The TRRAP transcription cofactor represses interferon-stimulated genes in colorectal cancer cells.

Transcription is essential for cells to respond to signaling cues and involves factors with multiple distinct activities. One such factor, TRRAP, functions as part of two large complexes, SAGA and TIP60, which have crucial roles during transcription activation. Structurally, TRRAP belongs to the phosphoinositide 3 kinase-related kinases (PIKK) family but is the only member classified as a pseudokinase.

The HSP90/R2TP assembly chaperone promotes cell proliferation in the intestinal epithelium.

The R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium and death within 10 days.

Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex.

RPAP3 and PIH1D1 are part of the HSP90 co-chaperone R2TP complex involved in the assembly process of many molecular machines. In this study, we performed a deep structural investigation of the HSP binding abilities of the two TPR domains of RPAP3. We combined 3D NMR, non-denaturing MS, and ITC techniques with Y2H, IP-LUMIER, FRET, and ATPase activity assays and explain the fundamental role played by the second TPR domain of RPAP3 in the specific recruitment of HSP90.

The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers.

Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control.

In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90-R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.

Drosophila Spag is the homolog of RNA polymerase II-associated protein 3 (RPAP3) and recruits the heat shock proteins 70 and 90 (Hsp70 and Hsp90) during the assembly of cellular machineries.

The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA(+)-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3.

HSP90 and the R2TP co-chaperone complex: building multi-protein machineries essential for cell growth and gene expression.

HSP90 (Heat Shock Protein 90) is an essential chaperone involved in the last folding steps of client proteins. It has many clients, and these are often recognized through specific adaptors. Recently, the conserved R2TP complex was identified as a key HSP90 co-chaperone.

CRM1 controls the composition of nucleoplasmic pre-snoRNA complexes to licence them for nucleolar transport.

Transport of C/D snoRNPs to nucleoli involves nuclear export factors. In particular, CRM1 binds nascent snoRNPs, but its precise role remains unknown. We show here that both CRM1 and nucleocytoplasmic trafficking are required to transport snoRNPs to nucleoli, but the snoRNPs do not transit through the cytoplasm.

HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II.

RNA polymerases are key multisubunit cellular enzymes. Microscopy studies indicated that RNA polymerase I assembles near its promoter. However, the mechanism by which RNA polymerase II is assembled from its 12 subunits remains unclear.

Massive translational repression of gene expression during mouse erythroid differentiation.

We took advantage of a mouse erythroid differentiation system to determine the relative contribution of transcriptional and translational control during this process. Comparison of expression data obtained with total cytoplasmic mRNAs or polysome-bound mRNAs (actively translated mRNAs) on Affymetrix high-density oligonucleotide microarrays revealed different characteristics of the two regulatory mechanisms. Indeed, mRNA expression from a vast majority of genes was affected, albeit most changes were relatively small and occurred at a low pace.

Cyclin B synthesis and rapamycin-sensitive regulation of protein synthesis during starfish oocyte meiotic divisions.

Translation of cyclin mRNAs represents an important event for proper meiotic maturation and post-fertilization mitoses in many species. Translational control of cyclin B mRNA has been described to be achieved through two separate but related mechanisms: translational repression and polyadenylation. In this paper, we evaluated the contribution of global translational regulation by the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-binding protein) on the cyclin B protein synthesis during meiotic maturation of the starfish oocytes.

The transcriptional cycle of HIV-1 in real-time and live cells.

RNA polymerase II (RNAPII) is a fundamental enzyme, but few studies have analyzed its activity in living cells. Using human immunodeficiency virus (HIV) type 1 reporters, we study real-time messenger RNA (mRNA) biogenesis by photobleaching nascent RNAs and RNAPII at specific transcription sites. Through modeling, the use of mutant polymerases, drugs, and quantitative in situ hybridization, we investigate the kinetics of the HIV-1 transcription cycle.

Nuclear envelope breakdown may deliver an inhibitor of protein phosphatase 1 which triggers cyclin B translation in starfish oocytes.

In vertebrates, enhanced translation of mRNAs in oocytes and early embryos entering M-phase is thought to occur through polyadenylation, involving binding, hyperphosphorylation and proteolytic degradation of Aurora-activated CPEB. In starfish, an unknown component of the oocyte nucleus is required for cyclin B synthesis following the release of G2/prophase block by hormonal stimulation. We have found that CPEB cannot be hyperphosphorylated following hormonal stimulation in starfish oocytes from which the nucleus has been removed.

TWE-PRIL; a fusion protein of TWEAK and APRIL.

TWEAK and APRIL are both members of the tumor necrosis factor family, which are involved in respectively angiogenesis and immune regulation. While TWEAK is processed at the cell surface, APRIL is processed inside the cell by a furin-convertase and is solely able to perform its function as a soluble factor. Recently, TWE-PRIL has been identified, which is an endogenous hybrid transcript between TWEAK and APRIL.

T cell activation in vivo targets diacylglycerol kinase alpha to the membrane: a novel mechanism for Ras attenuation.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid, leading to decreased and increased levels, respectively, of these two lipid messengers that play a central role in T cell activation. Nine DGK isoforms, grouped into five subtypes, are found in higher organisms; all contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. In this study, we have researched in vivo the regulation of DGK alpha, using a transgenic mouse model in which injection of an antigenic peptide activates the majority of peripheral T cells.

An endogenous hybrid mRNA encodes TWE-PRIL, a functional cell surface TWEAK-APRIL fusion protein.

TWEAK and APRIL are two recently identified tumour necrosis factor (TNF) ligand family members, implicated in angiogenesis and immune regulation, respectively. TWEAK is a transmembrane protein expressed on the cell surface, whereas APRIL acts solely as a secreted factor. In this report, using RACE, RT-PCR, cDNA library screening and an RNase protection assay, we characterize a hybrid transcript between TWEAK and APRIL mRNAs.

Regulation of mRNA expression in macrophages after Yersinia enterocolitica infection. Role of different Yop effectors.

The Yop virulon, which comprises a complete type III secretion system and secreted proteins, allows bacteria from the genus Yersinia to resist the nonspecific immune response of the host. This virulon, which is encoded by a plasmid called pYV in Yersinia enterocolitica, enables extracellular bacteria to inject six Yop effectors (YopE, -H, -T, -O, -P, -M) into the host cell. To investigate the role of YopP, YopM, and the other pYV-encoded factors on the expression of the host cell genes, we characterized the transcriptome alterations in infected mouse macrophages using the microarray technique.

Global and specific translational control by rapamycin in T cells uncovered by microarrays and proteomics.

Rapamycin has been shown to affect translation. We have utilized two complementary approaches to identify genes that are predominantly affected by rapamycin in Jurkat T cells. One was to compare levels of polysome-bound and total RNA using oligonucleotide microarrays complementary to 6,300 human genes.

Effects of rapeseed meal-glucosinolates on thyroid metabolism and feed utilization in rainbow trout.

Two rapeseed meals (RM1 and RM2), containing glucosinolates at a concentration of 26 and 40 micromol/g, respectively, were incorporated at increasing levels (10, 20, and 30% for RM1 and 30 and 50% for RM2) in diets of juvenile rainbow trout. Disturbances in the thyroid axis appeared after 14 days of feeding (with a dietary incorporation level of 10%). The dietary supplementation with T(3) or iodine induced an increase in plasma T(3) levels, compared to that in fish fed the RM diets, and reduced the deleterious effect of RM on growth.

Reliability of mRNA profiling: verification for samples with different complexities.

Normalization of mRNA profiling data remains an open issue, which turns critical when comparing divergent samples or mRNA populations with different complexities. To address this question, we generated samples with different RNA amounts and complexities by subcellular fractionation of cytoplasmic RNA into the mutually exclusive ribosome-free and polysome-bound RNA pools. For each of the 563 mRNAs analyzed, the hybridization signal corresponding to the cytoplasmic sample equals the sum of signals from the ribosome-free plus the polysome-bound targets (cytoplasmic mRNA = ribosome-free mRNA + polysome-bound mRNA).

Translation control: bridging the gap between genomics and proteomics?

mRNA profiling enables the expression levels of thousands of transcripts in a cell to be monitored simultaneously. Nevertheless, analyses in yeast and mammalian cells have demonstrated that mRNA levels alone are unreliable indicators of the corresponding protein abundances. This discrepancy between mRNA and protein levels argues for the relevance of additional control mechanisms besides transcription.

Isolation of translationally controlled mRNAs by differential screening.

Translationalregulation plays an important role in the control of gene expression. Changes in translation initiation rates are the most common translation-regulating mechanisms, resulting in alterations in mRNA loading of ribosomes. This differential mobilization of mRNAs onto polyribosomes was used in differential screening to directly identify cDNAs whose transcripts are translationally controlled during antigenic stimulation of primary human T lymphocytes.