Development of a rapid and inexpensive method to reveal natural antisense transcripts

Silvio Collani,Gianni Barcaccia

doi:10.1186/1746-4811-8-37

Abstract

BackgroundNatural antisense transcripts (NATs) are a group of RNAs encoded within a cell that have transcript complementarity to other RNA transcripts. NATs have been identified in multiple eukaryotes, including humans, mice, yeast and several plants, and are known to play crucial roles in gene regulation and modification via RNA interference, alternative splicing and genomic imprinting. NATs are also involved in several human diseases.ResultsWe describe a novel method to detect the occurrence of target NATs in specific plant tissues. This method differs from the others currently used in molecular biology laboratories for a number of reasons, particularly the simplicity and versatility of application, low cost and lower material requirement. We demonstrate that NATs can be detected by using diluted cDNA, avoiding the need for a large amount of RNA, thus differing from basic techniques, such as Northern blot hybridisation and reverse-transcription PCR amplification. Furthermore, our method also allows the precise detection of long NATs and their cloning into plasmid vectors for downstream applications. We also reported the first case of a tissue-specific NAT occurring in Oleaceae family and, the antisense orientation of this transcript, allows the splicing of two introns otherwise impossible in the sense orientation.ConclusionsThis method is the first that combines the polymerisation and cleavage activity of DNA polymerase and exonuclease enzymes, respectively, to discover NATs in living organisms. It may simplify the discovery of NATs in plants providing a new strategy for an easy identification and characterization of this group of RNA molecules. Furthermore, since NATs are found in multiple eukaryotes, our method can be easily applied to a wide range of organisms, including human, mice and yeast.

Highlights

Natural antisense transcripts (NATs) are a group of RNAs encoded within a cell that have transcript complementarity to other RNA transcripts
It is worth noting that, during RNA retro-transcription using gene-specific primers (GSPs), some GSPs fail to prime cDNA synthesis, even though they work well in PCR using DNA templates
To the best of our knowledge, this method is the first that combines the more amenable condition of using DNA instead of RNA with the polymerisation and cleavage activity of DNA polymerase and exonuclease enzymes, respectively, allowing for the precise detection of NATs in living organisms and providing several advantages compared to the conventional methods currently used

Summary

Introduction

Natural antisense transcripts (NATs) are a group of RNAs encoded within a cell that have transcript complementarity to other RNA transcripts. NATs have been identified in multiple eukaryotes, including humans, mice, yeast and several plants, and are known to play crucial roles in gene regulation and modification via RNA interference, alternative splicing and genomic imprinting. The cis-NATs, which are transcribed from the reverse-complementary strand of an annotated gene and, are fully or partially overlapping with their respective mRNAs, are known as antisense RNAs (asRNAs) [2]. NATs were found to be implicated in a wide range of aspects of gene expression in eukaryotes, including genomic imprinting, RNA interference, translational regulation, alternative splicing, X-inactivation and RNA editing [1,11,12,13,14,15]. It is known that NATs may be involved in several human diseases and in several responses to stress in plants [14,16]

Results

Discussion

Conclusion