Endogenous short interfering RNAs (endo-siRNAs) have recently emerged as versatile regulators of gene expression. They derive from double stranded intrinsic transcripts and are processed by Dicer and associate with Argonaute proteins. In Caenorhabditis elegans, endo-siRNAs are known as 22G and 26G RNAs and are involved in genome protection and gene regulation. Drosophila melanogaster endo-siRNAs are produced with the help of specific Dicer and Argonaute isoforms and play an essential role in transposon control and the protection from viral infections. Biological functions of endo-siRNAs in vertebrates include repression of transposable elements, chromatin organisation as well as gene regulation at transcriptional and post-transcriptional level.

Complex organisms, particularly mammals, have long generation times and produce small numbers of progeny that undergo increasingly entangled developmental programs. This reduces the ability of such organisms to explore evolutionary space, and, consequently, strategies that mitigate this problem likely have a strategic advantage. Here, we suggest that animals exploit the controlled shuffling of transposons to enhance genomic variability in conjunction with a molecular screening mechanism to exclude deleterious events. Accordingly, the removal of repressive DNA-methylation marks during male germ cell development is an evolved function that exploits the mutagenic potential of transposable elements. A wave of transcription during the meiotic phase of spermatogenesis produces the most complex transcriptome of all mammalian cells, including genic and noncoding sense–antisense RNA pairs that enable a genome-wide quality-control mechanism. Cells that fail the genomic quality test are excluded from further development, eventually resulting in a positively selected mature sperm population. We suggest that these processes, enhanced variability and stringent molecular quality control, compensate for the apparent reduced potential of complex animals to adapt and evolve.

Natural antisense transcripts (NATs) can interfere with the expression of complementary sense transcripts with exquisite specificity. We have previously cloned NATs of Slc34a loci (encoding Na-phosphate transporters) from fish and mouse. Here we report the cloning of a human SLC34A1-related NAT that represents an alternatively spliced PFN3 transcript (Profilin3). The transcript is predominantly expressed in testis. Phylogenetic comparison suggests two distinct mechanisms producing Slc34a-related NATs: Alternative splicing of a transcript from a protein coding downstream gene (Pfn3, human/mouse) and transcription from the bi-directional promoter (Rbpja, zebrafish). Expression analysis suggested independent regulation of the complementary Slc34a mRNAs. Analysis of randomly selected bi-directionally transcribed human/mouse loci revealed limited phylogenetic conservation and independent regulation of NATs. They were reduced on X chromosomes and clustered in regions that escape inactivation. Locus structure and expression pattern suggest a NATs-associated regulatory mechanisms in testis unrelated to the physiological role of the sense transcript encoded protein.

Natural antisense transcripts (NATs) are complementary to protein coding genes and potentially regulate their expression. Despite widespread occurrence of NATs in the genomes of higher eukaryotes, their biological role and mechanism of action is poorly understood. Zebrafish embryos offer a unique model system to study sense-antisense transcript interplay at whole organism level. Here, we investigate putative antisense transcript-mediated mechanisms by ectopically co-expressing the complementary transcripts during early zebrafish development. In zebrafish the gene Slc34a2a (Na-phosphate transporter) is bi-directionally transcribed, the NAT predominantly during early development up to 48 hours after fertilization. Declining levels of the NAT, Slc34a2a(as), coincide with an increase of the sense transcript. At that time, sense and antisense transcripts co-localize in the endoderm at near equal amounts. Ectopic expression of the sense transcript during embryogenesis leads to specific failure to develop a cerebellum. The defect is RNA-mediated and dependent on sense-antisense complementarity. Overexpression of a Slc34a2a paralogue (Slc34a2b) or the NAT itself had no phenotypic consequences. Knockdown of Dicer rescued the brain defect suggesting that RNA interference is required to mediate the phenotype. Our results corroborate previous reports of Slc34a2a-related endo-siRNAs in two days old zebrafish embryos and emphasize the importance of coordinated expression of sense-antisense transcripts. Our findings suggest that RNAi is involved in gene regulation by certain natural antisense RNAs.