Showing papers by "Phillip A. Sharp published in 2006"

Quantitative analysis of Argonaute protein reveals microRNA-dependent localization to stress granules

Abstract: Argonaute proteins associate with microRNAs (miRNAs) that bind mRNAs through partial base-pairings to primarily repress translation in animals. A fraction of Argonaute proteins and miRNAs biochemically cosediment with polyribosomes, yet another fraction paradoxically accumulates in ribosome-free processing bodies (PBs) in the cytoplasm. In this report, we give a quantitative account of the Argonaute protein localization and dynamics in living cells in different cellular states. We find that the majority of Argonaute is distributed diffusely in the cytoplasm, and, when cells are subjected to stress, Argonaute proteins accumulate to newly assembled structures known as stress granules (SGs) in addition to PBs. Argonaute proteins displayed distinct kinetics at different structures: exchange faster at SGs and much slower at PBs. Further, miRNAs are required for the Argonaute protein localization to SGs but not PBs. These quantitative kinetic data provide insights into miRNA-mediated repression.

Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion.

Abstract: The multiple isoforms of the transmembrane glycoprotein CD44 are produced by alternative RNA splicing. Expression of CD44 isoforms containing variable 5 exon (v5) correlates with enhanced malignancy and invasiveness of some tumors. Here we demonstrate that SRm160, a splicing coactivator, regulates CD44 alternative splicing in a Ras-dependent manner. Overexpression of SRm160 stimulates inclusion of CD44 v5 when Ras is activated. Conversely, small interfering RNA (siRNA)-mediated silencing of SRm160 significantly reduces v5 inclusion. Immunoprecipitation shows association of SRm160 with Sam68, a protein that also stimulates v5 inclusion in a Ras-dependent manner, suggesting that these two proteins interact to regulate CD44 splicing. Importantly, siRNA-mediated depletion of CD44 v5 decreases tumor cell invasion. Reduction of SRm160 by siRNA transfection downregulates the endogenous levels of CD44 isoforms, including v5, and correlates with a decrease in tumor cell invasiveness.

A positive feedback loop couples Ras activation and CD44 alternative splicing

Abstract: The Ras signaling pathway is important in both cell proliferation and tumor progression. Alternatively spliced isoforms of CD44 containing variable exon 6 (v6) can serve as coreceptors for growth factor receptors that activate Ras. Here we use v6-specific small interfering RNA (siRNA) to investigate the role of CD44 alternative splicing in Ras signaling. We identify a positive feedback loop in which Ras signaling promotes CD44v6 splicing, and CD44v6 then sustains late Ras signaling, which is important for cell cycle progression. These results are the first demonstration of a positive feedback loop linking signaling-dependent alternative splicing to mitogenic progression.

Function and localization of microRNAs in mammalian cells.

Abstract: microRNAs (miRNAs) represent a large set of master regulators of gene expression. They constitute 1‐4% of human genes and probably regulate 30% of protein-encoding genes. These small regulatory RNAs act at a posttranscriptional level—mediating translational repression and/or mRNA degradation—through their association with Argonaute protein and target mRNAs. In this paper, we discuss various mechanisms by which miRNAs regulate posttranscriptionally, including their subcellular localization. Recent results indicate that the majority of miRNA-targeted and thus translationally repressed mRNA is probably distributed in the diffuse cytoplasm, even though a small fraction is concentrated in subcellular compartments, such as processing bodies or stress granules; notably, the stress granule localization of Argonaute depends on the presence of miRNAs. Here we discuss the structural requirement of these subcellular compartments in light of their potential miRNA functions.

Characterization of the short RNAs bound by the P19 suppressor of RNA silencing in mouse embryonic stem cells.

Abstract: Studies of mammalian RNA interference (RNAi) have focused largely on the actions of microRNAs; however, in other organisms, endogenous short-interfering RNAs (siRNAs) are involved in silencing processes. To date, similar molecules have been difficult to characterize in mammalian cells. P19 is a plant suppressor of RNA silencing that binds with high affinity to siRNAs. Here, the short RNAs bound by P19 in mouse embryonic stem (ES) cells have been characterized. We show that P19 selectively immunoprecipitates endogenous short RNAs from ES cells. Cloning of immunoprecipitated RNA reveals a strong selection for short RNAs that are exact matches to ribosomal RNA (rRNA), with particular short rRNA species highly enriched in P19 immunoprecipitates. Complementary strands to the enriched rRNAs were not cloned, which was surprising because P19 was previously thought to bind only siRNAs. We show that P19 binds tightly to a noncanonical dsRNA substrate comprised of a short RNA annealed to a much longer partner, such that the double-stranded region between the two is 19 base pairs long. Binding to similar endogenous species might explain the association of P19 with short rRNAs in ES cells. Finally, we show that the P19-enriched rRNAs are not involved in canonical RNAi, as they exist in the absence of Dicer and do not function as post-transcriptional gene silencers. Our results support the previous observation that endogenous siRNAs are not abundant molecules in mouse ES cells.

Non-haem iron transport in the rat proximal colon

Abstract: Background Only 10% of dietary iron is absorbed in the duodenum which implies that 90% (approximately 9 mg day−1) reaches the lower small intestine and colon. Therefore the purpose of this study was to assess the iron transport capacity of the rat proximal colon and to determine whether iron absorption is regulated by changes in dietary iron content. Materials and methods Rats were fed for 14 days on either iron adequate (44 mg Fe kg−1 diet) or iron-deficient (< 0·5 mg Fe kg−1 diet) diets. The 59Fe transport across the colonic epithelium and its subsequent appearance in the blood were measured in vivo. In separate studies the colon was excised and used to measure divalent metal transporter expression. Results Divalent metal transporter (DMT1) was expressed at the apical membrane of the surface epithelium in rat proximal colon. In animals fed an iron-deficient diet, DMT1 mRNA and protein expression were increased. This was accompanied by a significant increase in tissue 59Fe uptake. Conclusions The proximal colon can absorb non-haem iron from the intestinal lumen. The purpose of this mechanism remains to be elucidated.

Analysis, Optimization and Probabilistic Assessment of an Airbag Landing System for the ExoMars Space Mission

Abstract: Vented airbag systems offer an attractive means of cushioning the landing impact of robotic planetary spacecraft. This type of airbag absorbs the impact kinetic energy by exhausting the inflation gas through vent patches in a controlled way that aims to bring the lander to rest with minimum rebound, limited deceleration and in an upright attitude. Such systems are characterised by highly non-linear behaviour. This, coupled with the difficulty of adequate terrestrial testing results in an analytical approach to design that relies on explicit finite element (FE) analysis. However, the simulation of an impact of a few tenths of a second duration typically requires tens of hours of CPU time, making it impractical to optimise a design using a trial end error approach and to perform the large number of analysis runs necessary for a probabilistic assessment of varied landing conditions. This paper presents a methodology for overcoming these problems with reference to a vented airbag design for the ESA ExoMars mission. The approach utilises the Moving Least Squares Method (MLSM) to fit high quality approximations to multi-dimensional response surfaces from a relatively small number of FE analysis runs. This method is well-adapted to highly non-linear and noisy response surfaces that are typical for this problem. The surrogate response surfaces were used to locate an optimum in the design parameter space and to perform 10,000 sample point Monte Carlo runs in a probabilistic assessment of reliability due to varying landing conditions.

19 The Biology of Short RNAs

Abstract: The RNA World is commonly discussed in the context of RNA catalysis. During this ancient stage of biology, however—where the genetic material consisted of RNA and many processes were catalyzed by RNA—RNA was almost certainly also the major regulatory molecule controlling both the flow of information and the rate of catalytic processes. Elsewhere in this volume, RNA is described as regulating many common processes such as translation, RNA splicing, and protein functions. These regulatory interactions use a combination of RNA features—primary sequence, secondary and tertiary structure—for recognition of other components for regulation. Given the complexity of known RNA molecules involved in regulation, it was a major surprise when simple RNAs of only about 22 nucleotides in length were found to have many general functions in the regulation of biological systems. These short RNAs are derived from double-stranded RNA (dsRNA) precursors, are recognized by multicomponent machinery in cells, and direct the regulation of gene expression through their primary sequence. The discovery of these RNA interference (RNAi)-related processes have radically changed concepts about the nature of regulatory factors in organisms. In theory, any linear sequence of RNA can be converted into a trans -acting regulatory factor by small RNA regulatory mechanisms. Small RNAs are now known to regulate expression of information in DNA sequences at the level of mRNA stability, mRNA translation, and transcription of RNA. Although it is possible that these processes utilizing short RNAs had their origins in the RNA World, it seems more likely that these...