Publications by authors named "Nagraj Sambrani"

14 Publications

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Adar RNA editing-dependent and -independent effects are required for brain and innate immune functions in Drosophila.

Nat Commun 2020 03 27;11(1):1580. Epub 2020 Mar 27.

Central European Institute of Technology, Masaryk University, Brno, Czech Republic.

ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a Drosophila Adar mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.
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http://dx.doi.org/10.1038/s41467-020-15435-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7101428PMC
March 2020

Membrane and synaptic defects leading to neurodegeneration in Adar mutant Drosophila are rescued by increased autophagy.

BMC Biol 2020 02 14;18(1):15. Epub 2020 Feb 14.

CEITEC Masaryk University, Kamenice 735/5, A35, CZ 62 500, Brno, Czech Republic.

Background: In fly brains, the Drosophila Adar (adenosine deaminase acting on RNA) enzyme edits hundreds of transcripts to generate edited isoforms of encoded proteins. Nearly all editing events are absent or less efficient in larvae but increase at metamorphosis; the larger number and higher levels of editing suggest editing is most required when the brain is most complex. This idea is consistent with the fact that Adar mutations affect the adult brain most dramatically. However, it is unknown whether Drosophila Adar RNA editing events mediate some coherent physiological effect. To address this question, we performed a genetic screen for suppressors of Adar mutant defects. Adar null mutant flies are partially viable, severely locomotion defective, aberrantly accumulate axonal neurotransmitter pre-synaptic vesicles and associated proteins, and develop an age-dependent vacuolar brain neurodegeneration.

Results: A genetic screen revealed suppression of all Adar mutant phenotypes tested by reduced dosage of the Tor gene, which encodes a pro-growth kinase that increases translation and reduces autophagy in well-fed conditions. Suppression of Adar phenotypes by reduced Tor is due to increased autophagy; overexpression of Atg5, which increases canonical autophagy initiation, reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar mutant phenotypes tested. Endosomal microautophagy (eMI) is another Tor-inhibited autophagy pathway involved in synaptic homeostasis in Drosophila. Increased expression of the key eMI protein Hsc70-4 also reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar mutant phenotypes tested.

Conclusions: These findings link Drosophila Adar mutant synaptic and neurotransmission defects to more general cellular defects in autophagy; presumably, edited isoforms of CNS proteins are required for optimum synaptic response capabilities in the brain during the behaviorally complex adult life stage.
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http://dx.doi.org/10.1186/s12915-020-0747-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020516PMC
February 2020

ADAR RNA editing in innate immune response phasing, in circadian clocks and in sleep.

Biochim Biophys Acta Gene Regul Mech 2019 03 31;1862(3):356-369. Epub 2018 Oct 31.

CEITEC, Masaryk University Brno, Kamenice 753/5, Pavilion A35, Brno CZ-62500, Czech Republic. Electronic address:

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in dsRNA. ADAR editing in pre-mRNAs recodes open reading frames and alters splicing, mRNA structure and interactions with miRNAs. Here, we review ADAR gene expression, splice forms, posttranslational modifications, subcellular localizations and functions of ADAR protein isoforms. ADAR1 edits cellular dsRNA to prevent aberrant activation of cytoplasmic antiviral dsRNA sensors; ADAR1 mutations lead to aberrant expression of interferon in Aicardi Goutières syndrome (AGS), a human congenital encephalopathy. We review related studies on mouse Adar1 mutant phenotypes, their rescues by preventing signaling from the antiviral RIG-I-like Sensors (RLRs), as well as Adar1 mechanisms in innate immune suppression and other roles of Adar1, including editing-independent effects. ADAR2, expressed primarily in CNS, edits glutamate receptor transcripts; regulation of ADAR2 activity in response to neuronal activity mediates homeostatic synaptic plasticity of vertebrate AMPA and kainite receptors. In Drosophila, synapses and synaptic proteins show dramatic decreases at night during sleep; Drosophila Adar, an orthologue of ADAR2, edits hundreds of mRNAs; the most conserved editing events occur in transcripts encoding synapse-associated proteins. Adar mutant flies exhibit locomotion defects associated with very increased sleep pressure resulting from a failure of homeostatic synaptic processes. A study on Adar2 mutant mice identifies a new role in circadian rhythms, acting indirectly through miRNAs such as let-7 to modulate levels of let-7 target mRNAs; ADAR1 also regulates let-7 miRNA processing. Drosophila ADAR, an orthologue of vertebrate ADAR2, also regulates let-7 miRNA levels and Adar mutant flies have a circadian mutant phenotype.
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http://dx.doi.org/10.1016/j.bbagrm.2018.10.011DOI Listing
March 2019

Enhancer identification and activity evaluation in the red flour beetle, .

Development 2018 04 5;145(7). Epub 2018 Apr 5.

Department of Biology, Miami University, Oxford, OH 45056, USA

Evolution of -regulatory elements (such as enhancers) plays an important role in the production of diverse morphology. However, a mechanistic understanding is often limited by the absence of methods for studying enhancers in species other than established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of using FAIRE (formaldehyde-assisted isolation of regulatory elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and at each stage. In addition, comparison of the FAIRE data with sets of computationally predicted (i.e. supervised -regulatory module-predicted) enhancers revealed a very high overlap between the two datasets. Second, using in the wing and in the embryo as case studies, we established the first universal reporter assay system that works in various contexts in , and in a cross-species context. Together, these advances will facilitate investigation of -evolution and morphological diversity in and other insects.
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http://dx.doi.org/10.1242/dev.160663DOI Listing
April 2018

Translational repression of the mRNA involves the RNA helicase Belle and RNA coating by Me31B and Trailer hitch.

RNA 2017 10 12;23(10):1552-1568. Epub 2017 Jul 12.

Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, 06099 Halle, Germany.

Translational repression of maternal mRNAs is an essential regulatory mechanism during early embryonic development. Repression of the mRNA, required for the formation of the anterior-posterior body axis, depends on the protein Smaug binding to two Smaug recognition elements (SREs) in the 3' UTR. In a comprehensive mass spectrometric analysis of the SRE-dependent repressor complex, we identified Smaug, Cup, Me31B, Trailer hitch, eIF4E, and PABPC, in agreement with earlier data. As a novel component, the RNA-dependent ATPase Belle (DDX3) was found, and its involvement in deadenylation and repression of was confirmed in vivo. Smaug, Cup, and Belle bound stoichiometrically to the SREs, independently of RNA length. Binding of Me31B and Tral was also SRE-dependent, but their amounts were proportional to the length of the RNA and equimolar to each other. We suggest that "coating" of the RNA by a Me31B•Tral complex may be at the core of repression.
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http://dx.doi.org/10.1261/rna.062208.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602113PMC
October 2017

The autoregulatory loop: A common mechanism of regulation of key sex determining genes in insects.

J Biosci 2016 Jun;41(2):283-94

Centre of Excellence for Genetics and Genomics of Silkmoths, Laboratory of Molecular Genetics, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500 001, India.

Sex determination in most insects is structured as a gene cascade, wherein a primary signal is passed through a series of sex-determining genes, culminating in a downstream double-switch known as doublesex that decides the sexual fate of the embryo. From the literature available on sex determination cascades, it becomes apparent that sex determination mechanisms have evolved rapidly. The primary signal that provides the cue to determine the sex of the embryo varies remarkably, not only among taxa, but also within taxa. Furthermore, the upstream key gene in the cascade also varies between species and even among closely related species. The order Insecta alone provides examples of astoundingly complex diversity of upstream key genes in sex determination mechanisms. Besides, unlike key upstream genes, the downstream double-switch gene is alternatively spliced to form functional sex-specific isoforms. This sex-specific splicing is conserved across insect taxa. The genes involved in the sex determination cascade such as Sex-lethal (Sxl) in Drosophila melanogaster, transformer (tra) in many other dipterans, coleopterans and hymenopterans, Feminizer (fem) in Apis mellifera, and IGF-II mRNA-binding protein (Bmimp) in Bombyx mori are reported to be regulated by an autoregulatory positive feedback loop. In this review, by taking examples from various insects, we propose the hypothesis that autoregulatory loop mechanisms of sex determination might be a general strategy. We also discuss the possible reasons for the evolution of autoregulatory loops in sex determination cascades and their impact on binary developmental choices.
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http://dx.doi.org/10.1007/s12038-016-9609-xDOI Listing
June 2016

Comparative developmental analysis of Drosophila and Tribolium reveals conserved and diverged roles of abrupt in insect wing evolution.

Dev Biol 2016 Jan 11;409(2):518-29. Epub 2015 Dec 11.

Department of Biology, Miami University, Oxford, OH 45056, USA. Electronic address:

Morphological innovation is a fundamental process in evolution, yet its molecular basis is still elusive. Acquisition of elytra, highly modified beetle forewings, is an important innovation that has driven the successful radiation of beetles. Our RNAi screening for candidate genes has identified abrupt (ab) as a potential key player in elytron evolution. In this study, we performed a series of RNA interference (RNAi) experiments in both Tribolium and Drosophila to understand the contributions of ab to the evolution of beetle elytra. We found that (i) ab is essential for proper wing vein patterning both in Tribolium and Drosophila, (ii) ab has gained a novel function in determining the unique elytron shape in the beetle lineage, (iii) unlike Hippo and Insulin, other shape determining pathways, the shape determining function of ab is specific to the elytron and not required in the hindwing, (iv) ab has a previously undescribed role in the Notch signal-associated wing formation processes, which appears to be conserved between beetles and flies. These data suggest that ab has gained a new function during elytron evolution in beetles without compromising the conserved wing-related functions. Gaining a new function without losing evolutionarily conserved functions may be a key theme in the evolution of morphologically novel structures.
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http://dx.doi.org/10.1016/j.ydbio.2015.12.006DOI Listing
January 2016

Distinct genetic requirements for BX-C-mediated specification of abdominal denticles.

Dev Dyn 2014 Jan 28;243(1):192-200. Epub 2013 Nov 28.

CNRS, Aix Marseille Université, Marseille, France.

Background: Hox genes encode transcription factors playing important role in segment specific morphogenesis along the anterior posterior axis. Most work in the Hox field aimed at understanding the basis for specialised Hox functions, while little attention was given to Hox common function. In Drosophila, genes of the Bithorax complex [Ultrabithorax (Ubx), abdominalA (abdA), and AbdominalB (AbdB)] all promote abdominal identity. While Ubx and AbdA share extensive sequence conservation, AbdB is highly divergent, questioning how it can perform similar functions as Ubx and AbdA.

Results: In this study, we investigate the genetic requirement for the specification of abdominal-type denticles by Ubx, AbdA, and AbdB. The impact of ectopic expression of Hox proteins in embryos mutant for Exd as well as of Wingless or Hedgehog signaling involved in intrasegmental patterning was analyzed. Results indicated that Ubx and AbdA do not require Exd, Wg, and Hh activity for specifying abdominal-type denticles, while AbdB does.

Conclusions: Our results support that distinct regulatory mechanisms underlie Ubx/AbdA- and AbdB-mediated specification of abdominal-type denticles, highlighting distinct strategies for achieving a similar biological output. This suggests that common function performed by distinct paralogue Hox proteins may also rely on newly acquired property, instead of conserved/ancestral properties.
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http://dx.doi.org/10.1002/dvdy.24081DOI Listing
January 2014

Distinct molecular strategies for Hox-mediated limb suppression in Drosophila: from cooperativity to dispensability/antagonism in TALE partnership.

PLoS Genet 2013 7;9(3):e1003307. Epub 2013 Mar 7.

Centre National de la Recherche Scientifique, Aix Marseille Université, Institut de Biologie du Développement de Marseille Luminy, UMR 7288, Parc Scientifique de Luminy, Marseille, France.

The emergence following gene duplication of a large repertoire of Hox paralogue proteins underlies the importance taken by Hox proteins in controlling animal body plans in development and evolution. Sequence divergence of paralogous proteins accounts for functional specialization, promoting axial morphological diversification in bilaterian animals. Yet functionally specialized paralogous Hox proteins also continue performing ancient common functions. In this study, we investigate how highly divergent Hox proteins perform an identical function. This was achieved by comparing in Drosophila the mode of limb suppression by the central (Ultrabithorax and AbdominalA) and posterior class (AbdominalB) Hox proteins. Results highlight that Hox-mediated limb suppression relies on distinct modes of DNA binding and a distinct use of TALE cofactors. Control of common functions by divergent Hox proteins, at least in the case studied, relies on evolving novel molecular properties. Thus, changes in protein sequences not only provide the driving force for functional specialization of Hox paralogue proteins, but also provide means to perform common ancient functions in distinct ways.
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http://dx.doi.org/10.1371/journal.pgen.1003307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591290PMC
June 2013

Antagonism versus cooperativity with TALE cofactors at the base of the functional diversification of Hox protein function.

PLoS Genet 2013 7;9(2):e1003252. Epub 2013 Feb 7.

CABD, CSIC/JA/Universidad Pablo de Olavide, Seville, Spain.

Extradenticle (Exd) and Homothorax (Hth) function as positive transcriptional cofactors of Hox proteins, helping them to bind specifically their direct targets. The posterior Hox protein Abdominal-B (Abd-B) does not require Exd/Hth to bind DNA; and, during embryogenesis, Abd-B represses hth and exd transcription. Here we show that this repression is necessary for Abd-B function, as maintained Exd/Hth expression results in transformations similar to those observed in loss-of-function Abd-B mutants. We characterize the cis regulatory module directly regulated by Abd-B in the empty spiracles gene and show that the Exd/Hth complex interferes with Abd-B binding to this enhancer. Our results suggest that this novel Exd/Hth function does not require the complex to bind DNA and may be mediated by direct Exd/Hth binding to the Abd-B homeodomain. Thus, in some instances, the main positive cofactor complex for anterior Hox proteins can act as a negative factor for the posterior Hox protein Abd-B. This antagonistic interaction uncovers an alternative way in which MEIS and PBC cofactors can modulate Abd-B like posterior Hox genes during development.
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http://dx.doi.org/10.1371/journal.pgen.1003252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567137PMC
June 2013

Selection of distinct Hox-Extradenticle interaction modes fine-tunes Hox protein activity.

Proc Natl Acad Sci U S A 2011 Feb 24;108(6):2276-81. Epub 2011 Jan 24.

Institut de Biologie du Développement de Marseille Luminy, Centre National de la Recherche Scientifique, Université de la Méditerranée, 13288 Marseille Cedex 09, France.

Hox genes encode transcription factors widely used for diversifying animal body plans in development and evolution. To achieve functional specificity, Hox proteins associate with PBC class proteins, Pre-B cell leukemia homeobox (Pbx) in vertebrates, and Extradenticle (Exd) in Drosophila, and were thought to use a unique hexapeptide-dependent generic mode of interaction. Recent findings, however, revealed the existence of an alternative, UbdA-dependent paralog-specific interaction mode providing diversity in Hox-PBC interactions. In this study, we investigated the basis for the selection of one of these two Hox-PBC interaction modes. Using naturally occurring variations and mutations in the Drosophila Ultrabithorax protein, we found that the linker region, a short domain separating the hexapeptide from the homeodomain, promotes an interaction mediated by the UbdA domain in a context-dependent manner. While using a UbdA-dependent interaction for the repression of the limb-promoting gene Distalless, interaction with Exd during segment-identity specification still relies on the hexapeptide motif. We further show that distinctly assembled Hox-PBC complexes display subtle but distinct repressive activities. These findings identify Hox-PBC interaction as a template for subtle regulation of Hox protein activity that may have played a major role in the diversification of Hox protein function in development and evolution.
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http://dx.doi.org/10.1073/pnas.1006964108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038764PMC
February 2011

Regulation of Hox activity: insights from protein motifs.

Adv Exp Med Biol 2010 ;689:3-16

Institute of Developmental Biology of Marseille Luminy, University of the Mediterranean, Marseille, France.

Deciphering the molecular bases of animal body plan construction is a central question in developmental and evolutionary biology. Genome analyses of a number of metazoans indicate that widely conserved regulatory molecules underlie the amazing diversity of animal body plans, suggesting that these molecules are reiteratively used for multiple purposes. Hox proteins constitute a good example of such molecules and provide the framework to address the mechanisms underlying transcriptional specificity and diversity in development and evolution. Here we examine the current knowledge of the molecular bases of Hox-mediated transcriptional control, focusing on how this control is encoded within protein sequences and structures. The survey suggests that the homeodomain is part of an extended multifunctional unit coordinating DNA binding and activity regulation and highlights the need for further advances in our understanding of Hox protein activity.
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http://dx.doi.org/10.1007/978-1-4419-6673-5_1DOI Listing
September 2010

A unique Extradenticle recruitment mode in the Drosophila Hox protein Ultrabithorax.

Proc Natl Acad Sci U S A 2007 Oct 17;104(43):16946-51. Epub 2007 Oct 17.

Institut de Biologie du Développement de Marseille Luminy, Centre National de la Recherche Scientifique, Université de la Méditerranée, Parc Scientifique de Luminy, 13288 Marseille Cedex 09, France.

Hox transcription factors are essential for shaping body morphology in development and evolution. The control of Hox protein activity in part arises from interaction with the PBC class of partners, pre-B cell transcription factor (Pbx) proteins in vertebrates and Extradenticle (Exd) in Drosophila. Characterized interactions occur through a single mode, involving a short hexapeptide motif in the Hox protein. This apparent uniqueness in Hox-PBC interaction provides little mechanistic insight in how the same cofactors endow Hox proteins with specific and diverse activities. Here, we identify in the Drosophila Ultrabithorax (Ubx) protein a short motif responsible for an alternative mode of Exd recruitment. Together with previous reports, this finding highlights that the Hox protein Ubx has multiple ways to interact with the Exd cofactor and suggests that flexibility in Hox-PBC contacts contributes to specify and diversify Hox protein function.
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http://dx.doi.org/10.1073/pnas.0705832104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2040397PMC
October 2007

Identification of a novel target of D/V signaling in Drosophila wing disc: Wg-independent function of the organizer.

Gene Expr Patterns 2004 Nov;5(1):113-21

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, India 500 007.

Growth and patterning during Drosophila wing development are mediated by signaling from its dorso-ventral (D/V) organizer. Wingless is expressed in the D/V boundary and functions as a morphogen to activate target genes at a distance. Wingless pathway and thereby D/V signaling is negatively regulated by the homeotic gene Ultrabithorax (Ubx) to mediate haltere development. In an enhancer-trap screen to identify genes that show differential expression between wing and haltere discs, we identified CG32062, which codes for a RNA-binding protein. In wing discs, CG32062 is expressed only in non-D/V cells. CG32062 expression in non-D/V cells is dependent on Notch-mediated signaling from the D/V boundary. However, CG32062 expression is independent of Wingless function, thus providing evidence for a second long-range signaling mechanism of the D/V organizer. In haltere discs, CG32062 is negatively regulated by Ubx. The non-cell autonomous nature of Ubx-mediated repression of CG32062 expression suggests that the novel component of D/V signaling is also negatively regulated during haltere specification.
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http://dx.doi.org/10.1016/j.modgep.2004.05.005DOI Listing
November 2004
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