In this Issue: Inflammation.

We live in a microbial world and are often confronted with infection and injury. Inflammation, the response that is unleashed to ward off these insults, has long been familiar to us. Indeed, the words that we still use to describe inflammation -redness and swelling with heat and pain -were coined in the first century A.D. by the Roman physician Cornelius Celsus: rubor et tumor cum calore et dolore. The word inflammation itself comes from the Latin inflammare: to set on fire. Research has taken us a long way from this first description of the overt symptoms of inflammation 2000 years ago to our current understanding of the molecular and cellular pathways that mediate the inflammatory process, regulate it, and lead to its resolution. We now know that inflammation can come in many forms and modalities depending on the context in which it is triggered, the trigger itself, and the tissues involved. Furthermore, we appreciate the contribution of inflammation to cancer and to chronic afflictions like neurodegenerative diseases and diabetes. In this year's Special Review Issue, we bring together Review articles and Essays that survey our current knowledge of inflammation and provide perspectives on the key questions and challenges ahead. We would like to thank the distinguished experts who contributed their time and effort as authors and reviewers to make this issue timely, comprehensive,and thought provoking. We hope that this collection of articles will be informative and inspiring, as we now harness the tools of modern biology in the continued effort to understand :"the old flame".

Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation.

MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.

Protein kinase Ctheta focusing at the cSMAC is a consequence rather than cause of TCR signaling and is dependent on the MEK/ERK pathway.

Correlation between protein kinase Ctheta focusing within the central supramolecular activation cluster (cSMAC) of the immunological synapse and optimal TCR/costimulatory receptor ligation was interpreted to imply that PKCtheta focusing is required for productive signaling. However, this notion has been called into question and competing data suggest that the cSMAC contributes to receptor down-modulation. The observation that PKCtheta focusing at the cSMAC is promoted by CD28 coligation, and also that it occurs late after proximal tyrosine phosphorylation events have been initiated, has led us to investigate an alternative possibility that PKCtheta focusing might be a consequence rather than a cause of productive integrated signaling. Indeed, we found that inhibition of the downstream signaling molecules MEK and PI3K (but not of calcineurin, NF-kappaB, JNK, or p38 MAPK) significantly prevented the focusing of PKCtheta at the cSMAC. It recently has been suggested that the cSMAC may be associated with TCR degradation and signal termination. Using MEK inhibition as a tool, we observed that absence of detectable PKCtheta focusing had no significant effect on TCR down-modulation or duration of CD3zeta phosphorylation. Our results suggest that PKCtheta focusing at the cSMAC occurs as a consequence of productive integrated downstream signaling at least at the level of MEK. If PKCtheta focusing accurately reflects the cSMAC as a whole, then our data also argue against the cSMAC as being required for proximal TCR signal termination.

RNA. Introduction.

Two scientists walk into a bar. After a pint and an exchange of pleasantries, one says to the other, "Where do you come from? Scientifically, I mean." The queried scientist responds, "Out of the RNA world." "Don't we all," the asker responds chuckling. Fifteen years ago, the joke would have been made with a nod to the notion that life arose from an RNA-based precursor, the so-called "RNA world." Yet had this conversation happened last week, the scientists would also be grinning in appreciation of the extent to which contemporary cellular biology is steeped in all things RNA. Ours is truly an RNA world.In this year's special review issue, the Cell editorial team has brought together articles focused on RNA in the modern world, providing perspectives on classical and emerging areas of inquiry. We extend our thanks to the many distinguished experts who contributed their time and effort as authors and reviewers to make the issue informative, thought-provoking, and timely. We hope that this collection of articles, written as we stand on the verge of a new wave of RNA biology, edifies and inspires by revealing the inner workings of these versatile molecules and by highlighting the next key questions that need to be addressed as we strive to understand the full functional scope of RNA in cells.

A role for the P-body component GW182 in microRNA function.

In animals, the majority of microRNAs regulate gene expression through the RNA interference (RNAi) machinery without inducing small-interfering RNA (siRNA)-directed mRNA cleavage. Thus, the mechanisms by which microRNAs repress their targets have remained elusive. Recently, Argonaute proteins, which are key RNAi effector components, and their target mRNAs were shown to localize to cytoplasmic foci known as P-bodies or GW-bodies. Here, we show that the Argonaute proteins physically interact with a key P-/GW-body subunit, GW182. Silencing of GW182 delocalizes resident P-/GW-body proteins and impairs the silencing of microRNA reporters. Moreover, mutations that prevent Argonaute proteins from localizing in P-/GW-bodies prevent translational repression of mRNAs even when Argonaute is tethered to its target in a siRNA-independent fashion. Thus, our results support a functional link between cytoplasmic P-bodies and the ability of a microRNA to repress expression of a target mRNA.

Purified Argonaute2 and an siRNA form recombinant human RISC.

Genetic, biochemical and structural studies have implicated Argonaute proteins as the catalytic core of the RNAi effector complex, RISC. Here we show that recombinant, human Argonaute2 can combine with a small interfering RNA (siRNA) to form minimal RISC that accurately cleaves substrate RNAs. Recombinant RISC shows many of the properties of RISC purified from human or Drosophila melanogaster cells but also has surprising features. It shows no stimulation by ATP, suggesting that factors promoting product release are missing from the recombinant enzyme. The active site is made up of a unique Asp-Asp-His (DDH) motif. In the RISC reconstitution system, the siRNA 5' phosphate is important for the stability and the fidelity of the complex but is not essential for the creation of an active enzyme. These studies demonstrate that Argonaute proteins catalyze mRNA cleavage within RISC and provide a source of recombinant enzyme for detailed biochemical studies of the RNAi effector complex.

RNA-interference-based functional genomics in mammalian cells: reverse genetics coming of age.

Sequencing of complete genomes has provided researchers with a wealth of information to study genome organization, genetic instability, and polymorphisms, as well as a knowledge of all potentially expressed genes. The identification of all genes encoded in the human genome opens the door for large-scale systematic gene silencing using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs). With the recent development of siRNA and shRNA expression libraries, the application of RNAi technology to assign function to cancer genes and to delineate molecular pathways in which these genes affect in normal and transformed cells, will contribute significantly to the knowledge necessary to develop new and also improve existing cancer therapy.

Argonaute2 is the catalytic engine of mammalian RNAi.

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.

Actin cytoskeleton regulates calcium dynamics and NFAT nuclear duration.

T-cell activation by antigen-presenting cells is accompanied by actin polymerization, T-cell receptor (TCR) capping, and formation of the immunological synapse. However, whether actin-dependent events are required for T-cell function is poorly understood. Herein, we provide evidence for an unexpected negative regulatory role of the actin cytoskeleton on TCR-induced cytokine production. Disruption of actin polymerization resulted in prolonged intracellular calcium elevation in response to anti-CD3, thapsigargin, or phorbol myristate acetate plus ionomycin, leading to persistent NFAT (nuclear factor of activated T cells) nuclear duration. These events were dominant, as the net effect of actin blockade was augmented interleukin 2 promoter activity. Increased surface expression of the plasma membrane Ca(2+) ATPase was observed upon stimulation, which was inhibited by cytochalasin D, suggesting that actin polymerization contributes to calcium export. Our results imply a novel role for the actin cytoskeleton in modulating the duration of Ca(2+)-NFAT signaling and indicate that actin dynamics regulate features of T-cell activation downstream of receptor clustering.

Differential Ras signaling via the antigen receptor and IL-2 receptor in primary T lymphocytes.

Ras can become activated via multiple distinct receptors in T lymphocytes. However, mechanistic studies of Ras signaling in normal T cells have been hampered by the lack of an efficient technology for gene transfer into resting post-thymic cells. We have overcome this limitation by utilizing adenoviral transduction of T cells from Coxsackie/adenovirus receptor transgenic mice. Unexpectedly, dominant negative Ras17N blocked activation of Ras and ERK in response to IL-2R engagement but not TCR/CD3 ligation. However, TCR-induced ERK activation was suppressed by inhibitors of PKC and PLC-gamma. This first biochemical study of DN Ras in normal quiescent T cells reveals a striking contrast in Ras signaling via two receptors, and suggests that the principal mechanism of TCR-induced Ras activation in normal T cells may be distinct from that utilized in T-lineage tumor cell lines.

Gene array and protein expression profiles suggest post-transcriptional regulation during CD8+ T cell differentiation.

Peripheral CD8(+) T cells circulate in a quiescent naive state until they are primed by specific antigen and differentiate into effector cells. In the effector state, CD8(+) T cells acquire cytolytic activity and produce increased levels of cytokines such as interferon-gamma. They also exhibit increased T cell receptor sensitivity, decreased CD28 dependence, and become inhibitable by CTLA-4 and other negative regulatory pathways. We hypothesized that one mechanism by which these two states are regulated is via differential expression of specific genes. To this end, basal gene expression profiles of naive and effector 2C TCR transgenic x RAG2(-/-) CD8(+) T cells were analyzed using Affymetrix arrays representing 11,000 genes. Of the 177 differentially expressed known genes, 68 were expressed at higher levels in effector cells, but 109 were more abundant in naive cells, supporting the notion that the naive state is not passive. Expression of genes related to metabolism, actin cytoskeletal dynamics, and effector function increased with priming, whereas expression of putative anti-proliferative genes decreased. Semiquantitative reverse transcription-PCR was utilized as a secondary validation for selected transcripts, and Western blot analysis was used to examine protein expression for molecules of interest. Surprisingly, for 24 genes examined, 12 showed discordant protein versus mRNA expression. In summary, our study indicates that: 1) not only does the expression of some genes in naive CD8(+) T cells become up-regulated upon priming, but the expression of other genes is down-regulated as well and 2) the complexities of T cell differentiation include regulation at the post-transcriptional level.