The Expression of Cold-Inducible RNA-Binding Protein mRNA in Sow Genital Tract Is Modulated by Natural Mating, But Not by Seminal Plasma.

The RNA-binding proteins (RBPs), some of them induced by transient receptor potential (TRP) ion channels, are crucial regulators of RNA function that can contribute to reproductive pathogenesis, including inflammation and immune dysfunction. This study aimed to reveal the influence of spermatozoa, seminal plasma, or natural mating on mRNA expression of RBPs and TRP ion channels in different segments of the internal genital tract of oestrous, preovulatory sows. Particularly, we focused on mRNA expression changes of the cold-inducible proteins (CIPs) and related TRP channels. Pre-ovulatory sows were naturally mated (NM) or cervically infused with semen (Semen-AI) or sperm-free seminal plasma either from the entire ejaculate (SP-TOTAL) or the sperm-rich fraction (SP-AI). Samples (cervix to infundibulum) were collected by laparotomy under general anaesthesia for transcriptomic analysis (GeneChip® Porcine Gene 1.0 ST Array) 24 h after treatments. The NM treatment induced most of the mRNA expression changes, compared to Semen-AI, SP-AI, and SP-TOTAL treatments including unique significative changes in CIRBP, RBM11, RBM15B, RBMS1, TRPC1, TRPC4, TRPC7, and TRPM8. The findings on the differential mRNA expression on RBPs and TRP ion channels, especially to CIPs and related TRP ion channels, suggest that spermatozoa and seminal plasma differentially modulated both protein families during the preovulatory phase, probably related to a still unknown early signalling mechanism in the sow reproductive tract.

Transcriptome profiling reveals the molecular processes for survival of Lysinibacillus fusiformis strain 15-4 in petroleum environments.

A bacterial strain designated Lysinibacillus fusiformis 15-4 was isolated from oil-free soil on the Qinghai-Tibet Plateau, which can grow well utilizing petroleum hydrocarbons as a carbon source at a lower temperature. To deeply characterize the molecular adaptations and metabolic processes of this strain when grown in a petroleum-containing environment, transcriptome analysis was performed. A total of 4664 genes and the expression of 3969 genes were observed in strain 15-4. When the strain was grown in petroleum-containing medium, 2192 genes were significantly regulated, of which 1312 (60%) were upregulated and 880 (40%) were downregulated. This strain degraded and adapted to petroleum via modulation of diverse molecular processes, including improvements in transporter activity, oxidoreductase/dehydrogenase activity, two-component system/signal transduction, transcriptional regulation, fatty acid catabolism, amino acid metabolism, and environmental stress responses. Many strain-specific genes were involved in the oxidation of hydrocarbon compounds, such as several luciferase family alkane monooxygenase genes, flavin-utilizing monooxygenase family genes, and flavoprotein-like family alkanesulfonate monooxygenase genes. Several cold shock protein genes were also induced suggesting adaptation to cold environments and the potential for petroleum degradation at low temperatures. The results obtained in this study may broaden our understanding of molecular adaptation of bacteria to hydrocarbon-containing environments and may provide valuable data for further study of L. fusiformis.

Cold shock protein RBM3 attenuates atrophy and induces hypertrophy in skeletal muscle.

RNA-binding motif protein 3 (RBM3), a stress-inducible RNA-binding protein that increases protein synthesis and confers cell protection in multiple cell types, has been identified as a possible regulator of skeletal muscle mass. Therefore, the primary aim of this study was to examine the impact of elevated RBM3 on skeletal muscle hypertrophy and resistance to atrophy. Plasmid-mediated overexpression of RBM3 in vitro and in vivo was used to assess the role of RBM3 in muscle. C2C12 myotubes overexpressing RBM3 were approximately 1.6 times larger than non-transfected myotubes, suggesting a role for RBM3 in hypertrophy. In addition, elevated RBM3 attenuated atrophy in myotubes exposed to dexamethasone. In agreement with in vitro results, overexpression of RBM3 in soleus muscle of F344/BN rats using electroporation techniques increased the cross sectional area of muscle fibers. Overexpression of RBM3 also attenuated muscle atrophy in rat soleus muscle undergoing disuse atrophy. These findings provide direct evidence for a novel role of RBM3 in inducing hypertrophy as well as attenuating atrophy.

In silico identification and in vivo characterization of small molecule therapeutic hypothermia mimetics.

Hypothermia has been proved to have a beneficial effect on several pathologies. CIRBP is one of the so termed cold-shock proteins involved in this process. In this work, we have detected small molecules capable of modulating the activity of CIRBP in the absence of a cold stimulus, by High Throughput Virtual Screening (HTVS) of the Diversity Set IV of the NCI and 15 compounds of our in-house data base. Fifteen compounds were selected from the HTVS to carry out a second screening through a cell-based Western blot assay. This assay, together with molecular modeling studies allowed us to select compound zr17-2 for an in vivo experiment, which showed an interesting increase of CIRBP expression in several organs of experimental animals. Therefore, we have demonstrated that the effect of hypothermia can be mimicked by small molecules, which can be developed as first-in-class new drugs for the treatment of several diseases.

The Small-RNA Profiles of Almond (Prunus dulcis Mill.) Reproductive Tissues in Response to Cold Stress.

Spring frost is an important environmental stress that threatens the production of Prunus trees. However, little information is available regarding molecular response of these plants to the frost stress. Using high throughput sequencing, this study was conducted to identify differentially expressed miRNAs, both the conserved and the non-conserved ones, in the reproductive tissues of almond tolerant H genotype under cold stress. Analysis of 50 to 58 million raw reads led to identification of 174 unique conserved and 59 novel microRNAs (miRNAs). Differential expression pattern analysis showed that 50 miRNA families were expressed differentially in one or both of almond reproductive tissues (anther and ovary). Out of these 50 miRNA families, 12 and 15 displayed up-regulation and down-regulation, respectively. The distribution of conserved miRNA families indicated that miR482f harbor the highest number of members. Confirmation of miRNAs expression patterns by quantitative real- time PCR (qPCR) was performed in cold tolerant (H genotype) alongside a sensitive variety (Sh12 genotype). Our analysis revealed differential expression for 9 miRNAs in anther and 3 miRNAs in ovary between these two varieties. Target prediction of miRNAs followed by differential expression analysis resulted in identification of 83 target genes, mostly transcription factors. This study comprehensively catalogued expressed miRNAs under different temperatures in two reproductive tissues (anther and ovary). Results of current study and the previous RNA-seq study, which was conducted in the same tissues by our group, provide a unique opportunity to understand the molecular basis of responses of almond to cold stress. The results can also enhance the possibility for gene manipulation to develop cold tolerant plants.

Activities of the peptidyl transferase center of ribosomes lacking protein L27.

The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome.

Cold shock CspA and CspB protein production during periodic temperature cycling in Escherichia coli.

BACKGROUND: Temperature is an important environmental factor which can dramatically affect biochemical processes in bacteria. Temperatures above optimal cause heat shock, while low temperatures induce cold shock. Since the physiological response of the bacterium Escherichia coli to slow temperature fluctuation is not well known, we investigated the effect of periodic temperature cycling between 37° and 8°C with a period of 2 h on proteome profile, cold shock CspA and CspB protein and gene production. RESULTS: Several proteins (i.e. succinyl-CoA synthetase subunit alpha, periplasmic oligopeptide-binding protein, maltose-binding periplasmic protein, outer membrane porin protein, flavodoxin-1, phosphoserine aminotransferase) were up or down regulated during temperature cycling, in addition to CspA and CspB production. The results indicate that transcription of cspA and cspB increased during each temperature downshift and consistently decreased after each temperature upshift. In sharp contrast CspA-FLAG and CspB-FLAG protein concentrations in the cell increased during the first temperature down-shift and remained unresponsive to further temperature fluctuations. The proteins CspA-FLAG and CspB-FLAG were not significantly degraded during the temperature cycling. CONCLUSION: The study demonstrated that slow periodic temperature cycling affected protein production compared to cells constantly incubated at 37°C or during classical cold shock. Bacterial cspA and cspB mRNA transcript levels fluctuated in synchrony with the temperature fluctuations. There was no corresponding pattern of CspA and CspB protein production during temperature cycling.

[Cold shock domain proteins in the extremophyte Thellungiella salsuginea (salt cress): gene structure and differential response to cold].

Four genes encoding cold shock domain (CSD) proteins have been identified in salt cress [Thellungiella salsuginea (halophila), an extremophyte currently recognized as a promising model for studying stress tolerance]. The deduced proteins prove highly homologous to those of Arabidopsis thaliana (up to 95% identity) and are accordingly enumerated TsCSDP1-TsCSDP4; after the N-proximal conserved CSD, they have respectively 6, 2, 7, and 2 zinc finger motifs evenly spaced by Gly-rich stretches. Much lower similarity (approximately 45%) is observed in the regions upstream of TATA-box promoters of TsCSDP1 vs. AtCSP1, with numerous distinctions in the sets of identifiable cis-regulatory elements. Plasmid expression of sCSDP1 rescues a cold-sensitive cup-lacking mutant of Escherichia coli, confirming that the protein is functional. In leaves of salt cress plants under normal conditions, the mRNA levels for the four TsCSDPs relate as 10: 27: 1: 31. Chilling to 4 degrees C markedly alters the gene expression; the 4-day dynamics are different for all four genes and quite dissimilar from those reported for their Arabidopsis homologues under comparable conditions. Thus, the much greater cold hardiness of Thellungiella vs. Arabidopsis cannot be explained by structural distinctions of its CSDPs, but rather may be due to expedient regulation of their expression at low temperature.