Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (SLC34A1/PFN3).

Natural antisense transcripts (NATs) constitute a significant group of regulatory, long noncoding RNAs. They are prominently expressed in testis but are also detectable in other organs. NATs are transcribed at low levels and co-expressed with related protein coding sense transcripts. Nowadays NATs are generally considered as regulatory, long noncoding RNAs without closer focus on the inevitable interference between sense and antisense expression. This work describes a cellular system where sense and antisense transcription of a specific locus (SLC34A1/PFN3) is induced using epigenetic modifiers and CRISPR-Cas9. The renal cell lines HEK293 and HKC-8 do not express SLC34A1/PFN3 under normal culture conditions. Five-day exposure to dexamethasone significantly stimulates sense transcript (SLC34A1) levels and antisense (PFN3) minimally; the effect is only seen in HEK293 cells. Enhanced expression is paralleled by reduced sense promoter methylation and an increase in activating histone marks. Expression is further modulated by cassettes that stimulate the expression of sense or antisense transcript but disrupt protein coding potential. Constitutive expression of a 5'-truncated SLC34A1 transcript increases sense expression independent of dexamethasone induction but also stimulates antisense expression. Concordant expression is confirmed with the antisense knock-in that also enhances sense expression. The antisense effect acts on transcription in cis since transient transfection with sense or antisense constructs fails to stimulate the expression of the opposite transcript. These results suggest that bi-directional transcription of the SLC34A1/PFN3 locus has a stimulatory influence on the expression of the opposite transcript involving epigenetic changes of the promoters. In perspective of extensive, previous research into bi-directionally transcribed SLC34A loci, the findings underpin a hypothesis where NATs display different biological roles in soma and germ cells. Accordingly, we propose that in somatic cells, NATs act like lncRNAs-with the benefit of close proximity to a potential target gene. In germ cells, however, recent evidence suggests different biological roles for NATs that require RNA complementarity and double-stranded RNA formation.

Widespread formation of double-stranded RNAs in testis.

The testis transcriptome is highly complex and includes RNAs that potentially hybridize to form double-stranded RNA (dsRNA). We isolated dsRNA using the monoclonal J2 antibody and deep-sequenced the enriched samples from testes of juvenile Dicer1 knockout mice, age-matched controls, and adult animals. Comparison of our data set with recently published data from mouse liver revealed that the dsRNA transcriptome in testis is markedly different from liver: In testis, dsRNA-forming transcripts derive from mRNAs including promoters and immediate downstream regions, whereas in somatic cells they originate more often from introns and intergenic transcription. The genes that generate dsRNA are significantly expressed in isolated male germ cells with particular enrichment in pachytene spermatocytes. dsRNA formation is lower on the sex (X and Y) chromosomes. The dsRNA transcriptome is significantly less complex in juvenile mice as compared to adult controls and, possibly as a consequence, the knockout of Dicer1 has only a minor effect on the total number of transcript peaks associated with dsRNA. The comparison between dsRNA-associated genes in testis and liver with a reported set of genes that produce endogenous siRNAs reveals a significant overlap in testis but not in liver. Testis dsRNAs also significantly associate with natural antisense genes-again, this feature is not observed in liver. These findings point to a testis-specific mechanism involving natural antisense transcripts and the formation of dsRNAs that feed into the RNA interference pathway, possibly to mitigate the mutagenic impacts of recombination and transposon mobilization.

Recent progress in chemical approaches for the development of novel neuraminidase inhibitors.

Influenza virus is the main cause of an infectious disease called influenza affecting the respiratory system including the throat, nose and lungs. Neuraminidase inhibitors are reagents used to block the enzyme called neuraminidase to prevent the influenza infection from spreading. Neuraminidase inhibitors are widely used in the treatment of influenza infection, but still there is a need to develop more potent agents for the more effective treatment of influenza. Complications of the influenza disease lead to death, and one of these complications is drug resistance; hence, there is an urgent need to develop more effective agents. This review focuses on the recent advances in chemical synthesis pathways used for the development of new neuraminidase agents along with the medicinal aspects of chemically modified molecules, including the structure-activity relationship, which provides further rational designs of more active small molecules.

Natural Antisense Transcripts at the Interface between Host Genome and Mobile Genetic Elements.

Non-coding RNAs are involved in epigenetic processes, playing a role in the regulation of gene expression at the transcriptional and post-transcriptional levels. A particular group of ncRNA are natural antisense transcripts (NATs); these are transcribed in the opposite direction to protein coding transcripts and are widespread in eukaryotes. Their abundance, evidence of phylogenetic conservation and an increasing number of well-characterized examples of antisense-mediated gene regulation are indicative of essential biological roles of NATs. There is evidence to suggest that they interfere with their corresponding sense transcript to elicit concordant and discordant regulation. The main mechanisms involved include transcriptional interference as well as dsRNA formation. Sense-antisense hybrid formation can trigger RNA interference, RNA editing or protein kinase R. However, the exact molecular mechanisms elicited by NATs in the context of these regulatory roles are currently poorly understood. Several examples confirm that ectopic expression of antisense transcripts trigger epigenetic silencing of the related sense transcript. Genomic approaches suggest that the antisense transcriptome carries a broader biological significance which goes beyond the physiological regulation of the directly related sense transcripts. Because NATs show evidence of conservation we speculate that they played a role in evolution, with early eukaryotes gaining selective advantage through the regulatory effects. With the surge of genome and transcriptome sequencing projects, there is promise of a more comprehensive understanding of the biological role of NATs and the regulatory mechanisms involved.

Molecular determinants of transport function in zebrafish Slc34a Na-phosphate transporters.

The epithelial Na+-coupled phosphate cotransporter family Slc34a (NaPi-II) is well conserved in vertebrates and plays an essential role in maintaining whole body levels of inorganic phosphate (Pi). A three-dimensional model of the transport protein has recently been proposed with defined substrate coordination sites. Zebrafish express two NaPi-II isoforms with high sequence identity but a 10-fold different apparent Km for Pi ([Formula: see text]). We took advantage of the two zebrafish isoforms to investigate the contribution of specific amino acids to Pi coordination and transport. Mutations were introduced to gradually transform the low-affinity isoform into a high-affinity transporter. The constructs were expressed in Xenopus laevis oocytes and functionally characterized. Becaue the cotransport of Pi and Na involves multiple steps that could all influence [Formula: see text], we performed a detailed functional analysis to characterize the impact of the mutations on particular steps of the transport cycle. We used varying concentrations of the substrates Pi and its slightly larger analog, arsenate, as well as the cosubstrate, Na+ Moreover, electrogenic kinetics were performed to assess intramolecular movements of the transporter. All of the mutations were found to affect multiple transport steps, which suggested that the altered amino acids induced subtle structural changes rather than coordinating Pi directly. The likely positions of the critical residues were mapped to the model of human Slc34a, and their localization in relation to the proposed substrate binding pockets concurs well with the observed functional data.