On the rules of engagement for microRNAs targeting protein coding regions

OnlinePublMiRNAs post-transcriptionally repress gene expression by binding to mRNA 3 UTRs, but the extent to which they act through protein coding regions (CDS regions) is less well established. MiRNA interaction studies show a substantial proportion of binding occurs in CDS regions, however sequencing studies show much weaker effects on mRNA levels than from 3 UTR interactions, presumably due to competition from the translating ribosome. Consequently, most target prediction algorithms consider only 3 UTR interactions. However, the consequences of CDS interactions may have been underestimated, with the reporting of a novel mode of miRNA-CDS interaction requiring base pairing of the miRNA 3 end, but not the canonical seed site, leading to repression of translation with little effect on mRNA turnover. Using extensive reporter, western blotting and bioinformatic analyses, we confirm that miRNAs can indeed suppress genes through CDS-interaction in special circumstances. However, in contrast to that previously reported, we find repression requires extensive base-pairing, including of the canonical seed, but does not strictly require base pairing of the 3 miRNA terminus and is mediated through reducing mRNA levels. We conclude that suppression of endogenous genes can occur through miRNAs binding to CDS, but the requirement for extensive basepairing likely limits the regulatory impacts to modest effects on a small subset of targets.Sunil Sapkota, Katherine A. Pillman, B. Kate Dredge, Dawei Liu, Julie M. Bracken, Saba Ataei Kachooei, Bradley Chereda, Philip A. Gregory, Cameron P. Bracken, and Gregory J. Goodal

Empowering Women through Participatory Action Research in Community-Based Disaster Risk Reduction Efforts

The role of women in community-based disaster risk reduction efforts (CBDRR) is an area of limited academic research and continues to be a thorny issue for policy and practice. This research paper describes a comparative case study of participatory action research (PAR) in CBDRR conducted in one rural and one urban tole (neighbourhood) of Kathmandu Valley, Nepal. PAR is not a method, rather it is a set of principles guiding research. The “Empowering Women through CBDRR” PAR was motivated by the National Society for Earthquake Technology-Nepal’s (NSET) desire to learn how to effectively empower women in disaster risk management on a local level and to enhance resilience to everyday hazards and risks as well as earthquakes. The hazards identified by residents in rural Bhainse were the supply of drinking water and landslides while the supply of drinking water and earthquakes were the perceived hazards in urban Tajhya Tole. The small-scale mitigation activities chosen and implemented by the female led disaster management committees in partnership with the local authorities and NSET addressed everyday risks (fire) that were important to the community or were related to livelihood concerns (landslide and drainage pipe). While there is clear evidence of women’s empowerment and capacity building, sustainability of initiatives is particularly dependent on the commitment of local authorities to incorporate the initiatives into local policies and actions. A gap remains between aspirations to practice empowerment of women and implementation. In many ways, ‘doing’ empowerment remains problematic in CBDRR

Insufficiently complex unique-molecular identifiers (UMIs) distort small RNA sequencing

The attachment of unique molecular identifiers (UMIs) to RNA molecules prior to PCR amplification and sequencing, makes it possible to amplify libraries to a level that is sufficient to identify rare molecules, whilst simultaneously eliminating PCR bias through the identification of duplicated reads. Accurate de-duplication is dependent upon a sufficiently complex pool of UMIs to allow unique labelling. In applications dealing with complex libraries, such as total RNA-seq, only a limited variety of UMIs are required as the variation in molecules to be sequenced is enormous. However, when sequencing a less complex library, such as small RNAs for which there is a more limited range of possible sequences, we find increased variation in UMIs are required, even beyond that provided in a commercial kit specifically designed for the preparation of small RNA libraries for sequencing. We show that a pool of UMIs randomly varying across eight nucleotides is not of sufficient depth to uniquely tag the microRNAs to be sequenced. This results in over de-duplication of reads and the marked under-estimation of expression of the more abundant microRNAs. Whilst still arguing for the utility of UMIs, this work demonstrates the importance of their considered design to avoid errors in the estimation of gene expression in libraries derived from select regions of the transcriptome or small genomes.Klay Saunders, Andrew G. Bert, B. Kate Dredge, John Toubia, Philip A. Gregory, Katherine A. Pillman, Gregory J. Goodall, Cameron P. Bracke

Genome-wide identification of miR-200 targets reveals a regulatory network controlling cell invasion

Published online 28.07.2014The microRNAs of the miR‐200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago‐HITS‐CLIP technology for transcriptome‐wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR‐200a and miR‐200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR‐200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR‐200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho‐ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR‐200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration.Cameron P Bracken, Xiaochun Li, Josephine A Wright, David M Lawrence, Katherine A Pillman, Marika Salmanidis, Matthew A Anderson, B Kate Dredge, Philip A Gregory, Anna Tsykin, Corine Neilsen, Daniel W Thomson, Andrew G Bert, Joanne M Leerberg, Alpha S Yap, Kirk B Jensen, Yeesim Khew‐Goodall, Gregory J Goodal

Role of glycemia in acute spinal cord injury: data from a rat experimental model and clinical experience.

While experimental and clinical evidence indicates that in brain injury blood glucose increases with injury severity and hyperglycemia worsens neurological outcome, the role of blood glucose in secondary mechanisms of neuronal damage after acute spinal cord injury has not yet been investigated. Data from spinal cord ischemia models suggests a deleterious effect of hyperglycemia, likely due to enhanced lactic acidosis, which is primarily dependent on the amount of glucose available to be metabolized. The purpose of this study is to summarize preliminary experimental and clinical observations on the role of blood glucose in acute spinal cord injury. Between 1995 and 1996 we used the New York University (NYU) rat spinal cord injury model to test the following hypotheses: 1) Blood glucose levels increase with injury severity. 2) Fasting protects from hyperglycemia and prevents secondary damage to the spinal cord. 3) Postinjury-induced hyperglycemia (dextrose 5% 2 gm/Kg) enhances spinal lesion volume. From a clinical perspective, we reviewed blood glucose records of 47 patients admitted to the Department of Neurosrgery in Verona, between 1991 and 1995, within 24 hours of acute spinal cord injury in order to determine: a) the incidence of hyperglycemia (> 140 mg/dl); b) the correlation between blood glucose and injury severity; and c) the role of methylprednisolone in affecting blood glucose. Results indicate that in a graded spinal cord injury model: 1) Early after injury, more severe contusions support significantly higher blood glucose levels. 2) Fasting overnight does not directly affect spinal cord lesion volume but influences blood gases, and we observed that a slightly systemic acidosis plays a minor neuroprotective role. Fasting also ensures more consistent normoglycemic baseline blood glucose values. 3) Postinjury-induced moderate hyperglycemia (160-190 mg/dl) does not significantly affect spinal cord injury. In the clinical study, we observed that during the first 24 hours after spinal cord injury: a) Glycemia ranges between 90 and 243 mg/dl (mean value 143 mg/dl), and close to 50% of the patients present blood glucose values higher than normal. b) Methylprednisolone administration is not associated to significantly higher blood glucose levels. c) There is a trend for larger glucose rises with more severe injury