notably, U4/U6 unwinding activity is not stimulated by fragments carrying alleles of prp8 that in humans confers an autosomal dominant form of retinitis pigmentosa. Because Brr2 activity must be restricted to prevent premature catalytic activation, our results have important implications for fidelity maintenance in the spliceosome

Nuclear DNA Replication in Trypanosomatids:There Are No Easy Methods for Solving Difficult Problems

In trypanosomatids, etiological agents of devastating diseases, replication is robust and finely controlled to maintain genome stability and function in stressful environments. However, these parasites encode several replication protein components and complexes that show potentially variant composition compared with model eukaryotes. This review focuses on the advances made in recent years regarding the differences and peculiarities of the replication machinery in trypanosomatids, including how such divergence might affect DNA replication dynamics and the replication stress response. Comparing the DNA replication machinery and processes of parasites and their hosts may provide a foundation for the identification of targets that can be used in the development of chemotherapies to assist in the eradication of diseases caused by these pathogens

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

The Effectiveness of Voluntary Jurisdiction in the ICJ: El Salvador v. Honduras, A Case in Point

This Comment discusses the positive aspects of consensual jurisdiction of the International Court of Justice by exploring the case that Honduras and El Salvador Voluntarily brought before a Chamber of the International Court of Justice. Part I discusses the history of the ICJ as well as the history of the dispute between Honduras and El Salvador. Part II examines the ICJ\u27s reasoning and conclusions in the El Salvador v. Honduras case. Part III argues that this case demonstrates the advantages of a court that offers greater flexibility without sacrificing its integrity and dedication to the development of international law. This Comment concludes that states should take advantage of the increasingly accessible World Court that has demonstrated its ability to deal with complex international disputes, territorial or otherwise