Andreas Werner

The Shrinkage-activated Na+ Conductance of Rat Hepatocytes and its Possible Correlation to rENaC

At moderate cell shrinkage, activation of Na+ channels is the most prominent mechanism of regulatory cell volume increase in rat hepatocytes. The amiloride sensitivity of these channels suggests a relation to the family of epithelial Na+ channels (ENaCs). The present study was performed to determine the pharmacological profile of shrinkage-activated Na+ channels and to test for ENaC expression in primary cultures of rat hepatocytes; in addition, the influence of the cell volume regulated serine/threonine kinase hSGK on activity and pharmacological profile of rENaC was examined in Xenopus oocytes. Conventional electrophysiology in hepatocytes reveals that the shrinkage-activated Na+ channel is inhibited by amiloride and EIPA with IC50 values of 6.0 and 0.12 μmol/l, respectively. Western blots and RT-PCR demonstrate that rat hepatocytes do express all three subunits (α, β, γ) of ENaC. Coexpression of hSGK with rENaC in Xenopus oocytes reveals that the kinase stimulates ENaC by a factor of 4. Moreover, hSGK decreases the affinity to amiloride (increase of IC50 from 0.12 to 0.26 μmol/l) and increases the affinity to EIPA (decrease of IC50 from 250 to 50 μmol/l). In conclusion, rat hepatocytes express ENaC, which is activated by the cell volume-sensitive kinase hSGK. ENaC may contribute to the Na+ channels activated by osmotic cell shrinkage in hepatocytes, whereby the relatively low amiloride and high EIPA sensitivity of the channel could at least be partially due to modification by SGK, which decreases the amiloride and increases the EIPA sensitivity of ENaC.

What do natural antisense transcripts regulate

In human and mouse up to 72% of all genomic loci show evidence of transcription from both sense and antisense strands. The benefit of the resulting natural antisense transcripts (NATs) remains unclear, largely because of a lack of significant correlation between gene ontology and antisense transcription. Here we suggest that a well defined group of NATs may be identified based on structural characteristics. Specifically, these NATs are processed transcripts that are complementary to the corresponding processed sense transcripts in exonic regions. Recent reports have established that co-expressed sense transcripts/NATs are processed into short RNAs. These so called endo-siRNAs are found in both sense and antisense orientation and were hypothesized to mediate pseudogene silencing. Here we propose that NATs are biologically important sources of endo-siRNAs. We also propose that endo-siRNAs are essential components of a regulatory network to control the mutagenic burden that unfolds on nucleic acid level without direct consequences on protein expression.

Natural Antisense Transcripts

The sequencing of whole genomes and the subsequent annotation of cDNAs revealed that about 20% of human and mouse genes overlap resulting in potential pairs of sense and antisense transcripts. An increasing number of experimentally identified antisense transcripts concurs with this predication. Characterization of overlapping transcripts in various species indicates that this form of RNA-mediated gene regulation represents a widespread phenomenon. However, the physiological relevance of natural antisense transcripts remains obscure. Genomic studies suggest that duplex formation between sense and antisense is required for biological function.Antisense transcripts play an established role in imprinting and X-chromosome inactivation and genomic rearrangements as observed in B- and T- leukocytes. Only a relatively small percentage of the predicted antisense transcripts are related to these biological phenomena that are also related to mono-allelic expression. Consequently, there are at least two categories of natural antisense transcripts that show significant differences with regard to their biological function as well as the potential mechanisms involved.

A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin

Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane. When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles.

Expression profiling of antisense transcripts on DNA arrays.

The majority of mouse genes are estimated to undergo bidirectional transcription; however, their tissue-specific distribution patterns and physiological significance are largely unknown. This is in part due to the lack of methodology to routinely assess the expression of natural antisense transcripts (NATs) on a large scale. Here we tested whether commercial DNA arrays can be used to monitor antisense transcription in mouse kidney and brain. We took advantage of the reversely annotated oligonucleotides on the U74 mouse genome array from Affymetrix that hybridize to NATs overlapping with the sense transcript in the area of the probe set. In RNA samples from mouse kidney and brain, 11.9% and 10.1%, respectively, of 5,652 potential NATs returned positive and about half of the antisense RNAs were detected in both tissues, which was similar to the fraction of sense transcripts expressed in both tissues. Notably, we found that the majority of NATs are related to the sense transcriptome since corresponding sense transcripts were detected for 92.5% (kidney) and 74.5% (brain) of the detected antisense RNAs. Antisense RNA transcription was confirmed by real-time PCR and included additional RNA samples from heart, thymus, and liver. The randomly selected transcripts showed tissue specific expression patterns and varying sense/antisense ratios. The results indicate that antisense transcriptomes are tissue specific, and although pairing of sense/antisense transcripts are known to result in rapid degradation, our data provide proof of principle that the sensitivity of commercial DNA arrays is sufficient to assess NATs in total RNA of whole organs.

Naturally occurring antisense RNA: function and mechanisms of action.

Purpose of reviewNatural antisense transcripts have recently emerged as important regulators of gene expression. The transcription of an antisense RNA can influence the output of the specific gene locus on a posttranscriptional level but may also help to establish a local epigenetic imprint. Recent findingsRecent advances in transcriptome sequencing have revealed widespread expression of complementary sense–antisense transcript pairs. The naturally occurring antisense transcripts can modulate the expression level of the sense transcripts or influence the sense mRNA processing. Given that both sense and antisense transcriptomes show tissue-specific regulation, these mechanisms may contribute to the physiological tuning of specific genes. An additional level of gene regulation by antisense transcripts has recently emerged: coexpressed sense and antisense transcripts can be cleaved and processed into single-stranded short RNAs (endo-siRNAs). Evidence suggests that these endo-siRNAs are linked to transcriptional silencing of the complementary transcripts. The impact of natural antisense transcripts may, therefore, not only feed forward to the protein level but also back to the genomic level. SummaryNatural antisense transcripts add a further level of regulation to gene expression. The novel insights into the biology of natural antisense transcripts and endo-siRNAs may lead to improved gene silencing strategies in biomedical research with subsequent use in clinical applications.

Processing of naturally occurring sense/antisense transcripts of the vertebrate Slc34a gene into short RNAs

Overlapping sense/antisense RNAs transcribed in opposite directions from the same genomic locus are common in vertebrates. The impact of antisense transcription on gene regulation and cell biology is largely unknown. We show that sense/antisense RNAs of an evolutionarily conserved phosphate transporter gene (Slc34a2a) are coexpressed in a short time window during embryonic development of zebrafish at 48 hours postfertilization (hpf). To address the mechanism by which coexpressed sense/antisense transcripts are processed, we injected sense/antisense RNAs in various combinations into Xenopus oocytes. In the cytoplasm RNAs were stable in whatever combination expressed. In the nucleus coinjected sense/antisense transcripts were degraded into short RNAs of approximately 23 bases within 4 h. A homologous transcript from toad or another isoform (Slc34a2b) from zebrafish failed to trigger processing. In oocytes that were primed with nuclear sense/antisense RNA coinjections, a reporter RNA was rapidly degraded. We produced evidence that the observed processing of complementary transcripts was not restricted to Xenopus oocytes, because Slc34a-related short RNAs were detected in zebrafish embryos by Northern blotting. Signals were observed at stages that showed coexpression of sense/antisense transcripts. Remarkably, strand-specific probes revealed that the orientation of short RNAs was developmentally regulated. In addition, RNA from zebrafish embryos 48 hpf was able to induce degradation of reporter constructs in Xenopus oocytes. Our findings may give important clues to understanding the physiological role of the widespread antisense transcription.

Contribution of natural antisense transcription to an endogenous siRNA signature in human cells

BackgroundEukaryotic cells express a complex layer of noncoding RNAs. An intriguing family of regulatory RNAs includes transcripts from the opposite strand of protein coding genes, so called natural antisense transcripts (NATs). Here, we test the hypothesis that antisense transcription triggers RNA interference and gives rise to endogenous short RNAs (endo-siRNAs).ResultsWe used cloned human embryonic kidney cells (HEK293) followed by short RNAseq to investigate the small genic RNA transcriptome. 378 genes gave rise to short RNA reads that mapped to exons of RefSeq genes. The length profile of short RNAs showed a broad peak of 20-24 nucleotides, indicative of endo-siRNAs. Collapsed reads mapped predominantly to the first and the last exon of genes (74%). RNAs reads were intersected with sequences occupied by RNAPII or bound to Argonaute (AGO1 by crosslinking, ligation, and sequencing of hybrids, CLASH). In the first exon, 94% of the reads correlated with RNAPII occupancy with an average density of 130 (relative units); this decreased to 65%/20 in middle exons and 54%/12 in the last exon. CLASH reads mapping to multi-exon genes showed little distribution bias with an average of about 5 CLASH reads overlapping with 60% of the endo-siRNA reads. However, endo-siRNAs (21-25 nt) intersecting with CLASH reads were enriched at the 5′end and decreased towards the 3′end.We then investigated the 378 genes with particular focus on features indicative for short RNA production; however, found that endo-siRNA numbers did not correlate with gene structures that favor convergent transcription. In contrast, our gene set was found notably over-represented in the NATsDB sense/antisense group as compared to non-overlapping and non-bidirectional groups. Moreover, read counts showed no correlation with the steady-state levels of the related mRNAs and the pattern of endo-siRNAs proved reproducible after an induced mutagenic insult.ConclusionsOur results suggest that antisense transcripts contribute to low levels of endo-siRNAs in fully differentiated human cells. A characteristic endo-siRNA footprint is being produced at sites of RNAPII transcription which is also related to AGO1. This endo-siRNA signature represents an intriguing finding and its reproducibility suggests that the production of endo-siRNAs is a regulated process with potential homoeostatic impact.

The functions of natural antisense transcripts

NATs (natural antisense transcripts) are widespread in eukaryotic genomes. Experimental evidence indicates that sense and antisense transcripts interact, suggesting a role for NATs in the regulation of gene expression. On the other hand, the transcription of a gene locus in both orientations and RNA hybrid formation can also lead to transcriptional interference, trigger an immune response or induce gene silencing. Tissue-specific expression of NATs and the compartmentalization of cells ensure that the regulatory impact of NATs prevails. Consequently, NATs are now acknowledged as important modulators of gene expression. New mechanisms of action and important biological roles of NATs keep emerging, making regulatory RNAs an exciting and quickly moving area of research.

Dietary P regulates phosphate transporter expression, phosphatase activity, and effluent P partitioning in trout culture

Phosphate utilization by fish is an important issue because of its critical roles in fish growth and aquatic environmental pollution. High dietary phosphorus (P) levels typically decrease the efficiency of P utilization, thereby increasing the amount of P excreted as metabolic waste in effluents emanating from rainbow trout aquaculture. In mammals, vitamin D3 is a known regulator of P utilization but in fish, its regulatory role is unclear. Moreover, the effects of dietary P and vitamin D3 on expression of enzymatic and transport systems potentially involved in phosphate utilization are little known. We therefore monitored production of effluent P, levels of plasma vitamin D3 metabolites, as well as expression of phosphatases and the sodium phosphate cotransporter (NaPi2) in trout fed semipu diets that varied in dietary P and vitamin D3 levels. Mean soluble P concentrations varied markedly with dietary P but not with vitamin D3, and constituted 40–70% of total effluent P production by trout. Particulate P concentrations accounted for 25–50% of effluent P production, but did not vary with dietary P or vitamin D3. P in settleable wastes accounted for <10% of effluent P. The stronger effect of dietary P on effluent P levels is paralleled by its striking effects on phosphatases and NaPi2. The mRNA abundance of the intestinal and renal sodium phosphate transporters increased in fish fed low dietary P; vitamin D3 had no effect. Low-P diets reduced plasma phosphate concentrations. Intracellular phytase activity increased but brushborder alkaline phosphatase activity decreased in the intestine, pyloric caeca, and gills of trout fed diets containing low dietary P. Vitamin D3 had no effect on enzyme activities. Moreover, plasma concentrations of 25-hydroxyvitamin D3 and of 1,25-dihydroxyvitamin D3 were unaffected by dietary P and vitamin D3 levels. The major regulator of P metabolism, and ultimately of levels of P in the effluent from trout culture, is dietary P.