scholarly journals Structural basis for piRNA-targeting

2020 ◽  
Author(s):  
Todd A. Anzelon ◽  
Saikat Chowdhury ◽  
Siobhan M. Hughes ◽  
Yao Xiao ◽  
Gabriel C. Lander ◽  
...  
Keyword(s):  
Transposable Elements ◽  
Target Recognition ◽  
Structural Basis ◽  
Seed Region ◽  
Active Conformation ◽  
Argonaute Proteins ◽  
Cellular Mrnas ◽  
Target Association ◽  
Animal Genomes

SummaryPiwi proteins use PIWI-interacting RNAs (piRNAs) to identify and silence the transposable elements (TEs) pervasively found in animal genomes. The Piwi targeting mechanism is proposed to be similar to targeting by Argonaute proteins, which employ microRNA (miRNA) guides to repress cellular mRNAs, but has not been characterized in detail. We present cryo-EM structures of a Piwi-piRNA complex with and without target RNAs and analysis of target recognition. Resembling Argonaute, Piwi identifies targets using the piRNA seed-region. However, Piwi creates a much weaker seed so that prolonged target association requires further piRNA-target pairing. Beyond the seed, Piwi creates wide central cleft wide for unencumbered piRNA-target pairing, enabling long-lived Piwi-piRNA-target interactions that are tolerant of mismatches. Piwi ensures targeting fidelity by blocking propagation of the piRNA-target duplex in the absence of faithful seed pairing, and by requiring extended piRNA-target pairing to reach an endonucleolytically active conformation. This mechanism allows Piwi to minimize off-targeting cellular mRNAs and adapt piRNA sequences to evolving genomic threats.

scholarly journals Beyond the seed: structural basis for supplementary microRNA targeting

2018 ◽  
Author(s):  
Jessica Sheu-Gruttadauria ◽  
Yao Xiao ◽  
Luca F. R. Gebert ◽  
Ian J. MacRae
Keyword(s):  
Mirna Target ◽  
Target Recognition ◽  
Structural Basis ◽  
Seed Region ◽  
Base Pairs ◽  
Argonaute Proteins ◽  
Target Rna ◽  
Mirna Seed ◽  
Mirna Targeting

AbstractmicroRNAs (miRNA) guide Argonaute proteins to mRNAs targeted for repression. Target recognition occurs primarily through the miRNA seed region, composed of guide (g) nucleotides g2–g8. However, nucleotides beyond the seed are also important for some known miRNA-target interactions. Here, we report the structure of human Argonaute2 (Ago2) engaged with a target RNA recognized through both miRNA seed and supplementary (g13–g16) regions. Ago2 creates a “supplementary chamber” that accommodates up to 5 miRNA-target base pairs. Seed and supplementary chambers are adjacent to each other, and can be bridged by an unstructured target loop of 1–15 nucleotides. Opening of the supplementary chamber may be constrained by tension in the miRNA 3' tail as increases in miRNA length stabilize supplementary interactions. Contrary to previous reports, we demonstrate optimal supplementary interactions can increase target affinity >20-fold. These results provide a mechanism for extended miRNA-targeting, suggest a function for 3' isomiRs in tuning miRNA targeting specificity, and indicate that supplementary interactions may contribute more to target recognition than is widely appreciated.

scholarly journals Mechanism of R-loop formation and conformational activation of Cas9

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Martin Jinek
Keyword(s):  
Dna Cleavage ◽  
Structural Basis ◽  
Seed Region ◽  
Loop Formation ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strand ◽  
Dna Substrate

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

scholarly journals Structural basis for virulence regulation in Vibrio cholerae by unsaturated fatty acid components of bile

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Justin T. Cruite ◽  
Gabriela Kovacikova ◽  
Kenzie A. Clark ◽  
Anne K. Woodbrey ◽  
Karen Skorupski ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Structural Model ◽  
Unsaturated Fatty Acids ◽  
Diarrheal Disease ◽  
Bacterial Pathogen ◽  
Virulence Gene ◽  
Structural Basis ◽  
Active Conformation ◽  
Structural Mechanism ◽  
Virulence Gene Regulation

AbstractThe AraC/XylS-family transcriptional regulator ToxT is the master virulence activator of Vibrio cholerae, the gram-negative bacterial pathogen that causes the diarrheal disease cholera. Unsaturated fatty acids (UFAs) found in bile inhibit the activity of ToxT. Crystal structures of inhibited ToxT bound to UFA or synthetic inhibitors have been reported, but no structure of ToxT in an active conformation had been determined. Here we present the 2.5 Å structure of ToxT without an inhibitor. The structure suggests release of UFA or inhibitor leads to an increase in flexibility, allowing ToxT to adopt an active conformation that is able to dimerize and bind DNA. Small-angle X-ray scattering was used to validate a structural model of an open ToxT dimer bound to the cholera toxin promoter. The results presented here provide a detailed structural mechanism for virulence gene regulation in V. cholerae by the UFA components of bile and other synthetic ToxT inhibitors.

scholarly journals Structural Basis for Competitive Inhibition of eIF4G-Mnk1 Interaction by the Adenovirus 100-Kilodalton Protein

2004 ◽  
Vol 78 (14) ◽  
pp. 7707-7716 ◽  
Author(s):  
Rafael Cuesta ◽  
Qiaoran Xi ◽  
Robert J. Schneider
Keyword(s):  
Protein Synthesis ◽  
Competitive Inhibition ◽  
Late Phase ◽  
Mrna Translation ◽  
Cellular Protein ◽  
Structural Basis ◽  
Binding Motif ◽  
Amino Acid Peptide ◽  
Cellular Mrnas ◽  
Cellular Protein Synthesis

ABSTRACT Translation of most cellular mRNAs involves cap binding by the translation initiation complex. Among this complex of proteins are cap-binding protein eIF4E and the eIF4E kinase Mnk1. Cap-dependent mRNA translation generally correlates with Mnk1 phosphorylation of eIF4E when both are bound to eIF4G. During the late phase of adenovirus (Ad) infection translation of cellular mRNA is inhibited, which correlates with displacement of Mnk1 from eIF4G by the viral 100-kDa (100K) protein and dephosphorylation of eIF4E. Here we describe the molecular mechanism for 100K protein displacement of Mnk1 from eIF4G and elucidate a structural basis for eIF4G interaction with Mnk1 and 100K proteins and Ad inhibition of cellular protein synthesis. The eIF4G-binding site is located in an N-terminal 66-amino-acid peptide of 100K which is sufficient to bind eIF4G, displace Mnk1, block eIF4E phosphorylation, and inhibit eIF4F (cap)-dependent cellular mRNA translation. Ad 100K and Mnk1 proteins possess a common eIF4G-binding motif, but 100K protein binds more strongly to eIF4G than does Mnk1. Unlike Mnk1, for which binding to eIF4G is RNA dependent, competitive binding by 100K protein is RNA independent. These data support a model whereby 100K protein blocks cellular protein synthesis by coopting eIF4G and cap-initiation complexes regardless of their association with mRNA and displacing or blocking binding by Mnk1, which occurs only on preassembled complexes, resulting in dephosphorylation of eIF4E.

scholarly journals Structural models of full-length JAK2 kinase

2019 ◽  
Author(s):  
Pelin Ayaz ◽  
Henrik M. Hammarén ◽  
Juuli Raivola ◽  
Dina Sharon ◽  
Stevan R. Hubbard ◽  
...  
Keyword(s):  
Drug Target ◽  
Structural Models ◽  
Full Length ◽  
Janus Kinase ◽  
Structural Basis ◽  
Constitutive Activity ◽  
Active Conformation ◽  
Dual Effect ◽  
Dynamics Simulations ◽  
Important Drug

AbstractThe protein JAK2 is a prototypical member of the Janus kinase family, and mediates signals from numerous cytokine receptors. The constitutively active V617F mutant of JAK2 is prevalent in many bone marrow disorders, blood cancers, and autoimmune diseases, and is an important drug target. Structures have been determined for each of the four individual domains making up JAK2, and for certain pairs of these domains, but no structure of full-length JAK2 is available, and thus the mechanisms underlying JAK2 regulation and the aberrant activity of the V617F mutant have been incompletely understood. Here we propose structural models of full-length JAK2 in both its active and inactive forms. Construction of these models was informed by long-timescale molecular dynamics simulations. Subsequent mutagenesis experiments showed that mutations at the putative interdomain interfaces modulated JAK2 activity. The models provide a structural basis for understanding JAK2 autoinhibition and activation, and suggest that the constitutive activity of the V617F mutant may arise from a dual effect of destabilizing the inactive conformation and stabilizing the active conformation.

scholarly journals On the folding of a structurally complex protein to its metastable active state

2018 ◽  
Vol 115 (9) ◽  
pp. 1998-2003 ◽  
Author(s):  
V. V. Hemanth Giri Rao ◽  
Shachi Gosavi
Keyword(s):  
Free Energy ◽  
Energy Barrier ◽  
Coarse Grained ◽  
Structural Basis ◽  
Free Energy Barrier ◽  
Active Conformation ◽  
Protease Inhibition ◽  
Stable Conformation ◽  
Folding Simulations ◽  
And Function

For successful protease inhibition, the reactive center loop (RCL) of the two-domain serine protease inhibitor, α1-antitrypsin (α1-AT), needs to remain exposed in a metastable active conformation. The α1-AT RCL is sequestered in a β-sheet in the stable latent conformation. Thus, to be functional, α1-AT must always fold to a metastable conformation while avoiding folding to a stable conformation. We explore the structural basis of this choice using folding simulations of coarse-grained structure-based models of the two α1-AT conformations. Our simulations capture the key features of folding experiments performed on both conformations. The simulations also show that the free energy barrier to fold to the latent conformation is much larger than the barrier to fold to the active conformation. An entropically stabilized on-pathway intermediate lowers the barrier for folding to the active conformation. In this intermediate, the RCL is in an exposed configuration, and only one of the two α1-AT domains is folded. In contrast, early conversion of the RCL into a β-strand increases the coupling between the two α1-AT domains in the transition state and creates a larger barrier for folding to the latent conformation. Thus, unlike what happens in several proteins, where separate regions promote folding and function, the structure of the RCL, formed early during folding, determines both the conformational and the functional fate of α1-AT. Further, the short 12-residue RCL modulates the free energy barrier and the folding cooperativity of the large 370-residue α1-AT. Finally, we suggest experiments to test the predicted folding mechanism for the latent state.

scholarly journals Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xue Huang ◽  
Wei Sun ◽  
Zhi Cheng ◽  
Minxuan Chen ◽  
Xueyan Li ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Metal Ion ◽  
Catalytic Domain ◽  
Dna Recognition ◽  
Structural Basis ◽  
Seed Region ◽  
Dna Complexes ◽  
Metal Ion Catalysis ◽  
Type V ◽  
Cas Proteins

Abstract To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2–crRNA–DNA complexes and a Cas12i2–crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1–8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9–15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.

Piwi-interacting RNAs: biological functions and biogenesis

2013 ◽  
Vol 54 ◽  
pp. 39-52 ◽  
Author(s):  
Kaoru Sato ◽  
Mikiko C. Siomi
Keyword(s):  
Nucleic Acid ◽  
Transposable Elements ◽  
Genetic Information ◽  
Molecular Mechanisms ◽  
Biological Functions ◽  
Next Generation ◽  
Argonaute Proteins ◽  
Non Coding Rnas ◽  
Germline Cells ◽  
Successive Generations

The integrity of the germline genome must be maintained to achieve successive generations of a species, because germline cells are the only source for transmitting genetic information to the next generation. Accordingly, the germline has acquired a system dedicated to protecting the genome from ‘injuries’ caused by harmful selfish nucleic acid elements, such as TEs (transposable elements). Accumulating evidence shows that a germline-specific subclass of small non-coding RNAs, piRNAs (piwi-interacting RNAs), are necessary for silencing TEs to protect the genome in germline cells. To silence TEs post-transcriptionally and/or transcriptionally, mature piRNAs are loaded on to germline-specific Argonaute proteins, or PIWI proteins, to form the piRISC (piRNA-induced silencing complex). The present chapter will highlight insights into the molecular mechanisms underlying piRISC-mediated silencing and piRNA biogenesis, and discuss a possible link with tumorigenesis, particularly in Drosophila.

scholarly journals Crystal Structure of GRIP1 PDZ6-Peptide Complex Reveals the Structural Basis for Class II PDZ Target Recognition and PDZ Domain-mediated Multimerization

2002 ◽  
Vol 278 (10) ◽  
pp. 8501-8507 ◽  
Author(s):  
Young Jun Im ◽  
Seong Ho Park ◽  
Seong-Hwan Rho ◽  
Jun Hyuck Lee ◽  
Gil Bu Kang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Target Recognition ◽  
Pdz Domain ◽  
Class Ii ◽  
Structural Basis ◽  
Peptide Complex

scholarly journals Endogenous siRNAs: regulators of internal affairs

2014 ◽  
Vol 42 (4) ◽  
pp. 1174-1179 ◽  
Author(s):  
Monica J. Piatek ◽  
Andreas Werner
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Drosophila Melanogaster ◽  
Transposable Elements ◽  
Essential Role ◽  
Viral Infections ◽  
Biological Functions ◽  
Small Interfering Rnas ◽  
Argonaute Proteins ◽  
Genome Protection

Endo-siRNAs (endogenous small-interfering RNAs) have recently emerged as versatile regulators of gene expression. They derive from double-stranded intrinsic transcripts and are processed by Dicer and associate with Argonaute proteins. In Caenorhabditis elegans, endo-siRNAs are known as 22G and 26G RNAs and are involved in genome protection and gene regulation. Drosophila melanogaster endo-siRNAs are produced with the help of specific Dicer and Argonaute isoforms and play an essential role in transposon control and the protection from viral infections. Biological functions of endo-siRNAs in vertebrates include repression of transposable elements and chromatin organization, as well as gene regulation at the transcriptional and post-transcriptional levels.

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