![Conserved basic residues in VP35 IID recognize the dsRNA backbone and the dsRNA blunt ends are "end-capped" by a pocket of hydrophobic residues that mimic RIG-1 like receptors of blunt-end dsRNA. [Leung, Structural Basis for dsRNA recognition and interferon antagonism by Ebola V35, Nature Structural & Molecular Biology; 2009]](https://www.researchgate.net/profile/Rick-Ricketson/publication/220028008/figure/fig7/AS:668342633394179@1536356709123/Conserved-basic-residues-in-VP35-IID-recognize-the-dsRNA-backbone-and-the-dsRNA-blunt_Q320.jpg)
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Computational Evidence for a Viral Encoded miRNA in the 5' UTR of the Zaire Ebolavirus Nucleoprotein Gene May Explain Its Differential Virulence: the Pandora Element
Abstract and Figures
The Zaire ebolavirus is one of the most virulent of human viral pathogens with a reported CFR of 90% resulting from complete disruption of the host immune response. Despite the relatively minor genetic differences between the species of the genera Ebolavirus, the case fatality rate (CFR) is significantly different. The Zaire Ebolavirus (EBOV) historically is the most lethal, with its sporadic outbreaks resulting in a CFR ranging from 60% (Minkébé, Gabon 1994) to 90% (Kéllé, Congo 2003). This is in sharp contrast to Reston ebolavirus (RESTV) which, despite its genetic similarity to EBOV and serologic evidence of human transmission, has yet to result in even minor symptoms of the disease.
The viral protein, VP35, has been shown to be a multifunctional virulence factor by antagonizing anti-viral signalling pathways via its interferon inhibitory domain (IID). However, it is unlikely the VP35 pathways could fully account for the observed differential virulence between the species. Viral encoded miRNAs have already been identified in many species and shown to modulate the expression and antiviral function of interferons (IFNs).
This paper summarizes the ab initio identification of a probable miRNA within the 5' UTR of the genomic Zaire ebolavirus nucleoprotein gene, termed the Pandora Element, and a possible mechanism by which it may associate with VP35 to achieve the observed disruption of the immune response. A structural characteristic of the RESTV Pandora domain will also be introduced that may also account for its lack of virulence. The associated hsa-miR-4999, hsa-mir-1302, and hsa-miR-2054 targets include XCL1 chemokine, ubiquitin specific peptidase 3, multiple signalling factors and interleukin.
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Supplementary resources (18)
ABSTRACT
Pathologies associated with Zika virus infection include partial progressive paralysis (Guillan Barre Syndrome) and microcephaly in Brazil and Panama. We identified an 85 nucleotide stem loop structure in the 3' end of Zika virus sfRNA (position (343-428) with the canonical structural and sequence characteristics of a miRNA. In comparison, the West Nile Virus has been previously demonstrated to contain a miRNA in its 3’ untranslated (UTR) sfRNA region. The West Nile Virus miRNA, KUN-mir-1, was found to be 81 nucleotides in length (492-573) and 58% conserved overall as to the corresponding Zika virus stem loop nucleotide sequence. The majority on the sequence homology was in the terminal loop (78%). The 40 nucleotide (nt) 3p arm demonstrated 60% homology, decreasing at the distal segment. The 5p arm demonstrates on 42% sequence homology.
The Zika virus stem loop in this study was identified to contain a hairpin structure in the 3’ end of the Zika virus sfRNA 3’ UTR segment predicted to contain a miRNA with significant homology to human hsa-mir-147a. Target analysis identified 241 human transcripts which include, but not limited to, neurofascin, synaptic vesicle glycoprotein 2A, neurofibromin 1, SAM and SH3 domain containing 1, neurogenin 2, in addition to multiple immune transcripts.
Autoantibodies to neurofascin have been reported in in Guillain-Barre Syndrome (GBS), but have not yet been reported in or after Zika virus infection. A significant 9 mer match exists between the Zika virus miRNA 3p arm and the Neurofascin 3’UTR seed region, CCACACA. In contrast to West Nile Virus, GATA4 was not predicted to be, nor was identified in the MirBaseDB, to be a target of the Zika virus miRNA.
These computational results indicate highly plausible mechanisms explaining the neurologic syndromes such a Guillan-Barre Syndrome and microcephaly associated with Zika virus infection, and warrant further investigation.
We describe an update of the miRBase database (http://www.mirbase.org/), the primary microRNA sequence repository. The latest miRBase release (v20, June 2013) contains 24 521 microRNA loci from
206 species, processed to produce 30 424 mature microRNA products. The rate of deposition of novel microRNAs and the number
of researchers involved in their discovery continue to increase, driven largely by small RNA deep sequencing experiments.
In the face of these increases, and a range of microRNA annotation methods and criteria, maintaining the quality of the microRNA
sequence data set is a significant challenge. Here, we describe recent developments of the miRBase database to address this
issue. In particular, we describe the collation and use of deep sequencing data sets to assign levels of confidence to miRBase
entries. We now provide a high confidence subset of miRBase entries, based on the pattern of mapped reads. The high confidence
microRNA data set is available alongside the complete microRNA collection at http://www.mirbase.org/. We also describe embedding microRNA-specific Wikipedia pages on the miRBase website to encourage the microRNA community
to contribute and share textual and functional information.
One of the cellular functions of the ribonuclease Dicer is to process microRNA precursors (pre-miRNAs) into mature microRNAs (miRNAs). Human Dicer performs this function in cooperation with its protein partners, AGO2, PACT and TRBP. The exact role of these accessory proteins in Dicer activity is still poorly understood. In this study, we used the northern blotting technique to investigate pre-miRNA cleavage efficiency and specificity after depletion of AGO2, PACT and TRBP by RNAi. The results showed that the inhibition of either Dicer protein partner substantially affected not only miRNA levels but also pre-miRNA levels, and it had a rather minor effect on the specificity of Dicer cleavage. The analysis of the Dicer cleavage products generated in vitro revealed the presence of a cleavage intermediate when pre-miRNA was processed by recombinant Dicer alone. This intermediate was not observed during pre-miRNA cleavage by endogenous Dicer. We demonstrate that AGO2, PACT and TRBP were required for the efficient functioning of Dicer in cells, and we suggest that one of the roles of these proteins is to assure better synchronization of cleavages triggered by two RNase III domains of Dicer.
RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small
interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts
to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular
weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature
of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find
that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt
3′ overhangs and the thermodynamic properties of 2–4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer
in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential
of an siRNA.
microRNAs (miRNAs) are small, endogenous RNAs of 20 approximately 25 nucleotides, processed from stem-loop regions of longer RNA precursors. Plant miRNAs act as negative regulators of target mRNAs predominately by slicing target transcripts, and a number of miRNAs play important roles in development. We analyzed a number of published datasets from Arabidopsis thaliana to characterize novel miRNAs, novel miRNA targets, and miRNA-regulated developmental changes in gene expression. These data include microarray profiling data and small RNA (sRNA) deep sequencing data derived from miRNA biogenesis/transport mutants, microarray profiling data of mRNAs in a developmental series, and computational predictions of conserved genomic stem-loop structures. Our conservative analyses identified five novel mature miRNAs and seven miRNA targets, including one novel target gene. Two complementary miRNAs that target distinct mRNAs were encoded by one gene. We found that genes targeted by known miRNAs, and genes up-regulated or down-regulated in miRNA mutant inflorescences, are highly expressed in the wild type inflorescence. In addition, transcripts upregulated within the mutant inflorescences were abundant in wild type leaves and shoot meristems and low in pollen and seed. Downregulated transcripts were abundant in wild type pollen and seed and low in shoot meristems, roots and leaves. Thus, disrupting miRNA function causes the inflorescence transcriptome to resemble the leaf and meristem and to differ from pollen and seed. Applications of our computational approach to other species and the use of more liberal criteria than reported here will further expand the number of identified miRNAs and miRNA targets. Our findings suggest that miRNAs have a global role in promoting vegetative to reproductive transitions in A. thaliana.
Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNA classes have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, their modes of target regulation and in the biological pathways they regulate. There is a growing realization that, despite their differences, these distinct small RNA pathways are interconnected, and that small RNA pathways compete and collaborate as they regulate genes and protect the genome from external and internal threats.
MicroRNAs (miRNAs) are small noncoding RNA gene products about 22 nt long that are processed by Dicer from precursors with a characteristic hairpin secondary structure. Guidelines are presented for the identification and annotation of new miRNAs from diverse organisms, particularly so that miRNAs can be reliably distinguished from other RNAs such as small interfering RNAs. We describe specific criteria for the experimental verification of miRNAs, and conventions for naming miRNAs and miRNA genes. Finally, an online clearinghouse for miRNA gene name assignments is provided by the Rfam database of RNA families.
Posttranscriptional gene silencing allows sequence-specific control of gene expression. Specificity is guaranteed by small antisense RNAs such as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Functional miRNAs derive from longer double-stranded RNA (dsRNA) molecules that are cleaved to pre-miRNAs in the nucleus and are transported by exportin 5 (Exp 5) to the cytoplasm. Adenovirus-infected cells express virus-associated (VA) RNAs, which are dsRNA molecules similar in structure to pre-miRNAs. VA RNAs are also transported by Exp 5 to the cytoplasm, where they accumulate. Here we show that small RNAs derived from VA RNAs (svaRNAs), similar to miRNAs, can be found in adenovirus-infected cells. VA RNA processing to svaRNAs requires neither viral replication nor viral protein expression, as evidenced by the fact that svaRNA accumulation can be detected in cells transfected with VA sequences. svaRNAs are efficiently bound by Argonaute 2, the endonuclease of the RNA-induced silencing complex, and behave as functional siRNAs, in that they inhibit the expression of reporter genes with complementary sequences. Blocking svaRNA-mediated inhibition affects efficient adenovirus production, indicating that svaRNAs are required for virus viability. Thus, svaRNA-mediated silencing could represent a novel mechanism used by adenoviruses to control cellular or viral gene expression.
Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21 nt small interfering RNAs (siRNAs) during RNA interference, and excises microRNAs from precursor hairpins. Dicer contains two domains related to the bacterial dsRNA-specific endonuclease, RNase III, which is known to function as a homodimer. Based on an X-ray structure of the Aquifex aeolicus RNase III, models of the enzyme interaction with dsRNA, and its cleavage at two composite catalytic centers, have been proposed. We have generated mutations in human Dicer and Escherichia coli RNase III residues implicated in the catalysis, and studied their effect on RNA processing. Our results indicate that both enzymes have only one processing center, containing two RNA cleavage sites and generating products with 2 nt 3' overhangs. Based on these and other data, we propose that Dicer functions through intramolecular dimerization of its two RNase III domains, assisted by the flanking RNA binding domains, PAZ and dsRBD.