Post-transcriptional Cleavage Research Articles

Full-Length Transcriptome Profiling of the Complete Mitochondrial Genome of Sericothrips houjii (Thysanoptera: Thripidae: Sericothripinae) Featuring Extensive Gene Rearrangement and Duplicated Control Regions.

The mitochondrial genome (mitogenome) of Thysanoptera has extensive gene rearrangement, and some species have repeatable control regions. To investigate the characteristics of the gene expression, transcription and post-transcriptional processes in such extensively gene-rearranged mitogenomes, we sequenced the mitogenome and mitochondrial transcriptome of Sericothrips houjii to analyze. The mitogenome was 14,965 bp in length and included two CRs contains 140 bp repeats between COIII-trnN (CR1) and trnT-trnP (CR2). Unlike the putative ancestral arrangement of insects, S. houjii exhibited only six conserved gene blocks encompassing 14 genes (trnL2-COII, trnD-trnK, ND2-trnW, ATP8-ATP6, ND5-trnH-ND4-ND4L and trnV-lrRNA). A quantitative transcription map showed the gene with the highest relative expression in the mitogenome was ND4-ND4L. Based on analyses of polycistronic transcripts, non-coding RNAs (ncRNAs) and antisense transcripts, we proposed a transcriptional model of this mitogenome. Both CRs contained the transcription initiation sites (TISs) and transcription termination sites (TTSs) of both strands, and an additional TIS for the majority strand (J-strand) was found within antisense lrRNA. The post-transcriptional cleavage processes followed the "tRNA punctuation" model. After the cleavage of transfer RNAs (tRNAs), COI and ND3 matured as bicistronic mRNA COI/ND3 due to the translocation of intervening tRNAs, and the 3' untranslated region (UTR) remained in the mRNAs for COII, COIII, CYTB and ND5. Additionally, isoform RNAs of ND2, srRNA and lrRNA were identified. In summary, the relative mitochondrial gene expression levels, transcriptional model and post-transcriptional cleavage process of S. houjii are notably different from those insects with typical mitochondrial gene arrangements. In addition, the phylogenetic tree of Thripidae including S. houjii was reconstructed. Our study provides insights into the phylogenetic status of Sericothripinae and the transcriptional and post-transcriptional regulation processes of extensively gene-rearranged insect mitogenomes.

Functional diversification of miR172 isoforms in tomato under abiotic stress

Plant gene families have expanded many folds as opposed to animals to compensate for being sessile as well as having unique features like ability to photosynthesise. While different protein families are well characterised in plants, similar knowledge on miRNA families is still in its infancy. The MIR172 family plays important role in various plant development processes including vegetative to reproductive phase change, floral patterning, nodulation, and fruit ripening as well as also in response to different environmental cues. However, in-depth analysis of this family in tomato (Solanum lycopersicum) is limited. In this study, we identified four new MIR172 loci (Sly-MIR172a1/a2/e/f) and two new isoforms, other than those reported at the miRBase repository. The MIR172 family has expanded by segmental duplication events and is conserved between the wild (S. pennellii and S. pimpinellifolium) and the cultivated tomato varieties. However, phylogenetic analysis showed that S. pennellii formed the most divergent member within each clade and S. pimpinellifolium is closer to the cultivated varieties. Additionally, investigations in 42 plant species highlighted that miR172a/b is the most abundant form in the plant kingdom. In addition to the classical target Apetala2 (AP2), degradome analysis identified SEC14p-like phosphatidylinositol transfer family protein (SEC14p) as a novel target of Sly-miR172 that was validated using precursor:effector and target:reporter transient assays. Further, we report dual mode of Sly-miR172-mediated silencing of targets Sly-AP2 and Sly-SEC14p by post-transcriptional transcript cleavage as well as translational repression. Different members of Sly-MIR172s:Sly-AP2s and Sly-MIR172s:Sly-SEC14p exhibit inverse expression correlation in response to different abiotic stresses, suggesting their role in stress response. Functional investigation of MIR172 showed that tomato plants performed better in different abiotic stresses (heat, drought, and salt) upon MIR172 overexpression or target knock-down by virus-induced-gene-silencing. Conversely, when miRNA is chelated using short-tandem-target-mimic, the plants exhibit sensitivity to these stresses. Thus, Sly-miR172 acts as a positive regulator while its targets Sly-AP2a and Sly-SEC14p as negative regulators of different abiotic stresses.

In vivo Polycystin-1 interactome using a novel Pkd1 knock-in mouse model.

PKD1 is the most commonly mutated gene causing autosomal dominant polycystic kidney disease (ADPKD). It encodes Polycystin-1 (PC1), a putative membrane protein that undergoes a set of incompletely characterized post-transcriptional cleavage steps and has been reported to localize in multiple subcellular locations, including the primary cilium and mitochondria. However, direct visualization of PC1 and detailed characterization of its binding partners remain challenging. We now report a new mouse model with HA epitopes and eGFP knocked-in frame into the endogenous mouse Pkd1 gene by CRISPR/Cas9. Using this model, we sought to visualize endogenous PC1-eGFP and performed affinity-purification mass spectrometry (AP-MS) and network analyses. We show that the modified Pkd1 allele is fully functional but the eGFP-tagged protein cannot be detected without signal amplification by secondary antibodies. Using nanobody-coupled beads and large quantities of tissue, AP-MS identified an in vivo PC1 interactome, which is enriched for mitochondrial proteins and components of metabolic pathways. These studies suggest this mouse model and interactome data will be useful to understand PC1 function, but that new methods and brighter tags will be required to track endogenous PC1.

Full-length transcriptome profiling of Aphidius gifuensis mitochondrial genome with gene rearrangement and control region duplication

Although mitochondrial gene rearrangement has been observed in many insect lineages, little is known about how it affects mitochondrial gene transcription. To address this question, we first constructed a quantitative transcription map for Aphidius gifuensis, a species of parasitoid wasp known to have a highly rearranged mitochondrial genome (mitogenome) and two potential control regions (CRs). Based on this transcription map, we assessed the models of the mitochondrial transcription and post-transcription cleavage. We found that the J and N strand of this mitogenome differ significantly in transcriptional regulation. On the J strand, we found two transcription initiation sites (TISs), five transcription termination sites (TTSs), and six polycistronic primary transcripts whereas only one TIS, one TTS and one polycistronic primary transcript can be found on the N strand. Most of the non-coding regions of both strands were transcribed into primary transcripts and cleaved after transcription. The proposed mode of transcription of A. gifuensis was similar to that of Drosophila, a model organism with no gene rearrangement. And two rearranged gene clusters (trnI-CR1-trnM-CR2-trnQ and trnW-trnY-trnC) seemed to have little effects on the mode of transcription. In addition, our results revealed the presence of TISs in CR1 and CR2, implying that both CRs maybe required for transcriptional regulation. Analysis of the post-transcriptional cleavage process showed that there were both “forward cleavage” and “reverse cleavage” models in A. gifuensis, and more than one way of cleavages were found in three regions. The incomplete transcripts suggested that the direction of mitochondrial RNA degradation was from 5′ to 3′ end and supported the view of polyadenylation-dependent RNA degradation. Our study provides insights into the transcriptional and post-transcriptional regulation processes of highly rearranged insect mitogenomes.

Redundant actions of neuropeptides encoded by the dh-pban gene for larval color pattern formation in the oriental armyworm Mythimnaseparata

The pyrokinin (PK)/pheromone biosynthesis-activating neuropeptide (PBAN) family, which is defined by a conserved C-terminal pentapeptide (FXPRLamide), is involved in many physiological processes in insects. In the oriental armyworm Mythimna separata, the larvae exhibit a variety of color patterns in response to changes in population density, which are caused by melanization and a reddish coloration hormone (MRCH), which is a member of the FXPRLamide neuropeptides. Interestingly, in some lepidopteran insects, MRCH is known as a PBAN, which activates the pheromone gland to produce sex pheromones. PBAN is encoded by a single gene, dh-pban, which encodes additional FXPRLamide neuropeptides, such as the diapause hormone (DH) and subesophageal ganglion neuropeptides (SGNPs). To determine the roles of the dh-pban gene, which produces multiple types of FXPRLamide neuropeptides after post-transcriptional cleavage of the precursor protein, we performed CRISPR/Cas9-mediated targeted mutagenesis in M. separata. We demonstrated that knockout armyworm larvae lost density-dependent cuticular melanization and retained yellow body color, even when reared under crowded conditions. Moreover, our rescue experiments using the synthetic peptides showed that not only PBAN but also β- and γ-SGNPs significantly induce the cuticular melanization in a dose dependent manner. Taken together, our results provide genetic evidence that neuropeptides encoded by the single dh-pban gene act redundantly to control density-dependent color pattern formation in M. separata.

Kinetic competition between theophylline sensitive ribozyme cleavage and termination in living cells by 3D orbital tracking-fluorescent cross-correlation spectroscopy.

Synthetic RNA-based regulatory tools such as riboswitches are promising candidates for the realization of complex engineered genetic circuits. However, previous efforts to implement such tools in synthetic biology have yielded inconsistent results. Often the quantification of performance is measured using protein expression, even though these devices act at the RNA level. To overcome this, we use in vivo RNA labeling of Hemoglobin B in Human Epithelial Kidney T-Rex Cells to determine two objectives. First, we will determine whether a theophylline-dependent hammerhead ribozyme riboswitch alters gene expression by inducing early termination of transcription or via post-transcriptional mRNA cleavage. The second objective will use a self-cleaving hammerhead ribozyme to separate transcription elongation from termination. This will allow for the determination of transcription termination kinetics. Quantification for both objectives will be completed using 3D Orbital Tracking-Fluorescent Cross-Correlation Spectroscopy (3DOT-FCCS). To determine the levels of post-transcription mRNA cleavage, we will use Raster Image Correlation Spectroscopy (RICS) to measure mRNA stability via mRNA lifetime. Preliminary data suggests that the theophylline aptamer stabilizes the ribozyme, correlating to an increased RNA dwell time and termination. However, when the aptamer is absent, there is a decrease. This indicates early termination and premature transcript release from the DNA template are occurring with the ribozyme. In the future, we will examine the transcribing order of Intron 1 and Intron 2, splicing behaviors, post-termination splicing using RICS, and RNA lifetime.

Full-Length Transcriptome Profiling of Coridius chinensis Mitochondrial Genome Reveals the Transcription of Genes with Ancestral Arrangement in Insects.

Coridius chinensis (Hemiptera: Dinidoridae) is a medicinal insect. Its mitochondrial gene arrangement is consistent with that of Drosophila melanogaster and Erthesina fullo, the two insects with well-studied mitochondrial transcription. To investigate whether the structural consistency of mitochondrial genes leads to similarities in transcription and post-transcriptional processing, we improved the gene annotation and constructed a quantitative transcription map for the C. chinensis mitochondrial genome (mitogenome) using full-length transcriptome sequencing. The size of this mitogenome was 16,214 bp and the proposed model of mitochondrial transcription was similar to that of Drosophila. Both strands were nearly entirely transcribed except for the antisense genes downstream of trnS2 on N strand. The expression of cytochrome c subunit genes is higher than that of NADH-dehydrogenase subunit genes. The post-transcriptional cleavage process followed the "tRNA punctuation" model, and both the "reverse cleavage" model in Drosophila and "forward cleavage" model in E. fullo were found in C. chinensis. In addition, we found that long non-coding RNAs from the control region contained tandem repeats. Polyadenylation was performed after CCA triplet at the 3' end of tRNA. The isoform diversity of lrRNA was identified. Our study sheds light on the transcriptional regulation and RNA processing of insect mitogenomes with the putative ancestral gene arrangement.

A conserved HSF:miR169:NF-YA loop involved in tomato and Arabidopsis heat stress tolerance.

Heat stress transcription factors (HSFs) and microRNAs (miRNAs) regulate different stress and developmental networks in plants. Regulatory feedback mechanisms are at the basis of these networks. Here, we report that plants improve their heat stress tolerance through HSF-mediated transcriptional regulation of MIR169 and post-transcriptional regulation of Nuclear Factor-YA (NF-YA) transcription factors. We show that HSFs recognize tomato (Solanum lycopersicum) and Arabidopsis MIR169 promoters using yeast one-hybrid/chromatin immunoprecipitation-quantitative PCR. Silencing tomato HSFs using virus-induced gene silencing (VIGS) reduced Sly-MIR169 levels and enhanced Sly-NF-YA9/A10 target expression. Further, Sly-NF-YA9/A10 VIGS knockdown tomato plants and Arabidopsis plants overexpressing At-MIR169d or At-nf-ya2 mutants showed a link with increased heat tolerance. In contrast, Arabidopsis plants overexpressing At-NF-YA2 and those expressing a non-cleavable At-NF-YA2 form (miR169d-resistant At-NF-YA2) as well as plants in which At-miR169d regulation is inhibited (miR169d mimic plants) were more sensitive to heat stress, highlighting NF-YA as a negative regulator of heat tolerance. Furthermore, post-transcriptional cleavage of NF-YA by elevated miR169 levels resulted in alleviation of the repression of the heat stress effector HSFA7 in tomato and Arabidopsis, revealing a retroactive control of HSFs by the miR169:NF-YA node. Hence, a regulatory feedback loop involving HSFs, miR169s and NF-YAs plays a critical role in the regulation of the heat stress response in tomato and Arabidopsis plants.

Identification of human mitochondrial RNA cleavage sites and candidate RNA processing factors

BackgroundThe human mitochondrial genome is transcribed as long strands of RNA containing multiple genes, which require post-transcriptional cleavage and processing to release functional gene products that play vital roles in cellular energy production. Despite knowledge implicating mitochondrial post-transcriptional processes in pathologies such as cancer, cardiovascular disease and diabetes, very little is known about the way their function varies on a human population level and what drives changes in these processes to ultimately influence disease risk. Here, we develop a method to detect and quantify mitochondrial RNA cleavage events from standard RNA sequencing data and apply this approach to human whole blood data from > 1000 samples across independent cohorts.ResultsWe detect 54 putative mitochondrial RNA cleavage sites that not only map to known gene boundaries, short RNA ends and RNA modification sites, but also occur at internal gene positions, suggesting novel mitochondrial RNA cleavage junctions. Inferred RNA cleavage rates correlate with mitochondrial-encoded gene expression across individuals, suggesting an impact on downstream processes. Furthermore, by comparing inferred cleavage rates to nuclear genetic variation and gene expression, we implicate multiple genes in modulating mitochondrial RNA cleavage (e.g. MRPP3, TBRG4 and FASTKD5), including a potentially novel role for RPS19 in influencing cleavage rates at a site near to the MTATP6-COX3 junction that we validate using shRNA knock down data.ConclusionsWe identify novel cleavage junctions associated with mitochondrial RNA processing, as well as genes newly implicated in these processes, and detect the potential impact of variation in cleavage rates on downstream phenotypes and disease processes. These results highlight the complexity of the mitochondrial transcriptome and point to novel mechanisms through which nuclear-encoded genes can potentially influence key mitochondrial processes.

Towards unveiling the nature of short SERPINA1 transcripts: Avoiding the main ORF control to translate alpha1-antitrypsin C-terminal peptides

Alternative ORFs in-frame with the known genes are challenging to reveal. Yet they may contribute significantly to proteome diversity. Here we focused on the individual expression of the SERPINA1 gene exon 5 leading to direct translation of alpha1-antitrypsin (AAT) C-terminal peptides. The discovery of alternative ways for their production may expand the current understanding of the serpin gene's functioning. We detected short transcripts expressed primarily in hepatocytes. We identified four variants of hepatocyte-specific SERPINA1 short transcripts and individually probed their potential to be translated in living cells. The long mRNA gave the full-length AAT-eGFP fusion, while in case of short transcripts we deduced four active SERPINA1 in-frame alternative ORFs encoding 10, 21, 153 and 169 amino acids AAT C-terminal oligo- and polypeptides. Unlike secretory AAT-eGFP fusion exhibiting classical AAT behavior, truncated AAT-fusions differ by intracellular retention and nuclear enrichment. Immunofluorescence on the endogenous AAT C-terminal epitope showed its accumulation in the cell nucleoli, indicating that short transcripts may be translated in vivo. FANTOM5 CAGE data on SERPINA1 suggest that short transcripts originate from the post-transcriptional cleavage of the spliced mRNA, initiated mainly from the hepatocyte-specific promoter. ConclusionShort SERPINA1 transcripts may represent a source for the direct synthesis of AAT C-terminal peptides with properties uncommon to AAT.

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Activity of the Carboxy-Terminal Peptide Region of the Teneurins and Its Role in Neuronal Function and Behavior in Mammals.

Teneurin C-terminal associated peptides (TCAPs) are an evolutionarily ancient family of 40- to 41-residue bioactive peptides located on the extracellular end of each of the four teneurin transmembrane proteins. TCAP-1 may exist as a tethered peptide at the teneurin-1 carboxy end or as an independent peptide that is either released via post-transcriptional cleavage from its teneurin-1 pro-protein or independently expressed as its own mRNA. In neurons, soluble TCAP-1 acts as a paracrine factor to regulate cellular activity and neuroplastic interactions. In vitro studies indicate that, by itself, synthetic TCAP-1 promotes neuron growth and protects cells from chemical insult. In vivo, TCAP-1 increases hippocampal neuron spine density, reduces stress-induced behavior and ablates cocaine-seeking behaviors. Together, these studies suggest that the physiological effects of TCAP-1 are a result of an inhibition of corticotropin-releasing factor (CRF) activity leading to increased energy production. This hypothesis is supported by in vivo functional positron emissions tomography studies, which demonstrate that TCAP-1 significantly increases glucose uptake in rat brain. Complimentary in vitro studies show that enhanced glucose uptake is the result of TCAP-1-induced insertion of the glucose transporter into the neuronal plasma membrane, leading to increased glucose uptake and ATP production. Interestingly, TCAP-1-mediated glucose uptake occurs through a novel insulin-independent pathway. This review will focus on examining the role of TCAP on neuronal energy metabolism in the central nervous system.

Defining the Transcriptional and Post-transcriptional Landscapes of Mycobacterium smegmatis in Aerobic Growth and Hypoxia.

The ability of Mycobacterium tuberculosis to infect, proliferate, and survive during long periods in the human lungs largely depends on the rigorous control of gene expression. Transcriptome-wide analyses are key to understanding gene regulation on a global scale. Here, we combine 5′-end-directed libraries with RNAseq expression libraries to gain insight into the transcriptome organization and post-transcriptional mRNA cleavage landscape in mycobacteria during log phase growth and under hypoxia, a physiologically relevant stress condition. Using the model organism Mycobacterium smegmatis, we identified 6,090 transcription start sites (TSSs) with high confidence during log phase growth, of which 67% were categorized as primary TSSs for annotated genes, and the remaining were classified as internal, antisense, or orphan, according to their genomic context. Interestingly, over 25% of the RNA transcripts lack a leader sequence, and of the coding sequences that do have leaders, 53% lack a strong consensus Shine-Dalgarno site. This indicates that like M. tuberculosis, M. smegmatis can initiate translation through multiple mechanisms. Our approach also allowed us to identify over 3,000 RNA cleavage sites, which occur at a novel sequence motif. To our knowledge, this represents the first report of a transcriptome-wide RNA cleavage site map in mycobacteria. The cleavage sites show a positional bias toward mRNA regulatory regions, highlighting the importance of post-transcriptional regulation in gene expression. We show that in low oxygen, a condition associated with the host environment during infection, mycobacteria change their transcriptomic profiles and endonucleolytic RNA cleavage is markedly reduced, suggesting a mechanistic explanation for previous reports of increased mRNA half-lives in response to stress. In addition, a number of TSSs were triggered in hypoxia, 56 of which contain the binding motif for the sigma factor SigF in their promoter regions. This suggests that SigF makes direct contributions to transcriptomic remodeling in hypoxia-challenged mycobacteria. Taken together, our data provide a foundation for further study of both transcriptional and posttranscriptional regulation in mycobacteria.

TaMIR1119, a miRNA family member of wheat (Triticum aestivum), is essential in the regulation of plant drought tolerance

Through regulating target genes via the mechanisms of posttranscriptional cleavage or translational repression, plant miRNAs involve diverse biological processes associating with plant growth, development, and abiotic stress responses. In this study, we functionally characterized TaMIR1119, a miRNA family member of wheat (Triticum aestivum), in regulating the drought adaptive response of plants. TaMIR1119 putatively targets six genes categorized into the functional classes of transcriptional regulation, RNA and biochemical metabolism, trafficking, and oxidative stress defense. Upon simulated drought stress, the TaMIR1119 transcripts abundance in roots was drastically altered, showing to be upregulated gradually within a 48-h drought regime and that the drought-induced transcripts were gradually restored along with a 48-h recovery treatment. In contrast, most miRNA target genes displayed reverse expression patterns to TaMIR1119, exhibiting a downregulated expression pattern upon drought and whose reduced transcripts were re-elevated along with a normal recovery treatment. These expression analysis results indicated that TaMIR1119 responds to drought and regulates the target genes mainly through a cleavage mechanism. Under drought stress, the tobacco lines with TaMIR1119 overexpression behaved improved phenotypes, showing increased plant biomass, photosynthetic parameters, osmolyte accumulation, and enhanced antioxidant enzyme (AE) activities relative to wild type. Three AE genes, NtFeSOD, NtCAT1;3, and NtSOD2;1, encoding superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) proteins, respectively, showed upregulated expression in TaMIR1119 overexpression lines, suggesting that they are involved in the regulation of AE activities and contribution to the improved cellular reactive oxygen species (ROS) homeostasis in drought-challenged transgenic lines. Our results indicate that TaMIR1119 plays critical roles in regulating plant drought tolerance through transcriptionally regulating the target genes that modulate osmolyte accumulation, photosynthetic function, and improve cellular ROS homeostasis of plants.

Identification of an Alternative rRNA Post-transcriptional Maturation of 26S rRNA in the Kingdom Fungi.

Despite of the integrity of their RNA, some desert truffles present a non-canonical profile of rRNA where 3.3 kb is absent, 1.8 kb is clear and a band of 1.6 kb is observed. A similar rRNA profile was identified in organisms belonging to different life kingdoms, with the exception of the Kingdom Fungi, as a result of a split LSU rRNA called hidden gap. rRNA profiles of desert truffles were analyzed to verify the presence of the non-canonical profile. The RNA of desert truffles and yeast were blotted and hybridized with probes complementary to LSU extremes. RACE of LSU rRNA was carried out to determine the LSU rRNA breakage point. LSU rRNA of desert truffles presents a post-transcriptional cleavage of five nucleotides that generates a hidden gap located in domain D7. LSU splits into two molecules of 1.6 and 1.8 kb. Similar to other organisms, a UAAU tract, downstream of the breakage point, was identified. Phylogenetic comparison suggests that during fungi evolution mutations were introduced in the hypervariable D7 domain, resulting in a sequence that is specifically post-transcriptionally cleaved in some desert truffles.

Potential role of orexin A binding the receptor 1 for orexins in normal and cryptorchid dogs

BackgroundCryptorchidism is one of the most common birth disorders of the male reproductive system identified in dogs and other mammals. This condition is characterised by the absence of one (unilateral) or both (bilateral) gonads from the scrotum. The peptides orexin A (OxA) and B (OxB) were obtained by post-transcriptional proteolytic cleavage of a precursor molecule, called prepro-orexin. These substances bind two types of G-coupled receptors called receptor 1 (OX1R) and 2 (OX2R) for orexins. OX1R is specific to OxA while OX2R binds the two peptides with equal affinity. Orexins modulate a great variety of body functions, such as the reproductive mechanism. The purpose of the present research was to study the presence of OxA and its receptor 1 and their possible involvement in the canine testis under healthy and pathological conditions.MethodsThis study was performed using adult male normal dogs and male dogs affected by unilateral cryptorchidism. Tissue samples were collected from testes and were divided into three groups: normal, contralateral and cryptic. The samples were used for immunohistochemistry, Western blot and in vitro tests for testosterone evaluation in normal and pathological conditions.ResultsOxA-immunoreactivity (IR) was described in interstitial Leydig cells of the normal gonad, and Leydig, Sertoli cells and gonocytes in the cryptic gonad. In the normal testis, OX1R-IR was described in Leydig cells, in pachytene and second spermatocytes and in immature and mature spermatids throughout the stages of the germ developing cycle of the male gonad. In the cryptic testis OX1R-IR was distributed in Leydig and Sertoli cells. The presence of prepro-orexin and OX1R was demonstrated by Western blot analysis. The incubation of fresh testis slices with OxA caused the stimulation of testosterone synthesis in the normal and cryptic gonad while the steroidogenic OxA-induced effect was cancelled by adding the selective OX1R antagonist SB-408124.ConclusionsThese results led us to hypothesise that OxA binding OX1R might be involved in the modulation of spermatogenesis and steroidogenesis in canine testis in healthy and pathological conditions.

MiR-21 Inhibitors Modulate Biological Functions of Gastric Cancer Cells via PTEN/PI3K/mTOR Pathway.

Gastric cancer is one of the most common malignancy in the world. microRNAs (miRNAs) are naturally occurring noncoding RNA that control gene expression by targeting messenger RNA (mRNA) for post-transcriptional repression or cleavage. This study focused on a specific miRNA, miR-21, which was overexpressed in gastric cancer and examined the effects of miR-21 inhibitor on biological functions of gastric cancer cells and its possible mechanism. Gastric cancer cells MKN74 were treated with miR-21 inhibitor, negative control, and blank control. Cell proliferation, colony formation, migration, and invasion were assessed. Real-time PCR and western blot were applied to examine the expression of phosphatase and tens in homolog deleted on chromosome ten (PTEN)/PI3K/mTOR pathway molecules. miR-21 inhibitor markedly suppressed proliferation, migration, invasion, and colony formation of gastric cancer cells. Anti-miR-21 treatment also reduced the expression ratio of B cell lymphoma 2 (Bcl-2)/Bax. Furthermore, miR-21 inhibition was associated with increased expression of PTEN, which in turn decreased the ratios of S235/236, S240/244, and p-AK/AKT in gastric cancer cells. Inhibiting miR-21 modulates biological functions of gastric cancer cells via PTEN/PI3K/mTOR pathway and miR-21 inhibitor may provide a novel therapeutic strategy for gastric cancer.

The multifaceted regulatory potential of tRNA-derived fragments

tRNAs have been discovered in the late 1950-ies of the last century and have subsequently been characterized biochemically, genetically and structurally as amino-acid delivering molecules for protein biosynthesis. First reports of stable tRNA-derived cleavage products have been reported in the late 1970-ies (1), but at that time they were mostly regarded as functionless degradation products. The advent of high-throughput sequencing techniques in the last years allowed the discovery of an unforeseen variety of small non-protein-coding RNAs (ncRNAs), including tRNA-derived RNA fragments (typically referred to as tdRs or tRFs), in all domains of life. Post-transcriptional RNA cleavage encompasses all sorts of transcripts including mRNAs as well as ncRNAs thus amplifying the biological repertoire of cellular RNomes significantly. Most of these small ncRNAs are involved in different steps of gene regulation. The most studied and well understood small ncRNA regulators are the ~22 nt long miRNA and siRNA involved in postranscriptional gene silencing, a process termed RNA interference (RNAi).

MiRNAs in mtDNA-less cell mitochondria.

The novel regulation mechanism in mtDNA-less cells was investigated. Very low mtDNA copy in mtDNA-less 206 ρ° cells was identified. But no 13 mitochondria-specific proteins were translated in 206 ρ° cells. Their mitochondrial respiration complexes V, III and II were 86.5, 29.4 and 49.6% of 143B cells, respectively. Complexes I and IV completely lack in 206 ρ° cells. Non-mitochondrial respiration to generate ATP in 206 ρ° cells was discovered. The expression levels of some mitochondrial RNAs including 12S rRNA, COX1, COX2, COX3, ND4 and ND5 were low. However, ND1, ND3 and Cyto b were not expressed in 206 ρ° cells. Unequal transcription of mitochondrial RNAs indicated the post-transcriptional cleavage and processing mechanisms in the regulation of mitochondrial gene expression in 206 ρ° cells. MicroRNAs (miRNAs) may modulate these mitochondrial RNA expression in these cells. RNA-induced silencing complex indeed within 206 ρ° cell mitochondria indicated miRNAs in 206 ρ° cell mitochondria. miRNA profile in mtDNA-less 206 ρ° cells was studied by next-generation sequencing of small RNAs. Several mitochondria-enriched miRNAs such as miR-181c-5p and miR-146a-5p were identified in 206 ρ° cell mitochondria. miR-181c-5p and miR-146a-5p had 23 and 19 potential targets on mitochondrial RNAs respectively, and these two miRNAs had multiple targets on mitochondria-associated messenger RNAs encoded by nuclear genes. These data provided the first direct evidence that miRNAs were imported into mitochondria and regulated mitochondrial RNA expressions.

Characterization and expression pattern analysis of microRNAs in wheat under drought stress

Plant microRNAs (miRNAs) play important roles in regulating plant growth, development, and responses to abiotic stresses. In this study, 38 miRNAs (TaMIRs) from wheat (Triticum aestivum L.), 36 from the miRBase database, and two from our previous work were characterized and subjected to an expression pattern analysis under normal conditions and a drought stress. A semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), real-time quantitative PCR (qPCR), and small RNA blot analyses revealed that two TaMIRs (TaMIR1120 and TaMIR1123) were root-predominant and two TaMIRs (TaMIR1121 and TaMIR1134) were leaf-predominant. Seven TaMIR precursors showed altered expressions after the drought; of these, TaMIR1136 was upregulated, whereas TaMIR156, TaMIR408, TaMIR1119, TaMIR1129, TaMIR1133, and TaMIR1139 were downregulated. These seven drought-responsive TaMIRs showed dose-dependent and typical temporal expression patterns during drought induction, and they gradually returned back under the normal growth conditions. The drought-responsive and the tissue-predominant TaMIRs had varying numbers of target genes. Randomly selected target genes exhibited opposite expression patterns to their corresponding TaMIRs suggesting that they were regulated by distinct TaMIRs through a post-transcriptional cleavage. The target genes regulated by drought-responsive and tissue-predominant TaMIRs are involved in various cellular processes, such as signal transduction, transcriptional regulation, primary and secondary metabolisms, development, and defense responses. These results provide a novel insight into the miRNA-mediated responses of wheat to drought stress.