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Natural 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 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.Natural 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.