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MicroRNAs-direct Argonaute proteins to repress complementary target mRNAs via mRNA degradation or translational inhibition. While mammalian miRNA targeting has been well studied, the principles by which Drosophila miRNAs bind their target RNAs remain to be fully characterized. Here, we use RNA Bind-n-Seq to systematically identify binding sites and measure their affinities for four highly expressed Drosophila miRNAs. Our results reveal a narrower range of binding site diversity in flies compared to mammals, with fly miRNAs favoring canonical seed-matched sites and exhibiting limited tolerance for imperfections within these sites. We also identified non-canonical site types, including nucleation-bulged and 3'-only sites, whose binding affinities are comparable to canonical sites. These findings establish a foundation for future computational models of Drosophila miRNA targeting, enabling predictions of regulatory outcomes in response to cellular signals, and advancing our understanding of miRNA-mediated regulation in flies., Competing Interests: Competing interests The authors declare no competing interests.

mRNA translation and decay are tightly interconnected processes both in the context of mRNA quality-control pathways and for the degradation of functional mRNAs. Cotranslational mRNA degradation through codon usage, ribosome collisions, and the recruitment of specific proteins to ribosomes is an important determinant of mRNA turnover. However, the extent to which translation-dependent mRNA decay (TDD) and translation-independent mRNA decay (TID) pathways participate in the degradation of mRNAs has not been studied yet. Here we describe a comprehensive analysis of basal and signal-induced TDD and TID in mouse primary CD4 + T cells. Our results indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner. Our analysis further identifies the length of untranslated regions, the density of ribosomes, and GC3 content as important determinants of TDD magnitude. Consistently, all transcripts that undergo changes in ribosome density within their coding sequence upon T cell activation display a corresponding change in their TDD level. Moreover, we reveal a dynamic modulation in the relationship between GC3 content and TDD upon T cell activation, with a reversal in the impact of GC3- and AU3-rich codons. Altogether, our data show a strong and dynamic interconnection between mRNA translation and decay in mammalian primary cells., (© 2024 Mercier et al.; Published by Cold Spring Harbor Laboratory Press.)

Binding affinity quantitatively describes the strength of a molecular interaction and is reported by the equilibrium dissociation constant (K D ). Here, we present a protocol to measure K D of mammalian microRNA-loaded Argonaute2 protein by double filter binding. We describe steps for radiolabeling target RNA, measuring concentration of binding-competent protein, setting up binding reactions, separating protein-bound RNA from protein-unbound RNA, preparing library for Illumina sequencing, and performing data analysis. Our protocol is easily applied to other RNA- or DNA-binding proteins. For complete details on the use and execution of this protocol, please refer to Jouravleva et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs‑PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer‑1⋅Loqs‑PB heterodimer. The Dicer-1 dsRBD and three Loqs‑PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer‑1⋅Loqs‑PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Argonautes are nucleic acid-guided proteins that perform numerous cellular functions across all domains of life. Little is known about how distinct evolutionary pressures have shaped each Argonaute's biophysical properties. We applied high-throughput biochemistry to characterize how Thermus thermophilus Argonaute (TtAgo), a DNA-guided DNA endonuclease, finds, binds, and cleaves its targets. We found that TtAgo uses biophysical adaptations similar to those of eukaryotic Argonautes for rapid association but requires more extensive complementarity to achieve high-affinity target binding. Using these data, we constructed models for TtAgo association rates and equilibrium binding affinities that estimate the nucleic acid- and protein-mediated components of the target interaction energies. Finally, we showed that TtAgo cleavage rates vary widely based on the DNA guide, suggesting that only a subset of guides cleaves targets on physiologically relevant timescales., Competing Interests: Declaration of interests P.D.Z. is a member of the scientific advisory boards of Alnylam Pharmaceuticals, Voyager Therapeutics, and ProQR. He is also a consultant for The RNA Medicines Company. W.J.G is a scientific co-founder of Protillion and a consultant, and equity holder, for Guardant Health, 10X Genomics, Ultima Genomics, and Quantapore., (Copyright © 2022 Elsevier Inc. All rights reserved.)

RNA Bind-n-Seq (RBNS) is a cost-effective, high-throughput method capable of identifying the sequence preferences of RNA-binding proteins and of qualitatively defining relative dissociation constants. Although RBNS is often described as an unbiased method, several factors may influence the outcome of the analysis. Here, we discuss these biases and present an analytical strategy to estimate absolute binding affinities from RBNS data, extend RBNS to kinetic studies, and develop a framework to compute relative association and dissociation rate constants. As proof of principle, we measured the equilibrium binding properties of mammalian Argonaute2 (AGO2) guided by eight microRNAs (miRNAs) and kinetic parameters for let-7a. The miRNA-binding site repertoires, dissociation constants, and kinetic parameters calculated from RBNS data using our methods correlate well with values measured by traditional ensemble and single-molecule approaches. Our data provide additional quantitative measurements for Argonaute-bound miRNA binding that should facilitate development of quantitative targeting rules for individual miRNAs., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal trimming and 2'-O-methylation. Both trimming and methylation influence piRNA stability. Our biochemical data show that multiple mechanisms destabilize unmethylated mouse piRNAs, depending on whether the piRNA 5' or 3' sequence is complementary to a trigger RNA. Unlike target-directed degradation of microRNAs, complementarity-dependent destabilization of piRNAs in mice and flies is blocked by 3' terminal 2'-O-methylation and does not require base pairing to both the piRNA seed and the 3' sequence. In flies, 2'-O-methylation also protects small interfering RNAs (siRNAs) from complementarity-dependent destruction. By contrast, pre-piRNA trimming protects mouse piRNAs from a degradation pathway unaffected by trigger complementarity. In testis lysate and in vivo, internal or 3' terminal uridine- or guanine-rich tracts accelerate pre-piRNA decay. Loss of both trimming and 2'-O-methylation causes the mouse piRNA pathway to collapse, demonstrating that these modifications collaborate to stabilize piRNAs., Competing Interests: Declaration of interests P.D.Z. is a member of the editorial board of Molecular Cell., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication., Competing Interests: Declaration of Interests P.D.Z., S.M.J., and H.Z. have submitted a patent application regarding novel uses of TtAgo (PCT/US2016/025724)., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Argonaute proteins loaded with microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC), which represses target RNA expression. Predicting the biological targets, specificity, and efficiency of both miRNAs and siRNAs has been hamstrung by an incomplete understanding of the sequence determinants of RISC binding and cleavage. We applied high-throughput methods to measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of mouse AGO2 RISC. We find that RISC readily tolerates insertions of up to 7 nt in its target opposite the central region of the guide. Our data uncover specific guide:target mismatches that enhance the rate of target cleavage, suggesting novel siRNA design strategies. Using these data, we derive quantitative models for RISC binding and target cleavage and show that our in vitro measurements and models predict knockdown in an engineered cellular system., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We also use the pipeline to extend our understanding of TtAgo by measuring the protein's binding kinetics at physiological temperatures and for target DNAs containing multiple, adjacent binding sites.

There is a well-established association between aging and the onset of metastasis. Although the mechanisms through which age impinges upon the malignant phenotype remain uncharacterized, the role of a senescent microenvironment has been emphasized. We reported previously that human epithelial cells that undergo telomere-driven chromosome instability (T-CIN) display global microRNA (miR) deregulation and develop migration and invasion capacities. Here, we show that post-crisis cells are not able to form tumors unless a senescent microenvironment is provided. The characterization of cell lines established from such tumors revealed that these cells have acquired cell autonomous tumorigenicity, giving rise to heterogeneous tumors. Further experiments demonstrate that explanted cells, while displaying differences in cell differentiation markers, are all endowed of enhanced stem cell properties including self-renewal and multilineage differentiation capacity. Treatments of T-CIN+ cells with senescence-conditioned media induce sphere formation exclusively in cells with senescence-associated tumorigenicity, a capacity that depends on miR-145 repression. These results indicate that the senescent microenvironment, while promoting further transdifferentiations in cells with genome instability, is able to propel the progression of premalignant cells towards a malignant, cell stem-like state., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Telomere shortening is a major source of chromosome instability (CIN) at early stages during carcinogenesis. However, the mechanisms through which telomere-driven CIN (T-CIN) contributes to the acquisition of tumor phenotypes remain uncharacterized. We discovered that human epithelial kidney cells undergoing T-CIN display massive microRNA (miR) expression changes that are not related to local losses or gains. This widespread miR deregulation encompasses a miR-200-dependent epithelial-to-mesenchymal transition (EMT) that confers to immortalized pre-tumoral cells phenotypic traits of metastatic potential. Remarkably, a miR signature of these cells, comprising a downregulation of miRs with conserved expression in kidney, was retrieved in poorly differentiated aggressive renal cell carcinomas. Our results reveal an unanticipated connection between telomere crisis and the activation of the EMT program that occurs at pre-invasive stages of epithelial cancers, through mechanisms that involve miR deregulation. Thus, this study provides a new rational into how telomere instability contributes to the acquisition of the malignant phenotype.

Helicobacter pylori secretes a unique virulence factor, Tipalpha, that enters gastric cells and both stimulates the production of the TNF-alpha and activates the NF-kappaB pathway. The structures of a truncated version of Tipalpha (TipalphaN34) in two crystal forms are presented here. Tipalpha adopts a novel beta(1)alpha(1)alpha(2)beta(2)beta(3)alpha(3)alpha(4) topology that can be described as a combination of three domains. A first region consists in a short flexible extension, a second displays a dodecin-like fold and a third is a helical bundle domain similar to the sterile alpha motif (SAM). Analysis of the oligomerisation states of TipalphaN34 in the crystals and in solution suggests that the disulfide bridges could hold together Tipalpha monomers during their secretion in the gastric environment.