MiR-CLIP reveals iso-miR selective regulation in the miR-124 targetome.

Many microRNAs regulate gene expression via atypical mechanisms, which are difficult to discern using native cross-linking methods. To ascertain the scope of non-canonical miRNA targeting, methods are needed that identify all targets of a given miRNA. We designed a new class of miR-CLIP probe, whereby psoralen is conjugated to the 3p arm of a pre-microRNA to capture targetomes of miR-124 and miR-132 in HEK293T cells. Processing of pre-miR-124 yields miR-124 and a 5'-extended isoform, iso-miR-124. Using miR-CLIP, we identified overlapping targetomes from both isoforms. From a set of 16 targets, 13 were differently inhibited at mRNA/protein levels by the isoforms. Moreover, delivery of pre-miR-124 into cells repressed these targets more strongly than individual treatments with miR-124 and iso-miR-124, suggesting that isomirs from one pre-miRNA may function synergistically. By mining the miR-CLIP targetome, we identified nine G-bulged target-sites that are regulated at the protein level by miR-124 but not isomiR-124. Using structural data, we propose a model involving AGO2 helix-7 that suggests why only miR-124 can engage these sites. In summary, access to the miR-124 targetome via miR-CLIP revealed for the first time how heterogeneous processing of miRNAs combined with non-canonical targeting mechanisms expand the regulatory range of a miRNA.

Site-Specific Labeling of MicroRNA Precursors: A Structure-Activity Relationship Study.

Functionalized oligoribonucleotides are essential tools in RNA chemical biology. Various synthetic routes have been developed over recent years to conjugate functional groups to oligoribonucleotides. However, the presence of the functional group on the oligoribonucleotide backbone can lead to partial or total loss of biological function. The limited knowledge concerning the positioning of functional groups therefore represents a hurdle for the development of oligoribonucleotide chemical tools. Here we describe a systematic investigation of site-specific labeling of pre-miRNAs to identify positions for the incorporation of functional groups, in order not to hinder their processing into active mature miRNAs.

Ligation of 2', 3'-cyclic phosphate RNAs for the identification of microRNA binding sites.

Identifying the targetome of a microRNA is key for understanding its functions. Cross-linking and immunoprecipitation (CLIP) methods capture native miRNA-mRNA interactions in cells. Some of these methods yield small amounts of chimeric miRNA-mRNA sequences via ligation of 5'-phosphorylated RNAs produced during the protocol. Here, we introduce chemically synthesized microRNAs (miRNAs) bearing 2'-, 3'-cyclic phosphate groups, as part of a new CLIP method that does not require 5'-phosphorylation for ligation. We show in a system that models miRNAs bound to their targets, that addition of recombinant bacterial ligase RtcB increases ligation efficiency, and that the transformation proceeds via a 3'-phosphate intermediate. By optimizing the chemistry underlying ligation, we provide the basis for an improved method to identify miRNA targetomes.

Pharmacodynamic and Pharmacokinetic Properties of Full Phosphorothioate Small Interfering RNAs for Gene Silencing In Vivo.

State-of-the-art small interfering RNA (siRNA) therapeutics such as givosiran and fitusiran are constructed from three variable components: a fully-modified RNA core that conveys metabolic stability, a targeting moiety that mediates target-cell uptake, and a linker. This structural complexity poses challenges for metabolite characterization and risk assessment after long-term patient exposure. In this study, we show that basic phosphorothioate modification of a siRNA targeting the oncoprotein Lin28B provides a useful increase in metabolic stability, without greatly compromising potency. We found that its stability in vitro matched that of nanoparticle-free patisiran in serum and surpassed it in liver tritosome extracts, although it did not reach the stability of the fitusiran siRNA core structure. Liver and kidney were the main sites of accumulation after its subcutaneous administration in mice. Despite the lack of a delivery agent-free antitumor effect, we anticipate our study to be a starting point to develop alternative siRNA scaffolds that can be degraded into naturally-occurring metabolites and help alleviate the aforementioned challenges. Furthermore, Lin28B is a promising target for cancers, and the development of such simplified siRNA analogs, possibly together with novel targeting units, holds potential.

Effectiveness of Hand Self-Shiatsu to Promote Sleep in Young People with Chronic Pain: a Case Series Design.

BACKGROUND: Sleep problems exist for up to 30% of young people, and increase in the case of those with chronic pain. Because exclusive pharmacological management of sleep problems for children with pain is contraindicated, the development of appropriate non-pharmacological sleep interventions is a significant, largely unmet, need. PURPOSE: This study examined whether the application of a standardized hand self-shiatsu (HSS) intervention within a population of young people with chronic pain would be associated with improved objectively and subjectively measured sleep. SETTING: The Pain Management Clinic of the Stollery Children's Hospital Hospital, a large tertiary care centre in Edmonton, Alberta and the University of Alberta. RESEARCH DESIGN: Sixteen young adults, aged 17 to 27, were recruited for a case series study. The intervention involved participants self-applying a standardized hand shiatsu protocol. Participants wore an actigraph for one week at baseline before learning the HSS technique, and then at four- and eight-week follow-up. At the same measurement points they completed validated self-report measures of their sleep quality and daytime fatigue. Each participant also completed a sleep log to supplement the actigraphy data and to collect their general impressions of the HSS experience. Data were analyzed with SPSS 23 software, using Freidman's test for analysis of variance. RESULTS: The objective data did not support the hypothesis that this standardized HSS protocol improves objectively measured sleep. However, standardized self-report measures demonstrated statistically significant improvement in perceived sleep disturbance (chi-squared test [χ2] = 8.034, p = .02), sleep-related impairment (χ2 = 7.614, p = .02), and daytime fatigue as measured by the PROMIS Fatigue SF 8-a (χ2 = 12.035, p = .002), and the Flinder's Fatigue Scale (χ2 = 11.93, p = .003). Qualitative sleep log information indicated wide-spread endorsement of HSS for the management of sleep difficulties. CONCLUSION: Contrary to objective findings, self-report data support the technique of HSS to improve sleep. Participants' comments reflected an overall high level of acceptance and appreciation for the HSS technique. Results highlight the importance of expanding the theory and practice related to sleep measurement to better integrate the qualitative domain.

EGF stimulates the activation of EGF receptors and the selective activation of major signaling pathways during mitosis.

Mitosis and epidermal growth factor (EGF) receptor (EGFR) are both targets for cancer therapy. The role of EGFR signaling in mitosis has been rarely studied and poorly understood. The limited studies indicate that the activation of EGFR and downstream signaling pathways is mostly inhibited during mitosis. However, we recently showed that EGFR is phosphorylated in response to EGF stimulation in mitosis. Here we studied EGF-induced EGFR activation and the activation of major signaling pathways downstream of EGFR during mitosis. We showed that EGFR was strongly activated by EGF during mitosis as all the five major tyrosine residues including Y992, Y1045, Y1068, Y1086, and Y1173 were phosphorylated to a level similar to that in the interphase. We further showed that the activated EGFR is able to selectively activate some downstream signaling pathways while avoiding others. Activated EGFR is able to activate PI3K and AKT2, but not AKT1, which may be responsible for the observed effects of EGF against nocodazole-induced cell death. Activated EGFR is also able to activate c-Src, c-Cbl and PLC-γ1 during mitosis. However, activated EGFR is unable to activate ERK1/2 and their downstream substrates RSK and Elk-1. While it activated Ras, EGFR failed to fully activate Raf-1 in mitosis due to the lack of phosphorylation at Y341 and the lack of dephosphorylation at pS259. We conclude that contrary to the dogma, EGFR is activated by EGF during mitosis. Moreover, EGFR-mediated cell signaling is regulated differently from the interphase to specifically serve the needs of the cell in mitosis.

Stereochemical bias introduced during RNA synthesis modulates the activity of phosphorothioate siRNAs.

An established means of improving the pharmacokinetics properties of oligoribonucleotides (ORNs) is to exchange their phosphodiester linkages for phosphorothioates (PSs). However, this strategy has not been pursued for small interfering RNAs (siRNAs), possibly because of sporadic reports that PS siRNAs show reduced inhibitory activity. The PS group is chiral at phosphorous (Rp/Sp centres), and conventional solid-phase synthesis of PS ORNs produces a population of diastereoisomers. Here we show that the choice of the activating agent for the synthesis of a PS ORN influences the Rp/Sp ratio of PS linkages throughout the strand. Furthermore, PS siRNAs composed of ORNs with a higher fraction of Rp centres show greater resistance to nucleases in serum and are more effective inhibitors in cells than their Sp counterparts. The finding that a stereochemically biased population of ORN diastereoisomers can be synthesized and exploited pharmacologically is important because uniform PS modification of siRNAs may provide a useful compromise of their pharmacokinetics and pharmacodynamics properties in RNAi therapeutics.