Cognate Target Research Articles

Structure and Biosynthesis of Crocagins: Polycyclic Posttranslationally Modified Ribosomal Peptides from Chondromyces crocatus.

Secondary metabolome mining efforts in the myxobacterial multiproducer of natural products, Chondromyces crocatus Cm c5, resulted in the isolation and structure elucidation of crocagins, which are novel polycyclic peptides containing a tetrahydropyrrolo[2,3-b]indole core. The gene cluster was identified through an approach combining genome analysis, targeted gene inactivation in the producer, and in vitro experiments. Based on our findings, we developed a biosynthetic scheme for crocagin biosynthesis. These natural products are formed from the three C-terminal amino acids of a precursor peptide and thus belong to a novel class of ribosomally synthesized and post-translationally modified peptides (RiPPs). We demonstrate that crocagin A binds to the carbon storage regulator protein CsrA, thereby inhibiting the ability of CsrA to bind to its cognate RNA target.

Small and Hungry: MicroRNAs in Micronutrient Homeostasis of Plants.

MicroRNAs are emerging players in plant development and response to stresses, both biotic and abiotic such as micronutrient deficiency. These small RNAs regulate cognate downstream targets either by transcript cleavage or translational inhibition. Micronutrient deficiencies lead to poor quality and yield of crops and impaired human health. Over the years several microRNAs have been identified which regulate expression of genes controlling uptake, mobilization and homeostasis of macronutrients such as nitrogen, phosphorus and sulfur to ensure sufficiency without toxicity. This review attempts to understand the roles played by micro RNAs involved in homeostasis of the micronutrients boron, manganese, zinc, copper, iron, molybdenum and nickel and the cross talk between them upon perception of nutritional stress. Notably and surprisingly, several micro RNAs are not specific for a particular micronutrient stress and the challenge remains to uncover ones (if any) that are directly relevant to the micronutrient. Current findings of this yet infant field could potentiate biotechnological applications towards biofortification, plant innate immunity and remedy heavy metal toxicity.

Glucocorticoid Receptor:MegaTrans Switching Mediates the Repression of an ERα-Regulated Transcriptional Program

The molecular mechanisms underlying the opposing functions of glucocorticoid receptors (GRs) and estrogen receptor α (ERα) in breast cancer development remain poorly understood. Here we report that, in breast cancer cells, liganded GR represses a large ERα-activated transcriptional program by binding, in trans, to ERα-occupied enhancers. This abolishes effective activation of these enhancers and their cognate target genes, and it leads to the inhibition of ERα-dependent binding of components of the MegaTrans complex. Consistent with the effects of SUMOylation on other classes of nuclear receptors, dexamethasone (Dex)-induced trans-repression of the estrogen E2 program appears to depend on GR SUMOylation, which leads to stable trans-recruitment of the GR-N-CoR/SMRT-HDAC3 corepressor complex on these enhancers. Together, these results uncover a mechanism by which competitive recruitment of DNA-binding nuclear receptors/transcription factors in trans to hot spot enhancers serves as an effective biological strategy for trans-repression, with clear implications for breast cancer and other diseases.

Three new active members of the I-OnuI family of homing endonucleases.

In vitro characterization of 3 LAGLIDADG-type homing endonucleases (HEs) (I-CcaI, I-CcaII, and I-AstI) that belong to the I-OnuI family showed that they are functional HEs that cleave their respective cognate target sites. These endonucleases are encoded within group ID introns and appear to be orthologues that have inserted into 3 different mitochondrial genes: rns, rnl, and cox3. The endonuclease activity of I-CcaI was tested using various substrates, and its minimum DNA recognition sequence was estimated to be 26 nt. This set of HEs may provide some insight into how these types of mobile elements can migrate into new locations. This study provides additional endonucleases that can be added to the catalog of currently available HEs that may have various biotechnology applications.

Aptamers as Valuable Molecular Tools in Neurosciences.

Aptamers are short nucleic acids that interact with a variety of targets with high affinity and specificity. They have been shown to inhibit biological functions of cognate target proteins, and they are identifiable by an in vitro selection process, also termed SELEX (Systematic Evolution of Ligands by EXponential enrichment). Being nucleic acids, aptamers can be synthesized chemically or enzymatically. The latter renders RNA aptamers compatible with the cell's own transcription machinery and, thus, expressable inside cells. The synthesis of aptamers by chemical approaches opens up the possibility of producing aptamers on a large scale and enables a straightforward access to introduce modifications in a site-specific manner (e.g., fluorophores or photo-labile groups). These characteristics make aptamers broadly applicable (e.g., as an analytical, diagnostic, or separation tool). In this TechSight, we provide a brief overview on aptamer technology and the potential of aptamers as valuable research tools in neurosciences.

Mechanisms of fast and stringent search in homologous pairing of double-stranded DNA.

Self-organization in the cell relies on the rapid and specific binding of molecules to their cognate targets. Correct bindings must be stable enough to promote the desired function even in the crowded and fluctuating cellular environment. In systems with many nearly matched targets, rapid and stringent formation of stable products is challenging. Mechanisms that overcome this challenge have been previously proposed, including separating the process into multiple stages; however, how particular in vivo systems overcome the challenge remains unclear. Here we consider a kinetic system, inspired by homology dependent pairing between double stranded DNA in bacteria. By considering a simplified tractable model, we identify different homology testing stages that naturally occur in the system. In particular, we first model dsDNA molecules as short rigid rods containing periodically spaced binding sites. The interaction begins when the centers of two rods collide at a random angle. For most collision angles, the interaction energy is weak because only a few binding sites near the collision point contribute significantly to the binding energy. We show that most incorrect pairings are rapidly rejected at this stage. In rare cases, the two rods enter a second stage by rotating into parallel alignment. While rotation increases the stability of matched and nearly matched pairings, subsequent rotational fluctuations reduce kinetic trapping. Finally, in vivo chromosome are much longer than the persistence length of dsDNA, so we extended the model to include multiple parallel collisions between long dsDNA molecules, and find that those additional interactions can greatly accelerate the searching.

Abstract A77: Efficient targeting of HER-2-positive cancers by Antibody-Coupled T cell Receptor (ACTR) engineered autologous T cells

Abstract HER2 gene amplification occurs in 20-30% of aggressive breast and gastric cancer diagnoses, often signifying poor prognosis. The current standard of care for HER2 amplified cancer is the HER2 targeting antibody trastuzumab with chemotherapy. In the setting of HER2 amplified breast cancer, the combination of HER2 targeting antibodies trastuzumab and pertuzumab with the chemotherapeutic docetaxel is the frontline therapy. Despite the success of targeting the HER2 pathway, there are still many patients who are refractory or relapse following HER2 targeting regimens. Outside of the setting of HER2 overexpression or gene amplification, targeting HER2-positive cancers has thus far been ineffective. Engineered autologous T-cells, including chimeric antigen receptors (CARs) and high affinity T-cell receptors (TCRs), have gained attention recently due to their potent efficacy, with overall response rates reaching 80% and examples of long lasting remissions, particularly in advanced lymphoma and leukemia. However, initial clinical attempts to target HER2-amplified breast cancer with CAR-T cell therapy met with either acute toxicities or lack of efficacy. The Antibody-Coupled T-cell Receptor (ACTR) platform is a universal, engineered T cell therapy technology developed to mediate anti-tumor activity in combination with tumor-targeting antibodies. The ACTR chimeric construct is composed of the high-affinity CD16 (FCGR3A) V158 variant with the signaling domains from CD3z and 4-1BB. ACTR is designed to engage the Fc domain of human IgG1 antibodies when opsonized to the cognate target cells and deliver an activation signal to the engineered T cells. ACTR T cells combined with either HER2 targeting trastuzumab or pertuzumab exhibited potent cytotoxic activity, cytokine response and proliferation on a HER2 amplified tumor cell lines. ACTR activity was specific to antibody treated cells, and had little activity on HER2 low or negative tumor lines. Furthermore, the effectiveness of ACTR T cells in a non-amplified HER2 (HER2 low) setting was tested in the presence of a combination of trastuzumab and pertuzumab. This multi-antibody combination increased the cytotoxicity by ACTR T cells, whereas trastuzumab or pertuzumab as single antibody combinations with ACTR had little effect, suggesting a potential therapeutic approach for this disease setting with high unmet medical need. Importantly, the ACTR platform decouples the targeting moiety from the engineered T cell allowing for an antibody dose response which may spare cytotoxicity on normal cells. The specific response to HER2 positive tumor cells in the presence of trastuzumab, pertuzumab or the combination of HER2 directed antibodies demonstrates the potential therapeutic activity of ACTR T cells and supports consideration of clinical testing of ACTR T cells in HER2-positive cancers. Furthermore, combining multiple targeting antibodies to achieve greater potency and efficacy is unique to the ACTR technology and demonstrates the potential for such activity across many other tumor targets and tumor targeting antibodies. Citation Format: Casey B. Judge, Rachel DeBarge, Eugene Choi, Katie O'Callaghan, Lindsay Edwards, Birgit Schultes, Seth Ettenberg, Heather A. Huet. Efficient targeting of HER-2-positive cancers by Antibody-Coupled T cell Receptor (ACTR) engineered autologous T cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A77.

Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity.

ABSTRACTAnalysis of the incorporation of cellular microRNAs (miRNAs) into highly purified HIV-1 virions revealed that this largely, but not entirely, mirrored the level of miRNA expression in the producer CD4+ T cells. Specifically, of the 58 cellular miRNAs detected at significant levels in the producer cells, only 5 were found in virions at a level 2- to 4-fold higher than that predicted on the basis of random cytoplasmic sampling. Of note, these included two miRNAs, miR-155 and miR-92a, that were reported previously to at least weakly bind HIV-1 transcripts. To test whether miRNA binding to the HIV-1 genome can induce virion incorporation, artificial miRNA target sites were introduced into the viral genome and a 10- to 40-fold increase in the packaging of the cognate miRNAs into virions was then observed, leading to the recruitment of up to 1.6 miRNA copies per virion. Importantly, this high level of incorporation significantly inhibited HIV-1 virion infectivity. These results suggest that target sites for cellular miRNAs can inhibit RNA virus replication at two distinct steps, i.e., during infection and during viral gene expression, thus explaining why a range of different RNA viruses appear to have evolved to avoid cellular miRNA binding to their genome.

Follicle-Stimulating Hormone Receptor Is Expressed by Most Ovarian Cancer Subtypes and Is a Safe and Effective Immunotherapeutic Target

To define the safety and effectiveness of T cells redirected against follicle-stimulating hormone receptor (FSHR)-expressing ovarian cancer cells. FSHR expression was determined by Western blotting, immunohistochemistry, and qPCR in 77 human ovarian cancer specimens from 6 different histologic subtypes and 20 human healthy tissues. The effectiveness of human T cells targeted with full-length FSH in vivo was determined against a panel of patient-derived xenografts. Safety and effectiveness were confirmed in immunocompetent tumor-bearing mice, using constructs targeting murine FSHR and syngeneic T cells. FSHR is expressed in gynecologic malignancies of different histologic types but not in nonovarian healthy tissues. Accordingly, T cells expressing full-length FSHR-redirected chimeric receptors mediate significant therapeutic effects (including tumor rejection) against a panel of patient-derived tumors in vivo In immunocompetent mice growing syngeneic, orthotopic, and aggressive ovarian tumors, fully murine FSHR-targeted T cells also increased survival without any measurable toxicity. Notably, chimeric receptors enhanced the ability of endogenous tumor-reactive T cells to abrogate malignant progression upon adoptive transfer into naïve recipients subsequently challenged with the same tumor. Interestingly, FSHR-targeted T cells persisted as memory lymphocytes without noticeable PD-1-dependent exhaustion during end-stage disease, in the absence of tumor cell immunoediting. However, exosomes in advanced tumor ascites diverted the effector activity of this and other chimeric receptor-transduced T cells away from targeted tumor cells. T cells redirected against FSHR+ tumor cells with full-length FSH represent a promising therapeutic alternative against a broad range of ovarian malignancies, with negligible toxicity even in the presence of cognate targets in tumor-free ovaries. Clin Cancer Res; 23(2); 441-53. ©2016 AACR.

MicroRNA sponging by a new hearty circRNA

The discovery of circular RNAs (circRNAs) as a widespread and abundant class of endogenous RNA in Eukarya (1-3) has sparked a previously unparalleled interest in this class of RNA molecules. CircRNAs, characterized by a covalently closed structure, are generated through back-splicing of one or more exons from protein-coding and non-coding genes and have been found in great number in diverse species such as human (1-3) and Drosophila (4). Almost in parallel with the realization of circRNAs as pervasive endogenous molecules, came the first functional characterization of circRNAs; two circRNAs, termed ciRS-7/CDR1as and Sry circRNA were found to bind to miRNAs and prevent these miRNAs from acting on their cognate targets (2,5).

Interrogation of Functional miRNA-Target Interactions by CRISPR/Cas9 Genome Engineering.

Post-transcriptional silencing by microRNAs (miRNAs) is a critical constituent of eukaryotic gene regulation. miRNAs are short (~22nt) noncoding RNAs capable of specifically targeting the miRNA-induced-silencing-complex (miRISC) to transcripts bearing a complementary miRNA response element (MRE). Although recent methodological advances have greatly improved our understanding of miRNA biogenesis and the mechanisms by which miRNAs repress their cognate targets, exploring the physiological relevance of direct miRNA-target interactions in vivo has remained an outstanding challenge. Here we describe the experimental protocol underlying a novel approach, which allows direct interrogation of specific miRNA-MRE interactions by CRISPR/Cas9-mediated genome engineering. In this instance, the CRISPR/Cas9 system is first used to catalyze homology-directed replacement of candidate MREs with molecular barcodes at endogenous loci. Subsequently, the effect of MRE mutation on transcript abundance (i.e., MRE activity) can be rapidly evaluated by routine quantitative PCR. This strategy enables functional investigation of a putative miRNA-target pair in a pool of transiently transfected cells, obviating the need for generation of clonal cell lines or transgenic animals. This protocol can be implemented in any cell line in less than 2 weeks, and can readily be scaled up for multiplex studies. To facilitate the conceptual workflow underlying this strategy, we also describe a genome-wide resource for automated design and computational evaluation of CRISPR/Cas9 guide RNAs targeting all predicted MREs in various species (miR-CRISPR).

Small RNA-Guided Transcriptional Gene Activation (RNAa) in Mammalian Cells.

Small RNA partnering with Argonaute (Ago) proteins plays important roles in diverse biological processes mainly by suppressing the expression of cognate target sequences. Mounting evidence reveals that the small RNA-Ago pathway can also positively regulate gene expression, a phenomenon termed as RNA activation (RNAa), which is evolutionarily conserved from Caenorhabditis elegans to human. In this chapter, I provide a general overview of mammalian RNAa phenomena and their basic characteristics and discuss recent advances toward understanding the nature of the molecular machinery responsible for RNAa and the development of RNAa-based research tools and therapeutics.

Drug Target Identification Using an iTRAQ-Based Quantitative Chemical Proteomics Approach-Based on a Target Profiling Study of Andrographolide.

Identifying the cellular binding targets of drugs and other bioactive small molecules is a crucial step for understanding their molecular mechanisms of action as well as potential off-target effects. The field of chemical proteomics is an emerging discipline in chemical biology using synthetic chemistry and high-throughput detection techniques to study small molecule-protein interactions. In this chapter, we describe a quantitative chemical proteomics protocol combining bioorthogonal click chemistry and quantitation by isobaric tags for relative and absolute quantification (iTRAQ) to identify the specific binding targets of drugs and bioactive small molecules such as natural products. A modified drug probe with a click chemistry-enabling addition is synthesized and used in live cell treatments where it undergoes covalent interactions with its cognate cellular targets. The probes are then ligated to biotin through click chemistry and enriched with avidin beads, followed by iTRAQ labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification and relative quantitation discriminating specific targets from nonspecific binding proteins. The presented protocol has been used to successfully profile prominent drugs and natural products including andrographolide, aspirin, curcumin, etc., and can be a powerful tool to study the molecular mechanisms of bioactive small molecules.

Antibodies raised against divalent type b flagellin and pilin provide effective immunotherapy against Pseudomonas aeruginosa infection of mice with burn wounds

Burn wound infections caused by multidrug-resistant Pseudomonas aeruginosa strains are a serious challenge to therapy because of the complex pathogenesis and paucity of new effective antibiotics. Therefore, there is renewed interest in developing antibody-based therapeutic strategies. Immunotherapy strategies typically target selected virulence factors that are expressed by the majority of clinical strains of P. aeruginosa, particularly because virulence factors mediate infection. Here we used a murine model of burn wound infection to evaluate the efficacy of antibodies raised against the divalent type b flagellin and PilA (flagellin b + PilA), as acute virulence factors, to prevent and treat infection. Antibodies to flagellin b + PilA exhibited superior synergistic effects that improved opsono-phagocytosis and cell invasion compared with antibodies to each monovalent flagellin b or PilA. Further, when used for prophylaxis, the antibodies against flagellin b + PilA and combined therapeutic and prophylactic regimens markedly improved the survival of mice infected with disparate P. aeruginosa strains from 91.6% to 100% compared with treatment using imipenem. Therefore, antibodies against flagellin b + PilA interfere with the activities of their respective cognate individual target antigens and enhance coverage against clinical strains of P. aeruginosa that may not express one of these two virulence factors.

Characterizing Semaphorin-Mediated Effects on Sensory and Motor Axon Pathfinding and Connectivity During Embryonic Development.

How are precise connectivity to peripheral targets and corresponding sensory-motor networks established during developmental innervation of the vertebrate extremities? The formation of functional sensory-motor circuits requires highly appropriate temporal and spatial regulation of axon growth which is achieved through the combination of different molecular mechanisms such as communication between heterotypic fiber systems, axon-environment, or axon-glia interactions that ensure proper fasciculation and accurate pathfinding to distal targets. Family members of the class 3 semaphorins and their cognate receptors, the neuropilins, were shown to govern various events during wiring of central and peripheral circuits, with mice lacking Sema3-Npn signaling showing deficits in timing of growth, selective fasciculation, guidance fidelity, and coupling of sensory axon growth to motor axons at developmental time points. Given the accuracy with which these processes have to interact in a stepwise manner, deficiency of the smallest cog in the wheel may impact severely on the faithful establishment and functionality of peripheral circuitries, ultimately leading to behavioral impairments or even cause the death of the animal. Reliable quantitative analyses of sensory-motor fasciculation, extension, and guidance of axons to their cognate target muscles and the skin during development, but also assessment of physiological and behavioral consequences at adult age, are therefore a necessity to extend our understanding of the molecular mechanisms of peripheral circuit formation. In this chapter we provide a detailed methodology to characterize class 3 semaphorin-mediated effects on peripheral sensory and motor axon pathfinding and connectivity during embryonic development.

Interaction of an Iridium(III) Complex with G-Quadruplex DNA and Its Application in Luminescent Switch-On Detection of Siglec-5.

Sialic acid (Sia) binding immunoglobulin (Ig)-like lectin-5 (Siglec-5) is a type-I transmembrane protein, and it has been demonstrated as a biomarker of granulocytic maturation and acute myeloid leukemia phenotype. Herein we aimed to construct a method that could sensitively detect Siglec-5 by taking advantage of the high affinity and selectivity of the K19 aptamer for its cognate target, and the selective interaction of luminescent iridium(III) transition metal complexes with G-quadruplex DNA. The iridium(III) complex 1 [Ir(tpyd)2(2,9-dmphen)]PF6 (where tpyd =2-(m-tolyl)pyridine; 2,9-dmphen =2,9-dimethyl-1,10-phenanthroline) was synthesized, and it displayed high luminescence for G-quadruplex DNA compared to dsDNA and ssDNA. Additionally, complex 1 exhibited a blue shift luminescence response to c-kit2 G-quadruplex, and the interaction between 1 and G-quadruplexes was discussed based on the results of G-tetrad assay, loop effect assay, and other assays. Then complex 1 was utilized to develop a G-quadruplex-based sensing platform for Siglec-5 in aqueous solution. Upon the addition of Siglec-5, the specific binding of the K19 aptamer sequence results in a conformational change that generates a split G-quadruplex structure, which is then recognized by the G-quadruplex-specific iridium(III) complex with an enhanced luminescent response. Futhermore, the use of the assay for detecting Siglec-5 in cellular debris was demonstrated.

Cognate Costs in Bilingual Speech Production: Evidence from Language Switching.

This study investigates cross-language lexical competition in the bilingual mental lexicon. It provides evidence for the occurrence of inhibition as well as the commonly reported facilitation during the production of cognates (words with similar phonological form and meaning in two languages) in a mixed picture naming task by highly proficient Welsh-English bilinguals. Previous studies have typically found cognate facilitation. It has previously been proposed (with respect to non-cognates) that cross-language inhibition is limited to low-proficient bilinguals; therefore, we tested highly proficient, early bilinguals. In a mixed naming experiment (i.e., picture naming with language switching), 48 highly proficient, early Welsh-English bilinguals named pictures in Welsh and English, including cognate and non-cognate targets. Participants were English-dominant, Welsh-dominant, or had equal language dominance. The results showed evidence for cognate inhibition in two ways. First, both facilitation and inhibition were found on the cognate trials themselves, compared to non-cognate controls, modulated by the participants' language dominance. The English-dominant group showed cognate inhibition when naming in Welsh (and no difference between cognates and controls when naming in English), and the Welsh-dominant and equal dominance groups generally showed cognate facilitation. Second, cognate inhibition was found as a behavioral adaptation effect, with slower naming for non-cognate filler words in trials after cognates than after non-cognate controls. This effect was consistent across all language dominance groups and both target languages, suggesting that cognate production involved cognitive control even if this was not measurable in the cognate trials themselves. Finally, the results replicated patterns of symmetrical switch costs, as commonly reported for balanced bilinguals. We propose that cognate processing might be affected by two different processes, namely competition at the lexical-semantic level and facilitation at the word form level, and that facilitation at the word form level might (sometimes) outweigh any effects of inhibition at the lemma level. In sum, this study provides evidence that cognate naming can cause costs in addition to benefits. The finding of cognate inhibition, particularly for the highly proficient bilinguals tested, provides strong evidence for the occurrence of lexical competition across languages in the bilingual mental lexicon.

Anti-regulatory T cells.

Our initial understanding of immune-regulatory cells was based on the discovery of suppressor cells that assure peripheral T-cell tolerance and promote immune homeostasis. Research has particularly focused on the importance of regulatory T cells (Tregs) for immune modulation, e.g. directing host responses to tumours or inhibiting autoimmunity development. However, recent studies report the discovery of self-reactive pro-inflammatory T cells-termed anti-regulatory T cells (anti-Tregs)-that target immune-suppressive cells. Thus, regulatory cells can now be defined as both cells that suppress immune reactions as well as effector cells that counteract the effects of suppressor cells and support immune reactions. Self-reactive anti-Tregs have been described that specifically recognize human leukocyte antigen-restricted epitopes derived from proteins that are normally expressed by regulatory immune cells, including indoleamine 2,3-dioxygenase (IDO), tryptophan 2,6-dioxygenase (TDO), programmed death-ligand 1 (PD-L1), and forkhead box P3 (Foxp3). These proteins are highly expressed in professional antigen-presenting cells under various physiological conditions, such as inflammation and stress. Therefore, self-reactive T cells that recognize such targets may be activated due to the strong activation signal given by their cognate targets. The current review describes the existing knowledge regarding these self-reactive anti-Tregs, providing examples of antigen-specific anti-Tregs and discussing their possible roles in immune homeostasis and their potential future clinical applications.

Abiotic Stress Tolerance in Soybean : Regulated by ncRNA

Plants respond through a cascade of reactions resulting in varied cellular environment leading to alterations in the patterns of protein expression resulting in phonotypic changes. Single cell genomics and global proteomics came out to be powerful tools and efficient techniques in studying stress tolerant plants. Non-coding RNAs are a distinct class of regulatory RNAs in plants and animals that control a variety of biological processes. Small ncRNAs play a vital role in post transcriptional gene regulation by either translational repression or by inducing mRNA cleavage. The major classes of small RNAs include microRNAs (miRNAs) and small interfering RNAs (siRNAs), which differ in their biogenesis. miRNAs control the expression of cognate target genes by binding to complementary sequences, resulting in cleavage or translational inhibition of the target RNAs. siRNAs too have a similar structure, function, and biogenesis like miRNAs but are derived from long double-stranded RNAs and can often direct DNA methylation at target sequences.In this review, we focus on the involvement of ncRNAs in comabting abiotic stresses of soybean. This review emphasis on previously known miRNAs as they play important role in several abiotic stresses like drought, salinity, chilling and heat stress by their diverse roles in mediating biological processes like gene expression, chromatin formation, defense of genome against invading viruses. This review attempts to elucidate the various kinds of non-coding RNAs explored, their discovery, biogenesis, functions, and response for different type of abiotic stresses and future aspects for crop improvement in the context of soybean, a representative grain legume.

The Staphylococcus aureus group II biotin protein ligase BirA is an effective regulator of biotin operon transcription and requires the DNA binding domain for full enzymatic activity.

Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins. However, the S. aureus BirA was reported to dimerize and bind DNA in the absence of biotin or biotinoyl-AMP (Soares da Costa et al. (2014) Mol Microbiol 91: 110-120). These in vitro results argued that the protein would be unable to respond to the levels of biotin or acceptor proteins and thus would lack the regulatory properties of the other characterized BirA proteins. We tested the regulatory function of the protein using an in vivo model system and examined its DNA binding properties in vitro using electrophoretic mobility shift and fluorescence anisotropy analyses. We report that the S. aureus BirA is an effective regulator of biotin operon transcription and that the prior data can be attributed to artifacts of mobility shift analyses. We also report that deletion of the DNA binding domain of the S. aureus BirA results in loss of virtually all of its ligation activity.