Detection of naturally occurring RNA aptamers using a metagenomic DNA library

Over the past few decades, the idea of RNA transcripts as passive genetic messengers within biological systems has changed significantly. RNAs, unlike DNA, possess an intrinsic capability to relay genetic information by actively participating in biochemical interactions that influence cellular activity. Riboswitches, or ligand-sensitive genetic elements, are functional RNAs within nascent and messenger RNA transcripts that consist of a structurally conserved aptamer domain and an expression platform capable of regulating gene expression at the level of transcription or translation. Despite the numerous examples of prokaryotic riboswitches, the thiamine pyrophosphate (TPP) riboswitch is the only known functional riboswitch in eukaryotes. The existence of multiple human adenosine aptamers encoded within the human genome suggest that eukaryotic riboswitch activity may be more prevalent than previously thought. To investigate the diversity of eukaryotic aptamers and elucidate their biological roles, we designed an in vitro selection scheme to select for naturally occurring RNA aptamers from a metagenomic DNA library and monitored the expression of the Ras-related protein 3C (RAB3C) adenosine aptamer by reverse transcriptase – quantitative polymerase chain reaction (RT-qPCR). Libraries enriched for adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) RNA aptamers were analyzed by deep sequencing and Apta-seq methods. Computational analysis of enriched libraries by deep sequencing showed that a significant fraction of sequences from both ATP and cAMP selections were repetitive elements. Sequence alignments and Apta-seq analysis found that these specific repetitive elements include endogenous retroviral elements and CG-rich repeats, and not simple repeats such as telomeric or ribosomal RNA elements. Binding activity assays showed that individual sequences isolated from the enriched libraries lost specificity to either ATP or cAMP. Binding activity was recovered upon reintroducing a fraction of the enriched library suggesting that ligand binding may require a binding partner. RT-qPCR assays revealed changes in 3' untranslated region (UTR) expression of the RAB3C gene in the presence of adenosine. Column binding assays using total RNA isolated from these expression assays showed that these human adenosine aptamers bind to ATP in the context of their natural transcripts. This result highlights the biological significance of naturally occurring aptamers in eukaryotes