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 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 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 Pairing to the microRNA 3′ region occurs through two alternative binding modes, with affinity shaped by nucleotide identity as well as pairing position

2021 ◽  
Author(s):  
Sean E. McGeary ◽  
Namita Bisaria ◽  
David P. Bartel
Keyword(s):  
Nucleotide Identity ◽  
Seed Region ◽  
List Type ◽  
Binding Modes ◽  
High Affinity ◽  
The Many ◽  
Insight Into ◽  
Mirna Seed ◽  
Mirna Targeting

ABSTRACTMicroRNAs (miRNAs), in association with Argonaute (AGO) proteins, direct repression by pairing to sites within mRNAs. Compared to pairing preferences of the miRNA seed region (nucleotides 2–8), preferences of the miRNA 3′ region are poorly understood, due to the sparsity of measured affinities for the many pairing possibilities. We used RNA bind-n-seq with purified AGO2–miRNA complexes to measure relative affinities of >1,000 3′-pairing architectures for each miRNA. In some cases, optimal 3′ pairing increased affinity by >500-fold. Some miRNAs had two high-affinity 3′-pairing modes—one of which included additional nucleotides bridging seed and 3′ pairing to enable high-affinity pairing to miRNA nucleotide 11. The affinity of binding and the position of optimal pairing both tracked with the occurrence of G or oligo(G/C) nucleotides within the miRNA. These and other results advance understanding of miRNA targeting, providing insight into how optimal 3′ pairing is determined for each miRNA.HIGHLIGHTSRNA bind-n-seq reveals relative affinities of >1,000 3′-pairing architecturesTwo distinct 3′-binding modes can enhance affinity, by >500-fold in some instancesG and oligo(G/C) residues help define the miRNA 3′ segment most critical for pairingSeed mismatch identity can influence the contribution of compensatory 3′ pairing

scholarly journals Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Nicole T Schirle ◽  
Jessica Sheu-Gruttadauria ◽  
Stanley D Chandradoss ◽  
Chirlmin Joo ◽  
Ian J MacRae
Keyword(s):  
Mirna Target ◽  
Water Molecules ◽  
Messenger Rnas ◽  
Argonaute Proteins ◽  
Microrna Targets ◽  
Human Genes ◽  
Post Transcriptional Regulation ◽  
Target Rna ◽  
Initial Binding ◽  
Pairing Potential

MicroRNAs (miRNAs) direct post-transcriptional regulation of human genes by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for repression. An enigmatic feature of many conserved mammalian miRNA target sites is that an adenosine (A) nucleotide opposite miRNA nucleotide-1 confers enhanced target repression independently of base pairing potential to the miRNA. In this study, we show that human Argonaute2 (Ago2) possesses a solvated surface pocket that specifically binds adenine nucleobases in the 1 position (t1) of target RNAs. t1A nucleotides are recognized indirectly through a hydrogen-bonding network of water molecules that preferentially interacts with the N6 amine on adenine. t1A nucleotides are not utilized during the initial binding of Ago2 to its target, but instead function by increasing the dwell time on target RNA. We also show that N6 adenosine methylation blocks t1A recognition, revealing a possible mechanism for modulation of miRNA target site potency.

scholarly journals SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jurre A. Steens ◽  
Yifan Zhu ◽  
David W. Taylor ◽  
Jack P. K. Bravo ◽  
Stijn H. P. Prinsen ◽  
...  
Keyword(s):  
Immune Response ◽  
Diagnostic Tool ◽  
Nasal Swab ◽  
Rna Binding ◽  
Seed Region ◽  
Base Pairing ◽  
Specific Detection ◽  
Type Iii ◽  
Single Host ◽  
Target Rna

AbstractCharacteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3′ end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5′ end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.

scholarly journals Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins

2021 ◽  
Author(s):  
Lu Li ◽  
Peike Sheng ◽  
Tianqi Li ◽  
Christopher J. Fields ◽  
Nicholas M. Hiers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ubiquitin Ligase ◽  
Transcriptional Repression ◽  
Mirna Target ◽  
Cross Linking ◽  
Base Pairing ◽  
Proapoptotic Protein ◽  
Nucleotide Addition ◽  
Mirna Degradation ◽  
Target Rna

Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA–target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3′ end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.

scholarly journals A DDX6-CNOT1 Complex and W-Binding Pockets in CNOT9 Reveal Direct Links between miRNA Target Recognition and Silencing

2014 ◽  
Vol 54 (5) ◽  
pp. 737-750 ◽  
Author(s):  
Ying Chen ◽  
Andreas Boland ◽  
Duygu Kuzuoğlu-Öztürk ◽  
Praveen Bawankar ◽  
Belinda Loh ◽  
...  
Keyword(s):  
Mirna Target ◽  
Target Recognition ◽  
Binding Pockets

scholarly journals Predicting effective microRNA target sites in mammalian mRNAs

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Vikram Agarwal ◽  
George W Bell ◽  
Jin-Wu Nam ◽  
David P Bartel
Keyword(s):  
Regulatory Networks ◽  
Quantitative Model ◽  
Seed Region ◽  
Microrna Target ◽  
Functional Sites ◽  
Uv Crosslinking ◽  
Gene Regulatory ◽  
Mirna Seed ◽  
Better Than

MicroRNA targets are often recognized through pairing between the miRNA seed region and complementary sites within target mRNAs, but not all of these canonical sites are equally effective, and both computational and in vivo UV-crosslinking approaches suggest that many mRNAs are targeted through non-canonical interactions. Here, we show that recently reported non-canonical sites do not mediate repression despite binding the miRNA, which indicates that the vast majority of functional sites are canonical. Accordingly, we developed an improved quantitative model of canonical targeting, using a compendium of experimental datasets that we pre-processed to minimize confounding biases. This model, which considers site type and another 14 features to predict the most effectively targeted mRNAs, performed significantly better than existing models and was as informative as the best high-throughput in vivo crosslinking approaches. It drives the latest version of TargetScan (v7.0; targetscan.org), thereby providing a valuable resource for placing miRNAs into gene-regulatory networks.

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