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Mitochondrial RNA editing in trypanosomes represents an attractive target for developing safer and more efficient drugs for treating infections with trypanosomes because this RNA editing pathway is not found in humans. Other workers have targeted several enzymes in this editing system, but not the RNA. Here, we target a universal domain of the RNA editing substrate, which is the U-helix formed between the oligo-U tail of the guide RNA and the target mRNA. We selected a part of the U-helix that is rich in G-U wobble base pairs as the target site for the virtual screening of 262,000 compounds. After chemoinformatic filtering of the top 5000 leads, we subjected 50 representative complexes to 50 nanoseconds of molecular dynamics simulations. We identified 15 compounds that retained stable interactions in the deep groove of the U-helix. The microscale thermophoresis binding experiments on these five compounds show low-micromolar to nanomolar binding affinities. The UV melting studies show an increase in the melting temperatures of the U-helix upon binding by each compound. These five compounds can serve as leads for drug development and as research tools to probe the role of the RNA structure in trypanosomal RNA editing. [ABSTRACT FROM AUTHOR]

Paraspeckles are nuclear ribonucleic complex formed of a long non-coding RNA, nuclear-enriched abundant transcript one (Neat1) and associated RNA-binding proteins (RBP) whose cellular known functions are to sequester in the nucleus both proteins and RNAs. However, how RNAs are bound to paraspeckles is largely unknown. It is highly likely that binding of RNAs may occur via interactions with RBPs and accordingly, two structures present in the 3ʹUTR of some RNAs have been shown to allow their association to paraspeckles via protein binding. However, Neat1 could also be involved in the targeting of RNAs through direct RNA–RNA interactions. Using an RNA pull-down procedure adapted to select only RNAs engaged in direct RNA–RNA interactions and followed by RNA-seq we showed that in a rat pituitary cell line, GH4C1 cells, 1791 RNAs were associated with paraspeckles by direct interaction with Neat1. Neat1 was actually found able to bind more than 30% of the total transcripts targeted by the paraspeckles, we have identified in this cell line in a previous study. Furthermore, given the biological processes in which direct RNAs targets of Neat1 were involved as determined by gene ontology analysis, it was proposed that Neat1 played a major role in paraspeckle functions such as circadian rhythms, mRNA processing, RNA splicing and regulation of cell cycle. Finally, we provided evidence that direct RNA targets of Neat1 were preferentially bound to the 5ʹ end of Neat1 demonstrating that they are located in the shell region of paraspeckles. [ABSTRACT FROM AUTHOR]

Small RNA (sRNA) molecules are key factors in the communication between hosts and their interacting pathogens, where they function as effectors that can modulate both host defense and microbial virulence/pathogenicity through a mechanism termed cross-kingdom RNA interference (ck-RNAi). Consistent with this recent knowledge, sRNAs and their double-stranded RNA precursor have been adopted to control diseases in crop plants, demonstrating a straight forward application of the new findings to approach agricultural problems. Despite the great interest in natural ck-RNAi, it is astonishing to find just a few additional examples in the literature since the first report was published in 2013. One reason might be that the identification of sRNA effectors is hampered both by technical challenges and lack of routine bioinformatics application strategies. Here, we suggest a practical procedure to find, characterize, and validate sRNA effectors in plant–microbe interaction. The aim of this review is not to present and discuss all possible tools, but to give guidelines toward the best established software available for the analysis.

Small RNA (sRNA) molecules are key factors in the communication between hosts and their interacting pathogens, where they function as effectors that can modulate both host defense and microbial virulence/pathogenicity through a mechanism termed cross-kingdom RNA interference (ck -RNAi). Consistent with this recent knowledge, sRNAs and their double-stranded RNA precursor have been adopted to control diseases in crop plants, demonstrating a straight forward application of the new findings to approach agricultural problems. Despite the great interest in natural ck -RNAi, it is astonishing to find just a few additional examples in the literature since the first report was published in 2013. One reason might be that the identification of sRNA effectors is hampered both by technical challenges and lack of routine bioinformatics application strategies. Here, we suggest a practical procedure to find, characterize, and validate sRNA effectors in plant–microbe interaction. The aim of this review is not to present and discuss all possible tools, but to give guidelines toward the best established software available for the analysis. [ABSTRACT FROM AUTHOR]

Argonaute proteins are widespread in prokaryotes and eukaryotes with diversified catalytic activities. Here, we describe an Argonaute from Marinitoga hydrogenitolerans (MhAgo) with all eight cleavage activities. Utilization of all four types of guides and efficient cleavage of single-stranded DNA (ssDNA) and RNA targets are revealed. The preference for the 5′-terminus nucleotides of 5′P guides, but no obvious preferences for that in 5′OH guides, is further uncovered. Moreover, the cleavage efficiency is heavily impaired by mismatches in the central and 3′-supplementary regions of guides, and the affinity between guides or guides/target duplex and MhAgo is proved as one of the factors affecting cleavage efficiency. Structural and mutational analyses imply some unknown distinctive structural features behind the cleavage activity of MhAgo. Meanwhile, 5′OH-guide RNA (gRNA)-mediated plasmid cleavage activity is unveiled. Conclusively, MhAgo is versatile, and its biochemical characteristics improve our understanding of pAgos and the pAgo-based techniques. [Display omitted] • Bacterial Argonaute MhAgo uses all four types of guides to target and cleave DNA and RNA • The preference of MhAgo to 5′ end of guides depends on types of guides and targets • MhAgo bears high specificity in the central and 3′-supplementary regions of guides • MhAgo can use 5′OH-gRNA to recognize and cleave plasmids Wang et al. report a pAgo from Marinitoga hydrogenitolerans (MhAgo) that can cleave ssDNA and RNA guided with four types of guides and has capacity in 5′OH-gRNA-directed cleavage of plasmid. The unique biochemical characteristics of MhAgo broaden our understanding of pAgos. [ABSTRACT FROM AUTHOR]

An efficient and highly versatile synthesis of two libraries 1(x,y) and 2-Ar(x,y,z) or 2-R2(x,y,w) based on the privileged benzimidazole scaffold is described. Our design is aimed at obtaining molecules, biased for binding to RNA targets, by incorporating functionalities, which are frequently found in natural RNA-ligands. The library construction was realized with the use of SPOS in high average yields and purity. Monitoring and quantitation of intermediates and final products were performed by the use of NMR spectroscopy using DMFu as an internal standard. [Copyright &y& Elsevier]

During development, cells must quickly switch from one cell state to the next to execute precise and timely differentiation. One method to ensure fast transitions in cell states is by controlling gene expression at the post-transcriptional level through action of RNA-binding proteins on mRNAs. The ability to accurately identify the RNA targets of RNA-binding proteins at specific stages is key to understanding the functional role of RNA-binding proteins during development. Here we describe an adapted formaldehyde RNA immunoprecipitation (fRIP) protocol to identify the in vivo RNA targets of a cytoplasmic RNA-binding protein, YTHDC2, from testis, during the first wave of spermatogenesis, at the stage when germ cells are shutting off the proliferative program and initiating terminal differentiation ( Bailey et al. , 2017 ). This protocol enables quick and efficient identification of endogenous RNAs bound to an RNA-binding protein, and facilitates the monitoring of stage-specific changes during development., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)

Study Question: Can novel meiotic RNA targets of DAZL (deleted in azoospermia-like) be identified in the human foetal ovary?, Summary Answer: SYCP1 (synaptonemal complex protein-1), TEX11 (testis expressed 11) and SMC1B (structural maintenance of chromosomes 1B) are novel DAZL targets in the human foetal ovary, thus DAZL may have previously unrecognised roles in the translational regulation of RNAs involved in chromosome cohesion and DNA recombination in the oocyte from the time of initiation of meiosis., What Is Known Already: The phenotype of Dazl deficiency in mouse is infertility in both sexes and DAZL has also been linked to infertility in humans. Few studies have explored targets of this RNA-binding protein. The majority of these investigations have been carried out in mouse, and have focussed on the male thus the basis for its central function in regulating female fertility is largely unknown., Study Design Size, Duration: We carried out RNA sequencing after immunoprecipitation of endogenous DAZL from human foetal ovarian tissue (17 weeks of gestation, obtained after elective termination of pregnancy) to identify novel DAZL targets involved in meiosis (n = 3 biological replicates)., Participants/materials, Setting, Methods: Using quantitative RT-PCR, we examined the expression of selected RNAs identified by our immunoprecipitation across gestation, and visualised the expression of potential target SMC1B in relation to DAZL, with a combination of in situ hybridisation and immunohistochemistry. 3' untranslated region (3'UTR)-luciferase reporter assays and polysome profile analysis were used to investigate the regulation of three RNA targets by DAZL, representing key functionalities: SYCP1, TEX11 and SMC1B., Main Results and the Role of Chance: We identified 764 potential RNA targets of DAZL in the human foetal ovary (false discovery rate 0.05 and log-fold change ≥ 2), with functions in synaptonemal complex formation (SYCP1, SYCP3), cohesin formation (SMC1B, RAD21), spindle assembly checkpoint (MAD2L1, TRIP13) and recombination and DNA repair (HORMAD1, TRIP13, TEX11, RAD18, RAD51). We demonstrated that the translation of novel targets SYCP1 (P = 0.004), TEX11 (P = 0.004) and SMC1B (P = 0.002) is stimulated by the presence of DAZL but not by a mutant DAZL with impaired RNA-binding activity., Large Scale Data: The raw data are available at GEO using the study ID: GSE81524., Limitations, Reasons for Caution: This analysis is based on identification of DAZL targets at the time when meiosis starts in the ovary: it may have other targets at other stages of oocyte development, and in the testis. Representative targets were validated, but detailed analysis was not performed on the majority of putative targets., Wider Implications of the Findings: These data indicate roles for DAZL in the regulation of several key functions in human oocytes. Through the translational regulation of novel RNA targets SMC1B and TEX11, DAZL may have a key role in regulating chromosome cohesion and DNA recombination; two processes fundamental in determining oocyte quality and whose establishment in foetal life may support lifelong fertility., Study Funding and Competing Interest(s): This study was supported by the UK Medical Research Council (grant no G1100357 to R.A.A. and an intramural MRC programme grant to I.R.A.). The authors declare no competing interests., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)