Deformability in the cleavage site of primary microRNA is not sensed by the double‐stranded RNA binding domains in the microprocessor component DGCR8

The prevalence of double‐stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA‐binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD‐containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8‐Core, consists of its two dsRBDs and a C‐terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N‐terminal dsRBD of DGCR8 in isolation nor the DGCR8‐Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single‐stranded segments flanking the stem. Extending DGCR8‐Core to include an N‐terminal heme‐binding region does not change our conclusions. Thus, our data suggest that although the DGCR8‐Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency. Proteins 2015; 83:1165–1179. © 2015 Wiley Periodicals, Inc.     

DRB2, DRB3 and DRB5 function in a non-canonical microRNA pathway in Arabidopsis thaliana

DOUBLE-STRANDED RNA BINDING (DRB) proteins have been functionally characterized in viruses, prokaryotes and eukaryotes and are involved in all aspects of RNA biology. Arabidopsis thaliana (Arabidopsis) encodes five closely related DRB proteins, DRB1 to DRB5. DRB1 and DRB4 are required by DICER-LIKE (DCL) proteins DCL1 and DCL4 to accurately and efficiently process structurally distinct double-stranded RNA (dsRNA) precursor substrates in the microRNA (miRNA) and trans-acting small-interfering RNA (tasiRNA) biogenesis pathways respectively. We recently reported that DRB2 is also involved in the biogenesis of specific miRNA subsets.1 Furthermore, the severity of the developmental phenotype displayed by the drb235 triple mutant plant, compared with those expressed by either drb2, drb3 and drb5 single mutants, or double mutant combinations thereof, indicates that DRB3 and DRB5 function in the same non-canonical miRNA pathway as DRB2. Through the use of our artificial miRNA (amiRNA) plant expression vector, pBlueGreen2,3 we demonstrate here that unlike DRB2, DRB3 and DRB5 are not involved in the dsRNA processing stages of the miRNA biogenesis pathway, but are required to mediate RNA silencing of target genes of DRB2-associated miRNAs.     

Biogenesis of MiRNA-195 and its role in biogenesis, the cell cycle, and apoptosis

microRNA-195(miR-195) is an important member of the micro-15/16/195/424/497 family, and which is activated in multiple diseases, such as cancers, heart failure, and schizophrenia. Mir-195 regulates a plethora of target proteins, which are involved in the cell cycle, apoptosis, proliferation. WEE1, CDK6, and Bcl-2 are confirmed target genes of miR-195 that are involved in miR-195-mediated cell-cycle and apoptosis effects. However, the mechanism of miR-195 action is not completely understood. This review summarizes recent the research progress regarding the roles of miR-195 in the cell cycle and in apoptosis.     

Penicillin-binding protein 5 can form a homo-oligomeric complex in the inner membrane of Escherichia coli

Penicillin-binding protein 5 (PBP5) is a DD-carboxypeptidase, which cleaves the terminal D-alanine from the muramyl pentapeptide in the peptidoglycan layer of Escherichia coli and other bacteria. In doing so, it varies the substrates for transpeptidation and plays a key role in maintaining cell shape. In this study, we have analyzed the oligomeric state of PBP5 in detergent and in its native environment, the inner membrane. Both approaches indicate that PBP5 exists as a homo-oligomeric complex, most likely as a homo-dimer. As the crystal structure of the soluble domain of PBP5 (i.e., lacking the membrane anchor) shows a monomer, we used our experimental data to generate a model of the homo-dimer. This model extends our understanding of PBP5 function as it suggests how PBP5 can interact with the peptidoglycan layer. It suggests that the stem domains interact and the catalytic domains have freedom to move from the position observed in the crystal structure. This would allow the catalytic domain to have access to pentapeptides at different distances from the membrane.     

Staphylococcal superantigen-like protein 10 binds to phosphatidylserine and apoptotic cells

Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins that have structural similarities to staphylococcal superantigens. Although SSLs do not have superantigenic activity, some of them have been reported to bind to host immune related molecules and they have been implicated in immune evasion by S. aureus. In this study, we showed that SSL10 is capable of binding to phospholipids. SSL10 bound to phosphatidylserine (PS) containing liposome, but not to phosphatidylcholine liposome. SSL10, but not SSL7, bound to PS containing liposome, suggesting that SSL10 specifically binds to PS. Analysis of PS binding ability among recombinant truncated SSL10 fragments revealed that the β-barrel in the N-terminal oligonucleotide/oligosaccharide-binding (OB)-fold domain contributes to PS binding capacity. Fluorescein isothiocyanate labeled OB-fold of SSL10 stained hydrogen peroxide treated Jurkat cells. Annexin V is widely utilized for detection of apoptosis. Unlike annexin V, the OB-fold domain of SSL10 also bound to apoptotic cells in the presence of EDTA, suggesting that the OB-fold of SSL10 recognizes PS and apoptotic cells in a Ca(2+) independent manner. These findings suggest SSL10 and its derived peptides may be a novel detection tool for apoptotic cells.     

Structural and functional characterization of CcmG from Pseudomonas aeruginosa,a key component of the bacterial cytochrome c maturation apparatus

The cytochrome c maturation process is carried out in the bacterial periplasm, where some specialized thiol‐disulfide oxidoreductases work in close synergy for the correct reduction of oxidized apocytochrome before covalent heme attachment. We present a structural and functional characterization of the soluble periplasmic domain of CcmG from the opportunistic pathogen P. aeruginosa (Pa‐CcmG), a component of the protein machinery involved in cyt c maturation in gram‐negative bacteria. X‐ray crystallography reveals that Pa‐CcmG is a TRX‐like protein; high‐resolution crystal structures show that the oxidized and the reduced forms of the enzyme are identical except for the active‐site disulfide. The standard redox potential was calculated to be E^0′ = ?0.213 V at pH 7.0; the pK_a of the active site thiols were pK_a = 6.13 ± 0.05 for the N‐terminal Cys74 and pK_a = 10.5 ± 0.17 for the C‐terminal Cys77. Experiments were carried out to characterize and isolate the mixed disulfide complex between Pa‐CcmG and Pa‐CcmH (the other redox active component of System I in P. aeruginosa). Our data indicate that the target disulfide of this TRX‐like protein is not the intramolecular disulfide of oxidized Pa‐CcmH, but the intermolecular disulfide formed between Cys28 of Pa‐CcmH and DTNB used for the in vitro experiments. This observation suggests that, in vivo, the physiological substrate of Pa‐CcmG may be the mixed‐disulfide complex between Pa‐CcmH and apo‐cyt. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Targeting Notch signalling by the conserved miR-8/200 microRNA family in development and cancer cells

Notch signalling is crucial for the correct development and growth of numerous organs and tissues, and when subverted it can cause cancer. Loss of miR-8/200 microRNAs (miRNAs) is commonly observed in advanced tumours and correlates with their invasion and acquisition of stem-like properties. Here, we show that this miRNA family controls Notch signalling activation in Drosophila and human cells. In an overexpression screen, we identified the Drosophila miR-8 as a potent inhibitor of Notch-induced overgrowth and tumour metastasis. Gain and loss of mir-8 provoked developmental defects reminiscent of impaired Notch signalling and we demonstrated that miR-8 directly inhibits Notch ligand Serrate. Likewise, miR-200c and miR-141 directly inhibited JAGGED1, impeding proliferation of human metastatic prostate cancer cells. It has been suggested that JAGGED1 may also be important for metastases. Although in metastatic cancer cells, JAGGED1 modestly regulated ZEB1, the miR-200c's target in invasion, studies in Drosophila revealed that only concurrent overexpression of Notch and Zfh1/ZEB1 induced tumour metastases. Together, these data define a new way to attenuate or boost Notch signalling that may have clinical interest.     

Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.     

RBRDetector: Improved prediction of binding residues on RNA‐binding protein structures using complementary feature‐ and template‐based strategies

Computational prediction of RNA‐binding residues is helpful in uncovering the mechanisms underlying protein‐RNA interactions. Traditional algorithms individually applied feature‐ or template‐based prediction strategy to recognize these crucial residues, which could restrict their predictive power. To improve RNA‐binding residue prediction, herein we propose the first integrative algorithm termed RBRDetector (RNA‐Binding Residue Detector) by combining these two strategies. We developed a feature‐based approach that is an ensemble learning predictor comprising multiple structure‐based classifiers, in which well‐defined evolutionary and structural features in conjunction with sequential or structural microenvironment were used as the inputs of support vector machines. Meanwhile, we constructed a template‐based predictor to recognize the putative RNA‐binding regions by structurally aligning the query protein to the RNA‐binding proteins with known structures. The final RBRDetector algorithm is an ingenious fusion of our feature‐ and template‐based approaches based on a piecewise function. By validating our predictors with diverse types of structural data, including bound and unbound structures, native and simulated structures, and protein structures binding to different RNA functional groups, we consistently demonstrated that RBRDetector not only had clear advantages over its component methods, but also significantly outperformed the current state‐of‐the‐art algorithms. Nevertheless, the major limitation of our algorithm is that it performed relatively well on DNA‐binding proteins and thus incorrectly predicted the DNA‐binding regions as RNA‐binding interfaces. Finally, we implemented the RBRDetector algorithm as a user‐friendly web server, which is freely accessible at http://ibi.hzau.edu.cn/rbrdetector . Proteins 2014; 82:2455–2471. © 2014 Wiley Periodicals, Inc.     

Alternative mRNA processing increases the complexity of microRNA-based gene regulation in Arabidopsis

During trans-splicing of discontinuous organellar introns, independently transcribed coding sequences are joined together to generate a continuous mRNA. The chloroplast psaA gene from Chlamydomonas reinhardtii encoding the P(700) core protein of photosystem I (PSI) is split into three exons and two group IIB introns, which are both spliced in trans. Using forward genetics, we isolated a novel PSI mutant, raa4, with a defect in trans-splicing of the first intron. Complementation analysis identified the affected gene encoding the 112.4 kDa Raa4 protein, which shares no strong sequence identity with other known proteins. The chloroplast localization of the protein was confirmed by confocal fluorescence microscopy, using a GFP-tagged Raa4 fusion protein. RNA-binding studies showed that Raa4 binds specifically to domains D2 and D3, but not to other conserved domains of the tripartite group II intron. Raa4 may play a role in stabilizing folding intermediates or functionally active structures of the split intron RNA.