Abstract
By coupling laser capture microdissection to nanoCAGE technology and next-generation sequencing we have identified the genome-wide collection of active promoters in the mouse Main Olfactory Epithelium (MOE). Transcription start sites (TSSs) for the large majority of Olfactory Receptors (ORs) have been previously mapped increasing our understanding of their promoter architecture. Here we show that in our nanoCAGE libraries of the mouse MOE we detect a large number of tags mapped in loci hosting Type-1 and Type-2 Vomeronasal Receptors genes (V1Rs and V2Rs). These loci also show a massive expression of Long Interspersed Nuclear Elements (LINEs). We have validated the expression of selected receptors detected by nanoCAGE with in situ hybridization, RT-PCR and qRT-PCR. This work extends the repertory of receptors capable of sensing chemical signals in the MOE, suggesting intriguing interplays between MOE and VNO for pheromone processing and positioning transcribed LINEs as candidate regulatory RNAs for VRs expression.
Highlights
Next-generation sequencing technologies have reshaped our understanding of the molecular constituents of cells and their regulatory elements
Two nanoCAGE libraries were synthesized from independent harvests and deeply sequenced using Illumina technology, yielding a total of 53,158,862 tags with a length of 25 bp and corresponding to the very 5 -end of Main Olfactory Epithelium (MOE) capped transcripts. 31,031,749 tags were confidently mapped to the mouse genome (Faulkner et al, 2008; Hashimoto et al, 2009)
The mapped nanoCAGE tags were clustered and aggregated into Tag Clusters (TCs) (Carninci et al, 2006), and the data were unified in publicly accessible tracks that can be uploaded in UCSC Genome Browser
Summary
Next-generation sequencing technologies have reshaped our understanding of the molecular constituents of cells and their regulatory elements. It is based on sequencing cDNA copies of the 5 ends of mRNAs, of which the integrity is inferred by the presence of their cap These sequences (“tags”) are sufficiently long to be aligned in most cases at a single location in the genome. The number of times a given tag is represented in a library gives an estimate of the expression level of the corresponding transcript. To expand this analysis to tiny amounts of ex vivo tissue and to the polyA− fraction of RNAs we have developed nanoCAGE, a technology that miniaturizes the requirement of CAGE for RNA material to the nanogram range and which can be used on fixed tissues (Plessy et al, 2010). We have used nanoCAGE to investigate the transcriptional landscape of the mouse Main Olfactory Epithelium (MOE) (Plessy et al, 2012)
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