Maf1 is an essential and specific mediator of transcriptional repression in the RNA polymerase (pol) III system. Maf1-dependent repression occurs in response to a wide range of conditions, suggesting that the protein itself is targeted by the major nutritional and stress-signaling pathways. We show that Maf1 is a substrate for cAMP-dependent PKA in vitro and is differentially phosphorylated on PKA sites in vivo under normal versus repressing conditions. PKA activity negatively regulates Maf1 function because strains with unregulated high PKA activity block repression of pol III transcription in vivo, and strains lacking all PKA activity are hyperrepressible. Nuclear accumulation of Maf1 is required for transcriptional repression and is regulated by two nuclear localization sequences in the protein. An analysis of PKA phosphosite mutants shows that the localization of Maf1 is affected via the N-terminal nuclear localization sequence. In particular, mutations that prevent phosphorylation at PKA consensus sites promote nuclear accumulation of Maf1 without inducing repression. These results indicate that negative regulation of Maf1 by PKA is achieved by inhibiting its nuclear import and suggest that a PKA-independent activation step is required for nuclear Maf1 to function in the repression of pol III transcription. Finally, we report a previously undescribed phenotype for Maf1 in tRNA genemediated silencing of nearby RNA pol II transcription.nuclear import ͉ phosphorylation ͉ tRNA biosynthesis T he action of all three nuclear RNA polymerases (pols) in the synthesis of rRNAs, ribosomal protein mRNAs, and tRNAs is coordinately regulated to control ribosome biogenesis and cell growth in response to nutrients and many other conditions (1). In Saccharomyces cerevisiae, Maf1 has been identified as an absolute and specific effector of repression in the pol III system (2). The diversity of conditions that signal repression, combined with the essential role of Maf1 in this process, suggests that the Maf1 protein is targeted by multiple signaling pathways.Genomewide localization of the pol III transcription apparatus has shown that nutrient deprivation and entry into stationary phase causes a significant decrease in polymerase occupancy on pol III genes (3, 4). This change is Maf1-dependent and is presumably a consequence of the direct interaction of Maf1 with the polymerase (5, 6). Consistent with this view, an in vitro system that recapitulates Maf1-dependent repression identified two steps that are inhibited as follows: polymerase recruitment to existing TFIIIB-DNA complexes and de novo assembly of the initiation factor TFIIIB onto DNA (5). In the latter step, Maf1 is thought to target the activity of TFIIIB via a direct interaction with one of its subunits, Brf1 (2, 5). However, the mechanism by which Maf1 inhibits TFIIIB-DNA assembly and transcription is not yet known.Maf1 is a phylogenetically conserved and structurally novel protein that lacks homology to any motifs of known function (6). However, three conserved doma...

In summary, MSC MVs increased alveolar fluid clearance and reduced lung protein permeability, and pretreatment with Poly (I:C) enhanced the antimicrobial activity of MVs in an ex vivo perfused human lung with severe bacteria pneumonia.

In Saccharomyces cerevisiae, Maf1 is essential for mediating the repression of transcription by RNA polymerase (pol) III in response to diverse cellular conditions. These conditions activate distinct signaling pathways that converge at or above Maf1. Thus, Maf1-dependent repression is thought to involve a common set of downstream inhibitory effects on the pol III machinery. Here we provide support for this view and define two steps in Maf1-dependent transcriptional repression. We show that chlorpromazine (CPZ)-induced repression of pol III transcription is achieved by inhibiting de novo assembly of transcription factor (TF) IIIB onto DNA as well as the recruitment of pol III to preassembled TFIIIB⅐DNA complexes. Additionally Brf1 was identified as a target of repression in extracts of CPZ-treated cells. Maf1-Brf1 and Maf1-pol III interactions were implicated in the inhibition of TFIIIB⅐DNA complex assembly and polymerase recruitment by recombinant Maf1. Co-immunoprecipitation experiments confirmed these interactions in yeast extracts and demonstrated that Maf1 does not differentially sequester Brf1 or pol III under repressing conditions. The results suggest that Maf1 functions by a non-stoichiometric mechanism to repress pol III transcription.Transcription of the large rRNAs by RNA polymerase (pol) 1 I and of 5 S rRNA and tRNAs by pol III is tightly co-regulated under essentially all conditions (1-3). This coordinate regulation is biologically important as it is conserved in all eukaryotes where transcription by pols I and III has been examined. The principal evolutionary imperatives that are thought to underlie this conserved regulation are the common function of rRNAs and tRNA in protein synthesis and the high energetic cost of their synthesis, which accounts for about 80% of nuclear gene transcription in actively growing cells (1, 3). The levels of pol I and pol III transcription are critical determinants of cell growth rate, and the deregulation of this transcription is a hallmark of cell transformation and tumorigenesis (4, 5). In addition, for single cell eukaryotes whose biological niche exposes them to periods when nutrients are in short supply and/or harsh environmental conditions, the ability to rapidly shut off the synthesis of rRNAs and tRNA is thought to be of vital importance for achieving metabolic economy (6) and hence is likely to impact cell survival.In higher eukaryotes, p53 and Rb and its relatives p107 and p130 play an important role in controlling pol I and pol III transcription and in coordinating the production of new protein synthetic capacity with cell proliferation (2, 5). These repressors function by binding directly to components of the pol I and pol III transcription machinery and thereby prevent proteinprotein interactions required for transcription. Specific components of this machinery are also substrates for phosphorylation by various kinases including extracellular signal-regulated kinase, casein kinase II, and cyclin-dependent kinases, which can either activate or repress tran...

Our previous study demonstrated that mesenchymal stem cell (MSC) microvesicles (MV) reduced lung inflammation, protein permeability, and pulmonary edema in endotoxin‐induced acute lung injury in mice. However, the underlying mechanisms for restoring lung protein permeability were not fully understood. In this current study, we hypothesized that MSC MV would restore protein permeability across injured human lung microvascular endothelial cells (HLMVEC) in part through the transfer of angiopoietin‐1 (Ang1) mRNA to the injured endothelium. A transwell coculture system was used to study the effect of MSC MV on protein permeability across HLMVECs injured by cytomix, a mixture of IL‐1β, TNF‐α, and IFN‐γ (50 ng/ml). Our result showed that cytomix significantly increased permeability to FITC‐dextran (70 kDa) across HLMVECs over 24 hours. Administration of MSC MVs restored this permeability in a dose dependent manner, which was associated with an increase in Ang1 mRNA and protein secretion in the injured endothelium. This beneficial effect was diminished when MSC MV was pretreated with an anti‐CD44 antibody, suggesting that internalization of MV into the HLMVEC was required for the therapeutic effect. Fluorescent microscopy showed that MSC MV largely prevented the reorganization of cytoskeleton protein F‐actin into “actin stress fiber” and restored the location of the tight junction protein ZO‐1 and adherens junction protein VE‐cadherin in injured HLMVECs. Ang1 siRNA pretreatment of MSC MV prior to administration to injured HLMVECs eliminated the therapeutic effect of MV. In summary, MSC MVs restored protein permeability across HLMVEC in part by increasing Ang1 secretion by injured HLMVEC. Stem Cells Translational Medicine 2018;7:615–624

Hot water extract prepared from the mycelial culture of mushroom Phellinus linteus stimulated polyclonal antibody production in an in vitro culture system. The active fraction PLP was purified from the extract ca. 1030-fold by ethanol precipitation followed by DEAE-cellulose and gel permeation chromatography. PLP contained 13.2% (w/w) peptide and 82.5% (w/w) carbohydrate. About 6.8% (w/w) of the total carbohydrate was uronic acid. The molecular weight distribution of PLP was found to be nearly homogeneous (153 kDa) in gel permeation HPLC analysis. Neutral sugar composition analysis revealed Ara (7.5%), Xyl (3.7%), Glc (21.1%), Gal (24.1%) and Man (44.2%). Uronic acid was identified as a glucuronic acid by gas chromatography. Ten amino acids were detected and Asp and Glu were the major components. In our assay system, the half-maximal concentration of PLP for B-lymphocyte stimulation was ca. 3 micrograms/ml. Partial acid hydrolysis as well as sodium periodate treatment of PLP decreased the activity significantly, suggesting that both the full molecular size and the sugar moiety were essential. However, proteinase K treatment for up to 48 h did not affect the activity.