scholarly journals Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production

2016 ◽  
Vol 113 (49) ◽  
pp. 14031-14036 ◽  
Author(s):  
Suresh K. Kandasamy ◽  
Ryuya Fukunaga
Keyword(s):  
Rna Silencing ◽  
Point Mutations ◽  
Binding Pocket ◽  
Rna Cleavage ◽  
High Fidelity ◽  
Small Interfering Rnas ◽  
Phosphate Binding ◽  
Micro Rnas ◽  
Paz Domain

The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22–24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5′-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5′-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.

scholarly journals Functional specialization of monocot DCL3 and DCL5 proteins through the evolution of the PAZ domain

2021 ◽  
Author(s):  
Shirui Chen ◽  
Wei Liu ◽  
Masahiro Naganuma ◽  
Yukihide Tomari ◽  
Hiro-oki Iwakawa
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Functional Differentiation ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Secondary Sirnas ◽  
Fixed End ◽  
Paz Domain ◽  
Reproductive Processes

Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt heterochromatic small interfering RNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs). The former small RNAs are linked to plant heterochromatin, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient "eudicot-type" DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 3′ overhangs and 5′ phosphates, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5 which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.

scholarly journals T cell lineage choice and differentiation in the absence of the RNase III enzyme Dicer

2005 ◽  
Vol 201 (9) ◽  
pp. 1367-1373 ◽  
Author(s):  
Bradley S. Cobb ◽  
Tatyana B. Nesterova ◽  
Elizabeth Thompson ◽  
Arnulf Hertweck ◽  
Eric O'Connor ◽  
...  
Keyword(s):  
T Cell ◽  
Mammalian Cells ◽  
Early Stage ◽  
Early Embryo ◽  
Specific Gene ◽  
Small Interfering Rnas ◽  
Developmentally Regulated ◽  
Micro Rnas ◽  
Wide Range

The ribonuclease III enzyme Dicer is essential for the processing of micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) from double-stranded RNA precursors. miRNAs and siRNAs regulate chromatin structure, gene transcription, mRNA stability, and translation in a wide range of organisms. To provide a model system to explore the role of Dicer-generated RNAs in the differentiation of mammalian cells in vivo, we have generated a conditional Dicer allele. Deletion of Dicer at an early stage of T cell development compromised the survival of αβ lineage cells, whereas the numbers of γδ-expressing thymocytes were not affected. In developing thymocytes, Dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences (constitutive heterochromatin), the maintenance of DNA methylation and X chromosome inactivation in female cells (facultative heterochromatin), and the stable shutdown of a developmentally regulated gene (developmentally regulated gene silencing). Most remarkably, given that one third of mammalian mRNAs are putative miRNA targets, Dicer seems to be dispensable for CD4/8 lineage commitment, a process in which epigenetic regulation of lineage choice has been well documented. Thus, although Dicer seems to be critical for the development of the early embryo, it may have limited impact on the implementation of some lineage-specific gene expression programs.

scholarly journals Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

2020 ◽  
Author(s):  
Tilak Kumar Gupta ◽  
Sven Klumpe ◽  
Karin Gries ◽  
Steffen Heinz ◽  
Wojciech Wietrzynski ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Point Mutations ◽  
Binding Pocket ◽  
Thylakoid Membranes ◽  
Lipid Binding ◽  
Structural Basis ◽  
Amphipathic Helices ◽  
Cryo Electron Microscopy

AbstractVesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the ring’s lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

scholarly journals Ligand control of interaction in vivo between ecdysteroid receptor and ultraspiracle ligand-binding domain

2004 ◽  
Vol 378 (3) ◽  
pp. 779-784 ◽  
Author(s):  
Thomas BERGMAN ◽  
Vincent C. HENRICH ◽  
Uwe SCHLATTNER ◽  
Markus LEZZI
Keyword(s):  
Ligand Binding ◽  
Fusion Proteins ◽  
Point Mutations ◽  
Binding Pocket ◽  
Binding Studies ◽  
Global Features ◽  
Ecdysteroid Receptor ◽  
Binding Domains ◽  
Dimerization Interface

Ecdysteroids (Ecs) enhance the formation of Ec receptor–ultraspiracle protein (EcR–USP) heterodimers which regulate gene transcription. To study EcR–USP heterodimerization, fusion proteins were constructed from the LBDs (ligand-binding domains) of Drosophila EcR or USP and the activation or DNA-binding region of GAL4 respectively. Reporter gene (lacZ) activation was fully dependent on the co-expression of the two fusion proteins and thus constitutes a reliable measure for the interaction in vivo between the two LBDs in the yeast cell. To identify structures involved in heterodimerization, a total of 27 point mutations were generated in the EcR and USP LBDs at selected sites. Heterodimerization and its inducibility by ligand were mainly affected by mutations in the dimerization interface and in the ligand-binding pocket of EcR respectively. However, also mutations not located in these structures or even in the LBD of USP influenced ligand-dependent heterodimerization. Together with previously reported ligand-binding studies, the existence of such local, intra- and inter-molecular mutation effects suggest that ligand-induced dimerization results from a synergistic interaction between ligand-binding and heterodimerization functions in EcR LBD, and that it depends on global features of the LBDs of EcR and USP and on their mutual surface compatibility.

scholarly journals Engineering Candida tenuis Xylose Reductase for Improved Utilization of NADH: Antagonistic Effects of Multiple Side Chain Replacements and Performance of Site-Directed Mutants under Simulated In Vivo Conditions

2005 ◽  
Vol 71 (10) ◽  
pp. 6390-6393 ◽  
Author(s):  
Barbara Petschacher ◽  
Bernd Nidetzky
Keyword(s):  
Xylose Reductase ◽  
Binding Pocket ◽  
Kinetic Mechanism ◽  
Side Chain ◽  
Multiple Site ◽  
Wild Type ◽  
Structural Effects ◽  
Phosphate Binding ◽  
And Performance

ABSTRACT Six single- and multiple-site variants of Candida tenuis xylose reductase that were engineered to have side chain replacements in the coenzyme 2′-phosphate binding pocket were tested for NADPH versus NADH selectivity (R sel) in the presence of physiological reactant concentrations. The experimental R sel values agreed well with predictions from a kinetic mechanism describing mixed alternative coenzyme utilization. The Lys-274→Arg and Arg-280→His substitutions, which individually improved wild-type R sel 50- and 20-fold, respectively, had opposing structural effects when they were combined in a double mutant.

scholarly journals Diverging Affinity of Tospovirus RNA Silencing Suppressor Proteins, NSs, for Various RNA Duplex Molecules

2010 ◽  
Vol 84 (21) ◽  
pp. 11542-11554 ◽  
Author(s):  
Esther Schnettler ◽  
Hans Hemmes ◽  
Rik Huismann ◽  
Rob Goldbach ◽  
Marcel Prins ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Micro Rna ◽  
Impatiens Necrotic Spot Virus ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Cell Extracts ◽  
Green Fluorescent

ABSTRACT The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

scholarly journals Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kathrin Nowak ◽  
Florian Rosenthal ◽  
Tobias Karlberg ◽  
Mareike Bütepage ◽  
Ann-Gerd Thorsell ◽  
...  
Keyword(s):  
Protein Identification ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Salt Bridge ◽  
Binding Pocket ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Adp Ribosylation ◽  
Macro Domain

Abstract Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

scholarly journals Molecular Bases of Viral RNA Targeting by Viral Small Interfering RNA-Programmed RISC

2007 ◽  
Vol 81 (8) ◽  
pp. 3797-3806 ◽  
Author(s):  
Vitantonio Pantaleo ◽  
György Szittya ◽  
József Burgyán
Keyword(s):  
Rna Silencing ◽  
Protein Complexes ◽  
Plant Viruses ◽  
Viral Rna ◽  
Rna Cleavage ◽  
Small Interfering Rnas ◽  
Virus Induced Gene Silencing ◽  
Rna Targeting ◽  
Show Evidence ◽  
Target Rna

ABSTRACT RNA silencing is conserved in a broad range of eukaryotes and operates in the development and maintenance of genome integrity in many organisms. Plants have adapted this system for antiviral defense, and plant viruses have in turn developed mechanisms to suppress RNA silencing. RNA silencing-related RNA inactivation is likely based on target RNA cleavage or translational arrest. Although it is widely assumed that virus-induced gene silencing (VIGS) promotes the endonucleolytic cleavage of the viral RNA genome, this popular assumption has never been tested experimentally. Here we analyzed the viral RNA targeting by VIGS in tombusvirus-infected plants, and we show evidence that antiviral response of VIGS is based on viral RNA cleavage by RNA-induced silencing effector complex (RISC) programmed by virus-specific small interfering RNAs (siRNAs). In addition, we found that the RISC-mediated cleavages do not occur randomly on the viral genome. Indeed, sequence analysis of cloned cleavage products identified hot spots for target RNA cleavage, and the regions of specific RISC-mediated cleavages are asymmetrically distributed along the positive- and negative-sense viral RNA strands. In addition, we identified viral siRNAs containing high-molecular-mass protein complexes purified from the recovery leaves of the silencing suppressor mutant virus-infected plants. Strikingly, these large nucleoproteins cofractionated with microRNA-containing complexes, suggesting that these nucleoproteins are silencing related effector complexes.

RNA-dependent RNA polymerases in RNA silencing

2011 ◽  
Vol 392 (4) ◽  
Author(s):  
Yoshiko Maida ◽  
Kenkichi Masutomi
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Rna Polymerases ◽  
Telomerase Reverse Transcriptase ◽  
Structural Homology ◽  
Small Interfering Rnas ◽  
Human Telomerase Reverse Transcriptase ◽  
Human Telomerase ◽  
Traditional Approaches

Abstract RNA-dependent RNA polymerases (RdRPs) synthesize double-stranded RNAs that are processed into small RNAs and mediate gene silencing. Viral RdRPs and cellular RdRPs show little structural homology to each other. Cellular RdRPs play key roles in RNA silencing by producing complementary strands for target RNAs via Dicer-dependent and -independent mechanisms. Although the existence of a functional mammalian homolog of RdRP has long been predicted, traditional approaches to identify such enzymes were unsuccessful. Recently, human telomerase reverse transcriptase, a polymerase closely related to viral RdRPs, has been shown to function as an RdRP and contributes to RNA silencing in vivo. These findings suggest that endogenous small interfering RNAs are produced by several mechanisms in eukaryotes.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

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