A role for human Dicer in pre-RISC loading of siRNAs

RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3′ overhangs and the thermodynamic properties of 2–4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA

Defining the Earliest Transcriptional Steps of Chondrogenic Progenitor Specification during the Formation of the Digits in the Embryonic Limb

The characterization of genes involved in the formation of cartilage is of key importance to improve cell-based cartilage regenerative therapies. Here, we have developed a suitable experimental model to identify precocious chondrogenic events in vivo by inducing an ectopic digit in the developing embryo. In this model, only 12 hr after the implantation of a Tgfβ bead, in the absence of increased cell proliferation, cartilage forms in undifferentiated interdigital mesoderm and in the course of development, becomes a structurally and morphologically normal digit. Systematic quantitative PCR expression analysis, together with other experimental approaches allowed us to establish 3 successive periods preceding the formation of cartilage. The “pre-condensation stage”, occurring within the first 3 hr of treatment, is characterized by the activation of connective tissue identity transcriptional factors (such as Sox9 and Scleraxis) and secreted factors (such as Activin A and the matricellular proteins CCN-1 and CCN-2) and the downregulation of the galectin CG-8. Next, the “condensation stage” is characterized by intense activation of Smad 1/5/8 BMP-signaling and increased expression of extracellular matrix components. During this period, the CCN matricellular proteins promote the expression of extracellular matrix and cell adhesion components. The third period, designated the “pre-cartilage period”, precedes the formation of molecularly identifiable cartilage by 2–3 hr and is characterized by the intensification of Sox 9 gene expression, along with the stimulation of other pro-chondrogenic transcription factors, such as HifIa. In summary, this work establishes a temporal hierarchy in the regulation of pro-chondrogenic genes preceding cartilage differentiation and provides new insights into the relative roles of secreted factors and cytoskeletal regulators that direct the first steps of this process in vivo

Cross-species genomic landscape comparison of human mucosal melanoma with canine oral and equine melanoma.

Mucosal melanoma is a rare and poorly characterized subtype of human melanoma. Here we perform a cross-species analysis by sequencing tumor-germline pairs from 46 primary human muscosal, 65 primary canine oral and 28 primary equine melanoma cases from mucosal sites. Analysis of these data reveals recurrently mutated driver genes shared between species such as NRAS, FAT4, PTPRJ, TP53 and PTEN, and pathogenic germline alleles of BRCA1, BRCA2 and TP53. We identify a UV mutation signature in a small number of samples, including human cases from the lip and nasal mucosa. A cross-species comparative analysis of recurrent copy number alterations identifies several candidate drivers including MDM2, B2M, KNSTRN and BUB1B. Comparison of somatic mutations in recurrences and metastases to those in the primary tumor suggests pervasive intra-tumor heterogeneity. Collectively, these studies suggest a convergence of some genetic changes in mucosal melanomas between species but also distinctly different paths to tumorigenesis

Transverse Line Formation in Protein-Deprived Rhesus Monkeys

Transverse lines (Harris lines) in long bones are density anomalies thought to be associated with intervals of growth disturbance and subsequent recovery. In this study, radiographs of infant rhesus monkeys, Macaca mulatto, were examined for evidence of transferse lines in the distal ends of the left radius. The objective was to document the frequency of early postnatal transverse line formation, i.e. the ap­pearance of birth lines. Other variables considered were animal sex and birthweight. The effects of different infant diets were also evaluated to determine whether the availability of protein affected the frequency of line formation.Thirty monkeys were placed on a protein-restricted diet at birth. Data for the experimental animals were compared with that of controls (n = 55) maintained on a diet adequate in protein. A higher mean number and higher percentage of transverse lines occurred in controls than in protein-restricted infants. No sex differences were present. Animal birthweight was not correlated with the occurrence of transverse lines.At age 120 days, 10 protein-restricted animals were placed on the higher protein diet of controls. Sequential x-rays were then examined to determine whether this transition was followed by the appearance of transverse lines. Recovery from the protein-restricted diet was not reflected by the formation of a line

Protein Deprivation and Perinatal Weight Changes in Rhesus Monkeys

Forty-six infant rhesus monkeys of both sexes were fed a for­mula containing 3.35%, 6.7%, or 13.4% protein as casein. Their weight gain and food consumption were compared with those of 53 monkeys fed a commercial formula simulating human milk. All groups passed through a three-day weight-loss stage and a transitional stage lasting about one week, after which they entered a rather permanent growth stage. Males ate more than the females did, but only because they weighed more. The lowest- protein diet had no significant deleterious effect on weight gain during the transition phase, and it had no effect on the females’ gain during the growth stage. Males fed the lowest protein diet, in contrast, gained little during the growth stage. The reduction in weight gain seems due to a loss in food efficiency, not to a reduction in consumption

A revised sedimentary and biostratigraphical architecture for the Type Llandovery area, Central Wales

The global standard for the Llandovery Series (early Silurian) in centralWales is re-assessed in the light of detailed geological surveying, biostratigraphical sampling and a rigorous examination of published datasets. A new sedimentary and biostratigraphical architecture is presented. Key graptolite, brachiopod, acritarch and, for the first time, chitinozoan assemblages are critically assessed. Upper Hirnantian to Aeronian strata record events that followed the Late Ordovician glacial maximum and comprise a series of progradational sequences bounded by flooding surfaces, but inferred still to be glacioeustatic in origin. Significant faunal renewals associated with many of the flooding levels underpin their potential for international recognition. Compound non-sequences are a feature of proximal parts of the system where erosion associated with fault footwall uplift was an important process. Extensive slump sheets contribute to further stratal loss and displacement in distal facies. A re-assessment of the Aeronian Stage GSSP reveals shortcomings with the biostratigraphical criteria used in its selection. Telychian portions of the succession display the disrupting effects of intra- Wenlock synsedimentary sliding; hence the relevance of key published fossil assemblages and the criteria used to erect the stage GSSP are undermined. However, the Llandovery area remains one of the best studied early Silurian successions in the world. This, together with regional considerations, supports the retention of the series standard in mid Wales where the contiguous deep-water basinal succession affords internationally cited exposure of richly graptolitic facies for the whole series and, significantly, for the post-sedgwickii Biozone interval

Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver

shRNA overexpression from viral gene therapy vectors can trigger cytotoxicity leading to organ failure and lethality in mice and rats. This process likely involves saturation of endogenous cellular RNAi factors including exportin-5 (Xpo-5). Here, we have shown that Xpo-5 overexpression enhanced shRNA efficiency in the liver of adult mice but increased hepatotoxicity. We identified the 4 members of the human Argonaute (Ago) protein family as downstream factors involved in saturation of endogenous cellular RNAi, all of which were able to interact with shRNAs in cells and mice. In Ago/shRNA coexpression studies, Ago-2 (Slicer) was the primary rate-limiting determinant of both in vitro and in vivo RNAi efficacy, toxicity, and persistence. In adult mice, vector-based Ago-2/Xpo-5 coexpression enhanced U6-driven shRNA silencing of exogenous and endogenous hepatic targets, reduced hepatotoxicity, and extended RNAi stability by more than 3 months. Use of weaker RNA polymerase III promoters to minimize shRNA expression likewise alleviated in vivo toxicity and permitted greater than 95% persistent knockdown of hepatitis B virus and other transgenes in mouse liver for more than 1 year. Our studies substantiate that abundant small RNAs can overload the endogenous RNAi pathway and reveal possible strategies for reducing hepatotoxicity of short- and long-term clinical gene silencing in humans