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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.

Carlile M, Nalbant P, Preston-Fayers K, McHaffie GS, Werner A. Processing of naturally occurring sense/antisense transcripts of the vertebrate Slc34a gene into short RNAs. Physiol Genomics 34: 95-100, 2008. First published April 15, 2008 doi:10.1152/physiolgenomics.00004.2008.-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 ϳ23 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. natural antisense RNA; RNA interference-related mechanism; zebrafish; sodium-phosphate cotransporter; gene regulation IN MOUSE, up to 72% of all genomic loci show evidence of sense and antisense transcription, and comparable numbers have been suggested for other organisms (13, 37). The majority of these natural antisense transcripts (NATs) are fully processed and overlap in exonic regions. NATs are underrepresented on mouse and human X chromosomes compared with autosomes (4, 14). This bias, however, is far less pronounced if the transcripts lack exonic overlaps (i.e., the potentially overlapping sequences are removed during splicing). These findings imply that antisense transcripts included in these studies are subjected to comparable evolutionary restrictions and may be processed by related mechanisms; furthermore, they indicate that the formation of RNA-RNA hybrids seems essential for the processing of these NATs (33).The biochemical function of NATs has been assessed in various organisms, and evidence for RNA interference (2), RNA editing (38), RNA splicing (11), transcriptional interference (23), or direct R...

Natural antisense transcripts (NATs) are important regulators of gene expression. Recently, a link between antisense transcription and the formation of endo-siRNAs has emerged. We investigated the bi-directionally transcribed Na/phosphate cotransporter gene (Slc34a1) under the aspect of endo-siRNA processing. Mouse Slc34a1 produces an antisense transcript that represents an alternative splice product of the Pfn3 gene located downstream of Slc34a1. The antisense transcript is prominently found in testis and in kidney. Co-expression of in vitro synthesized sense/antisense transcripts in Xenopus oocytes indicated processing of the overlapping transcripts into endo-siRNAs in the nucleus. Truncation experiments revealed that an overlap of at least 29 base-pairs is required to induce processing. We detected endo-siRNAs in mouse tissues that co express Slc34a1 sense/antisense transcripts by northern blotting. The orientation of endo-siRNAs was tissue specific in mouse kidney and testis. In kidney where the Na/phosphate cotransporter fulfils its physiological function endo-siRNAs complementary to the NAT were detected, in testis both orientations were found. Considering the wide spread expression of NATs and the gene silencing potential of endo-siRNAs we hypothesized a genome-wide link between antisense transcription and monoallelic expression. Significant correlation between random imprinting and antisense transcription could indeed be established. Our findings suggest a novel, more general role for NATs in gene regulation.

.-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. natural antisense transcript; DNA array; RNA; antisense transcriptome THE NUMBER OF PREDICTED ANTISENSE transcripts in mammalian genomes has risen considerably in the last five years from a few hundred to Ͼ50% of all genes in the latest report (15,26). These estimates are based on large-scale cDNA sequencing and expressed sequence tag (EST) database mining with a strong bias toward fully processed transcripts. Antisense RNA synthesis involves RNA polymerase II and general processing of the primary transcript by capping, polyadenylation, and splicing. The biological functions of these transcripts as well as many mechanistic aspects of antisense mediated gene regulation remain speculative. A recently suggested concept that integrates functional and genomic aspects predicts at least two different categories of antisense transcripts (32,33). A group of long noncoding transcripts are related to monoallelic expression as observed in imprinting, X chromosome inactivation, or allelic exclusion in leukocytes. Examples of such noncoding transcripts include the antisense RNA Air, Tsix/Xist, or, to a certain extent, the intergenic transcripts in V(D)J recombination (19,28). These transcripts are likely to recruit silencing factors [or in the case of V(D)J allow recruitment of a recombination machinery] but do not require an overlap with the cognate sense transcripts (2, 13, 27). The events ar...