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.

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.

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.

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.

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.