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

Abstract

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.

Biological Functions of Natural Antisense Transcripts

“…3C). Endo-siRNAs were reported in various species, including plants (Borsani et al, 2005;Zhang et al, 2012;Li et al, 2013;Yu et al, 2016), fungi (Lee et al, 2010) and animals (Tam et al, 2008;Watanabe et al, 2008;Okamura et al, 2011;Werner et al, 2014;Ling et al, 2016). The mechanism of endo-siRNA maturation from NATderived dsRNA that is understood the most, suggests a Dicer-dependent character of dsRNA processing to short siRNA, followed by Argonaute loading into the molecular silencing machinery, RNA-induced silencing complex (RISC) (Czech & Hannon, 2011;Kwak & Tomari, 2012).…”

“…Genome-wide analyses using RNA-Seq and singlestranded RNA-Seq protocols, strengthened by parallel analyses of small RNA or degradome sequencing, were proposed in recent years to expand our knowledge of the NATs involved in the endogenous RNA interference Li et al, 2013;Werner et al, 2014;Yu et al, 2016). These studies revealed that nearly 4% of the Arabidopsis thaliana cis-NATs produce putative endo-siRNAs, and approximately 200 of these endo-siRNAs exhibit relatively high expression levels ≥ 10 RPKM (Reads Per Kilobase per Million mapped reads) (Li et al, 2013).…”

Transpositional shuffling and quality control in male germ cells to enhance evolution of complex organisms

“…Interestingly, both endo-siRNAs and pachytene piRNAs originate from protein-coding genes that are significantly enriched in antisense transcripts, suggesting that the two branches of RNA interference may be intertwined via common testis-specific effector proteins. 19,36 The molecular basis and genomic background of the mechanism in Figure 2 is explained in more detail in other papers. 33,37 In the first step of the proposed sequence of events (Fig.…”

RNA interference in early branching metazoans

“…Plants and brown algae also possess miRNA systems, however all three are considered analogous, having independently evolved following co-option of components of the shared eukaryotic endo-siRNA system (Jones- Rhoades et al 2006, Axtell et al 2011, Tarver et al 2012. Of the three RNAi systems, endo-siRNAs have received the least attention in animals, possibly due to their perceived lesser importance in vertebrates, although a renewed interest appears to be developing (Flemr et al 2013, Werner et al 2014, García-López et al 2014, Svoboda 2014. In fact, the capacity for artificial knockdown of target gene expression by hijacking the machinery of the endosiRNA pathway has long been known to be an effective strategy for experimental RNA interference in some vertebrate cell types (Wianny and Zernicka-Goetz 2000, Svoboda et al 2000, Elbashir et al 2001.…”