LSm1-7 complexes bind to specific sites in viral RNA genomes and regulate their translation and replication

LSm1-7 complexes promote cellular mRNA degradation, in addition to translation and replication of positive-strand RNA viruses such as the Brome mosaic virus (BMV). Yet, how LSm1-7 complexes act on their targets remains elusive. Here, we report that reconstituted recombinant LSm1-7 complexes directly bind to two distinct RNA-target sequences in the BMV genome, a tRNA-like structure at the 3′-untranslated region and two internal A-rich single-stranded regions. Importantly, in vivo analysis shows that these sequences regulate the translation and replication of the BMV genome. Furthermore, both RNA-target sequences resemble those found for Hfq, the LSm counterpart in bacteria, suggesting conservation through evolution. Our results provide the first evidence that LSm1-7 complexes interact directly with viral RNA genomes and open new perspectives in the understanding of LSm1-7 functions.     

Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I

Mammalian cleavage factor I (CF I_m) is composed of two polypeptides of 25 kDa and either a 59 or 68 kDa subunit (CF I_m25, CF I_m59, CF I_m68). It is part of the cleavage and polyadenylation complex responsible for processing the 3′ ends of messenger RNA precursors. To investigate post-translational modifications in factors of the 3′ processing complex, we systematically searched for enzymes that modify arginines by the addition of methyl groups. Protein arginine methyltransferases (PRMTs) are such enzymes that transfer methyl groups from S-adenosyl methionine to arginine residues within polypeptide chains resulting in mono- or dimethylated arginines. We found that CF I_m68 and the nuclear poly(A) binding protein 1 (PABPN1) were methylated by HeLa cell extracts in vitro. By fractionation of these extracts followed by mass spectral analysis, we could demonstrate that the catalytic subunit PRMT5, together with its cofactor WD45, could symmetrically dimethylate CF I_m68, whereas pICln, the third polypeptide of the complex, was stimulatory. As sites of methylation in CF I_m68 we could exclusively identify arginines in a GGRGRGRF or “GAR” motif that is conserved in vertebrates. Further in vitro assays revealed a second methyltransferase, PRMT1, which modifies CF I_m68 by asymmetric dimethylation of the GAR motif and also weakly methylates the C-termini of both CF I_m59 and CF I_m68. The results suggest that native—as compared with recombinant—protein substrates may contain additional determinants for methylation by specific PRMTs. A possible involvement of CF I_m methylation in the context of RNA export is discussed.     

RioK1, a new interactor of protein arginine methyltransferase 5 (PRMT5), competes with pICln for binding and modulates PRMT5 complex composition and substrate specificity

Protein arginine methylation plays a critical role in differential gene expression through modulating protein-protein and protein-DNA/RNA interactions. Although numerous proteins undergo arginine methylation, only limited information is available on how protein arginine methyltransferases (PRMTs) identify their substrates. The human PRMT5 complex consists of PRMT5, WD45/MEP50 (WD repeat domain 45/methylosome protein 50), and pICln and catalyzes the symmetrical arginine dimethylation of its substrate proteins. pICln recruits the spliceosomal Sm proteins to the PRMT5 complex for methylation, which allows their subsequent loading onto snRNA to form small nuclear ribonucleoproteins. To understand how the PRMT5 complex is regulated, we investigated its biochemical composition and identified RioK1 as a novel, stoichiometric component of the PRMT5 complex. We show that RioK1 and pICln bind to PRMT5 in a mutually exclusive fashion. This results in a PRMT5-WD45/MEP50 core structure that either associates with pICln or RioK1 in distinct complexes. Furthermore, we show that RioK1 functions in analogy to pICln as an adapter protein by recruiting the RNA-binding protein nucleolin to the PRMT5 complex for its symmetrical methylation. The exclusive interaction of PRMT5 with either pICln or RioK1 thus provides the first mechanistic insight into how a methyltransferase can distinguish between its substrate proteins.     

Prematurely induced arousal from hibernation alters key aspects of warming in golden-mantled ground squirrels, Callospermophilus lateralis

Hibernation researchers have long been interested in the transitions between the dissimilar states of torpor and euthermy. Natural arousal from torpor occurs spontaneously with highly predictable timing. However, animals can also be induced to arouse prematurely in response to various disturbances. While many investigations have used natural and induced arousals synonymously, direct comparisons of these two types of arousal have been limited. We address the question of whether natural and prematurely induced arousals generate the same patterns of warming at the level of the whole organism. We compare the effects of ambient temperature on the dynamics of natural versus induced arousals. Arousal duration, maximum rewarming rate, and the variance associated with increases in body temperature differed between natural and induced arousals. Prematurely inducing arousal also alters the duration of the interbout aroused (IBA) period. We recommend that careful consideration be given to experimental design and data interpretation related to the arousal phase of a torpor bout.     

Hematopoiesis and the inosine modification in transfer RNA

Human promyelocytic leukemia (HL-60) cells were used to begin to evaluate the role in hematopoiesis of inosine biosynthesis in the tRNA anticodon wobble position; a reaction involving the enzymatic insertion of performed hypoxanthine. Dimethyl sulfoxide (DMSO) and hypoxanthine were found to induce the differentiation of HL-60 cells in a synergistic manner, and the induced differentiation was independent of changes in the purine catabolic enzymes adenosine deaminase and purine nucleoside phosphorylase. The short-term exposure of HL-60 cells to DMSO plus hypoxanthine resulted in enhanced leucine incorporation, and a model is presented showing how the inosine modification reaction in tRNA may be involved. A means by which hypoxanthine insertion into tRNA may modulate the synthesis of regulatory proteins (e.g., lymphokines and cell surface receptors) is also outlined.     

Design and testing of a novel measuring system for use in dental biomechanics--principles and examples of measurements with the hexapod measuring system]

A novel measuring set-up based on a hexapod system for use in dental biomechanics is described. It was specially developed to measure force/deflection characteristics of different dental materials and devices. The functionability and suitability of the system for use in experimental biomechanics were investigated in two different studies. In a first study the micro mobility of prosthetic telescopic crowns prior to and after simulated wear was determined to investigate the influence of wear processes on the stability of the anchorage elements and thus of prostheses. This study investigated the ability of the setup to load a specimen with high forces or torques of up to 100 Newton. The second study looked at the force/deflection characteristics of orthodontic anchorage pins used in orthodontics to additionally stabilize the anchorage unit, for example during molar movement. In this study specimens were loaded with small forces of less than 10 Newton, as are typically used in orthodontics. Using the setup, the deflection behaviour of these devices under high and low loading was measured at a resolution of approximately one micrometer or one angular second.     

Effects of quinine on K+ transport in heart mitochondria

Quinine inhibits the respiration-dependent extrusion of K⁺ from Mg²⁺-depleted heart mitochondria and the passive osmotic swelling of these mitochondria in K⁺ and Na⁺ acetate at alkaline pH. These observations concur with those of Nakashima and Garlid (J. Biol. Chem. 257, 9252, 1982) using rat liver mitochondria. Quinine also inhibits the respiration-dependent contraction of heart mitochondria swollen passively in Na⁺ or K⁺ nitrate and the increment of elevated respiration associated with the extrusion of ions from these mitochondria. Quinine, at concentrations up to 0.5 mM, inhibits the respiration-dependent⁴²K⁺/K⁺ exchange seen in the presence of mersalyl, but higher levels of the drug produce increased membrane permeability and net K⁺ loss from the matrix. These results are all consistent with an inhibition of the putative mitochondrial K⁺/H⁺ antiport by quinine. However, quinine has other effects on the mitochondrial membrane, and possible alternatives to this interpretation are discussed.     

Anti-Insulin Immune Responses Are Detectable in Dogs with Spontaneous Diabetes

Diabetes mellitus occurs spontaneously in dogs. Although canine diabetes shares many features with human type-1 diabetes, there are differences that have cast doubt on the immunologic origin of the canine disease. In this study, we examined whether peripheral immune responses directed against islet antigens were present in dogs with diabetes. Routine diagnostics were used to confirm diabetic status, and serum samples from dogs with (N = 15) and without (N = 15) diabetes were analyzed for the presence of antibodies against islet antigens (insulin, glutamic acid decarboxylase, insulinoma-associated protein tyrosine phosphatase, and islet beta-cell zinc cation efflux transporter) using standard radioassays. Interferon-γ production from peripheral blood T cells stimulated by porcine insulin and by human insulin was tested using Elispot assays. Anti-insulin antibodies were detectable in a subset of diabetic dogs receiving insulin therapy. Pre-activated T cells and incipient insulin-reactive T cells in response to porcine or human insulin were identified in non-diabetic dogs and in dogs with diabetes. The data show that humoral and cellular anti-insulin immune responses are detectable in dogs with diabetes. This in turn provides support for the potential to ethically use dogs with diabetes to study the therapeutic potential of antigen-specific tolerance.