Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Processing of procollagen III by meprins: new players in extracellular matrix assembly?

Meprins ? and ?, a subgroup of zinc metalloproteinases belonging to the astacin family, are known to cleave components of the extracellular matrix, either during physiological remodeling or in pathological situations. In this study we present a new role for meprins in matrix assembly, namely the proteolytic processing of procollagens. Both meprins ? and ? release the N- and C-propeptides from procollagen III, with such processing events being critical steps in collagen fibril formation. In addition, both meprins cleave procollagen III at exactly the same site as the procollagen C-proteinases, including bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family. Indeed, cleavage of procollagen III by meprins is more efficient than by BMP-1. In addition, unlike BMP-1, whose activity is stimulated by procollagen C-proteinase enhancer proteins (PCPEs), the activity of meprins on procollagen III is diminished by PCPE-1. Finally, following our earlier observations of meprin expression by human epidermal keratinocytes, meprin ? is also shown to be expressed by human dermal fibroblasts. In the dermis of fibrotic skin (keloids), expression of meprin ? increases and meprin ? begins to be detected. Our study suggests that meprins could be important players in several remodeling processes involving collagen fiber deposition.

Self-assembly of focal point oligo-catechol ethylene glycol dendrons on titanium oxide surfaces: adsorption kinetics, surface characterization, and nonfouling properties

This work covers the synthesis of second-generation, ethylene glycol dendrons covalently linked to a surface anchor that contains two, three, or four catechol groups, the molecular assembly in aqueous buffer on titanium oxide surfaces, and the evaluation of the resistance of the monomolecular adlayers against nonspecific protein adsorption in contact with full blood serum. The results were compared to those of a linear poly(ethylene glycol) (PEG) analogue with the same molecular weight. The adsorption kinetics as well as resulting surface coverages were monitored by ex situ spectroscopic ellipsometry (VASE), in situ optical waveguide lightmode spectroscopy (OWLS), and quartz crystal microbalance with dissipation (QCM-D) investigations. The expected compositions of the macromolecular films were verified by X-ray photoelectron spectroscopy (XPS). The results of the adsorption study, performed in a high ionic strength ("cloud-point") buffer at room temperature, demonstrate that the adsorption kinetics increase with increasing number of catechol binding moieties and exceed the values found for the linear PEG analogue. This is attributed to the comparatively smaller and more confined molecular volume of the dendritic macromolecules in solution, the improved presentation of the catechol anchor, and/or their much lower cloud-point in the chosen buffer (close to room temperature). Interestingly, in terms of mechanistic aspects of "nonfouling" surface properties, the dendron films were found to be much stiffer and considerably less hydrated in comparison to the linear PEG brush surface, closer in their physicochemical properties to oligo(ethylene glycol) alkanethiol self-assembled monolayers than to conventional brush surfaces. Despite these differences, both types of polymer architectures at saturation coverage proved to be highly resistant toward protein adsorption. Although associated with higher synthesis costs, dendritic macromolecules are considered to be an attractive alternative to linear polymers for surface (bio)functionalization in view of their spontaneous formation of ultrathin, confluent, and nonfouling monolayers at room temperature and their outstanding ability to present functional ligands (coupled to the termini of the dendritic structure) at high surface densities.

Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA

The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3' untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cdelta (PKCdelta), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCdelta-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCdelta represents an important novel mode of HuR activation implied in renal COX-2 regulation.

Microsomal triglyceride transfer protein polymorphism (-493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C

BACKGROUND: Hepatic steatosis may promote progression of chronic hepatitis C (CHC). Microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of ApoB lipoprotein and is implicated in hepatitis C virus (HCV)-related steatosis. The MTP -493G/T polymorphism may promote liver fat accumulation, but its role in HCV-related steatosis is still unclear. METHODS: Two hundred ninety-eight CHC patients were studied and genotyped for MTP -493G/T variants. Hepatic MTP mRNA expression and activity were determined in a subgroup. RESULTS: Patients with grades 2/3 steatosis were older, had a higher body mass index (BMI), more advanced fibrosis and lower MTP mRNA expression and carried more often HCV genotype 3 and the MTP T allele. Age, BMI, HCV-3 and MTP T allele [odds ratio (OR) 2.05; 95% confidence interval (CI) 1.2-3.53; P=0.009] were independent risk factors for steatosis grades 2/3, and in HCV genotype non-3 patients, the MTP T allele was the strongest predictor for steatosis grade 2/3 (OR 2.17; 95% CI 1.22-3.86; P=0.008). Moreover, TT carriers had higher high-density lipoprotein (65.6+/-14.6 vs 56.1+/-16.2 mg/dl; P=0.003) and apolipoprotein AI (1.80+/-0.3 vs 1.60+/-0.3 g/L; P=0.005) levels than G allele carriers. CONCLUSIONS: Chronic hepatitis C patients with the MTP -493T allele reveal higher grades of steatosis, indicating a relevant contribution to liver fat accumulation, particularly in HCV non-3 patients.

β1 integrins regulate myoblast fusion and sarcomere assembly

The mechanisms that regulate the formation of multinucleated muscle fibers from mononucleated myoblasts are not well understood. We show here that extracellular matrix (ECM) receptors of the beta1 integrin family regulate myoblast fusion. beta1-deficient myoblasts adhere to each other, but plasma membrane breakdown is defective. The integrin-associated tetraspanin CD9 that regulates cell fusion is no longer expressed at the cell surface of beta1-deficient myoblasts, suggesting that beta1 integrins regulate the formation of a protein complex important for fusion. Subsequent to fusion, beta1 integrins are required for the assembly of sarcomeres. Other ECM receptors such as the dystrophin glycoprotein complex are still expressed but cannot compensate for the loss of beta1 integrins, providing evidence that different ECM receptors have nonredundant functions in skeletal muscle fibers.

Toward an assembly line for U7 snRNPs: interactions of U7-specific Lsm proteins with PRMT5 and SMN complexes.

The survival of motor neurons (SMN) complex mediates the assembly of small nuclear ribonucleoproteins (snRNPs) involved in splicing and histone RNA processing. A crucial step in this process is the binding of Sm proteins onto the SMN protein. For Sm B/B', D1, and D3, efficient binding to SMN depends on symmetrical dimethyl arginine (sDMA) modifications of their RG-rich tails. This methylation is achieved by another entity, the PRMT5 complex. Its pICln subunit binds Sm proteins whereas the PRMT5 subunit catalyzes the methylation reaction. Here, we provide evidence that Lsm10 and Lsm11, which replace the Sm proteins D1 and D2 in the histone RNA processing U7 snRNPs, associate with pICln in vitro and in vivo without receiving sDMA modifications. This implies that the PRMT5 complex is involved in an early stage of U7 snRNP assembly and hence may have a second snRNP assembly function unrelated to sDMA modification. We also show that the binding of Lsm10 and Lsm11 to SMN is independent of any methylation activity. Furthermore, we present evidence for two separate binding sites in SMN for Sm/Lsm proteins. One recognizes Sm domains and the second one, the sDMA-modified RG-tails, which are present only in a subset of these proteins.

The Draft Assembly of the Radically Organized Stylonychia lemnae Macronuclear Genome.

Stylonychia lemnae is a classical model single-celled eukaryote, and a quintessential ciliate typified by dimorphic nuclei: A small, germline micronucleus and a massive, vegetative macronucleus. The genome within Stylonychia's macronucleus has a very unusual architecture, comprised variably and highly amplified "nanochromosomes," each usually encoding a single gene with a minimal amount of surrounding noncoding DNA. As only a tiny fraction of the Stylonychia genes has been sequenced, and to promote research using this organism, we sequenced its macronuclear genome. We report the analysis of the 50.2-Mb draft S. lemnae macronuclear genome assembly, containing in excess of 16,000 complete nanochromosomes, assembled as less than 20,000 contigs. We found considerable conservation of fundamental genomic properties between S. lemnae and its close relative, Oxytricha trifallax, including nanochromosomal gene synteny, alternative fragmentation, and copy number. Protein domain searches in Stylonychia revealed two new telomere-binding protein homologs and the presence of linker histones. Among the diverse histone variants of S. lemnae and O. trifallax, we found divergent, coexpressed variants corresponding to four of the five core nucleosomal proteins (H1.2, H2A.6, H2B.4, and H3.7) suggesting that these ciliates may possess specialized nucleosomes involved in genome processing during nuclear differentiation. The assembly of the S. lemnae macronuclear genome demonstrates that largely complete, well-assembled highly fragmented genomes of similar size and complexity may be produced from one library and lane of Illumina HiSeq 2000 shotgun sequencing. The provision of the S. lemnae macronuclear genome sets the stage for future detailed experimental studies of chromatin-mediated, RNA-guided developmental genome rearrangements.

Elevated temperature differentially affects virulence, VirB protein accumulation, and T-pilus formation in different Agrobacterium tumefaciens and Agrobacterium vitis strains.

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect. T-pilus assembly and virulence protein accumulation were monitored in Agrobacterium tumefaciens strain C58 at different temperatures ranging from 20 degrees C to growth-inhibitory 37 degrees C. Incubation at 28 degrees C but not at 26 degrees C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20 degrees C. Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins. Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28 degrees C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26 degrees C. Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20 degrees C and not at higher temperatures. Shift of virulence-induced agrobacteria from 20 to 28 or 37 degrees C had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different A. tumefaciens and Agrobacterium vitis strains grown at 20 and 28 degrees C. Levels of many virulence proteins were strongly reduced at 28 degrees C compared to 20 degrees C, and T-pilus assembly did not occur in all strains except "temperature-resistant" Ach5 and Chry5. Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.

Unique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component, Lsm11, in histone RNA processing.

A set of seven Sm proteins assemble on the Sm-binding site of spliceosomal U snRNAs to form the ring-shaped Sm core. The U7 snRNP involved in histone RNA 3' processing contains a structurally similar but biochemically unique Sm core in which two of these proteins, Sm D1 and D2, are replaced by Lsm10 and by another as yet unknown component. Here we characterize this factor, termed Lsm11, as a novel Sm-like protein with apparently two distinct functions. In vitro studies suggest that its long N-terminal part mediates an important step in histone mRNA 3'-end cleavage, most likely by recruiting a zinc finger protein previously identified as a processing factor. In contrast, the C-terminal part, which comprises two Sm motifs interrupted by an unusually long spacer, is sufficient to assemble with U7, but not U1, snRNA. Assembly of this U7-specific Sm core depends on the noncanonical Sm-binding site of U7 snRNA. Moreover, it is facilitated by a specialized SMN complex that contains Lsm10 and Lsm11 but lacks Sm D1/D2. Thus, the U7-specific Lsm11 protein not only specifies the assembly of the U7 Sm core but also fulfills an important role in U7 snRNP-mediated histone mRNA processing.

Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation

valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. We show that vls encodes a divergent WD domain protein and that the three available EMS-induced point mutations cause premature stop codons in the vls ORF. We have generated a null allele that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and our phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules.

Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes.

In Xenopus oocytes in vitro transcribed mouse U7 RNA is assembled into small nuclear ribonucleoproteins (snRNPs) that are functional in histone RNA 3' processing. If the special Sm binding site of U7 (AAUUUGUCUAG, U7 Sm WT) is converted into the canonical Sm sequence derived from the major snRNAs (AAUUUUUGGAG, U7 Sm OPT) the RNA assembles into a particle which accumulates more efficiently in the nucleus, but which is non-functional. U7 RNA with a heavily mutated Sm binding site (AACGCGUCAUG, U7 Sm MUT) is deficient in nuclear accumulation and function. By UV cross-linking U7 Sm WT RNA can be linked to three proteins, i.e. the common snRNP proteins G and B/B' and an apparently U7-specific protein of 40 kDa. As a result of altering the Sm binding site, U7 Sm OPT RNA cannot be cross-linked to the 40 kDa protein and no cross-links are obtained with U7 Sm MUT RNA. The fact that the Sm site also interacts with at least one U7-specific protein is so far unique to U7 RNA and may provide an explanation for the atypical sequence of this site. All described RNA-protein interactions, including that with the 40 kDa protein, already occur in the cytoplasm. An additional cytoplasmic photoadduct obtained with U7 Sm WT and U7 Sm OPT, but not U7 Sm MUT, RNAs is indicative of a protein of 60-80 kDa. The m7G cap structure of U7 Sm WT and U7 Sm OPT RNA becomes hypermethylated. However, the 3mG cap enhances, but is not required for, nuclear accumulation. Finally, U7 Sm WT RNA is functional in histone RNA processing even when bearing an ApppG cap.

C9ORF72 Regulates Stress Granule Formation and Its Deficiency Impairs Stress Granule Assembly, Hypersensitizing Cells to Stress

Hexanucleotide repeat expansions in the C9ORF72 gene are causally associated with frontotemporal lobar dementia (FTLD) and/or amyotrophic lateral sclerosis (ALS). The physiological function of the normal C9ORF72 protein remains unclear. In this study, we characterized the subcellular localization of C9ORF72 to processing bodies (P-bodies) and its recruitment to stress granules (SGs) upon stress-related stimuli. Gain of function and loss of function experiments revealed that the long isoform of C9ORF72 protein regulates SG assembly. CRISPR/Cas9-mediated knockdown of C9ORF72 completely abolished SG formation, negatively impacted the expression of SG-associated proteins such as TIA-1 and HuR, and accelerated cell death. Loss of C9ORF72 expression further compromised cellular recovery responses after the removal of stress. Additionally, mimicking the pathogenic condition via the expression of hexanucleotide expansion upstream of C9ORF72 impaired the expression of the C9ORF72 protein, caused an abnormal accumulation of RNA foci, and led to the spontaneous formation of SGs. Our study identifies a novel function for normal C9ORF72 in SG assembly and sheds light into how the mutant expansions might impair SG formation and cellular-stress-related adaptive responses.