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NanoCAGE analysis of the mouse olfactory epithelium identifies the expression of vomeronasal receptors and of proximal LINE elements.

Pascarella G, Lazarevic D, Plessy C, Bertin N, Akalin A, Vlachouli C, Simone R, Faulkner GJ, Zucchelli S, Kawai J, Daub CO, Hayashizaki Y, Lenhard B, Carninci P, Gustincich S - Front Cell Neurosci (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Area of Neuroscience, International School for Advanced Studies (SISSA) Trieste, Italy ; RIKEN Yokohama Institute, Center for Life Science Technologies, Division of Genomic Technologies Tsurumi-ku, Yokohama, Japan.

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.

No MeSH data available.


Related in: MedlinePlus

Validation of nanoCAGE data by RT-PCR confirms the expression in the MOE of V1Rs, V2Rs, and key components of the pheromone transduction pathway. (A) RT-PCR validation was carried out starting from the same total RNA sample of the MOE used for the nanoCAGE workflow. V1Rs and V2Rs to be validated were chosen by interest or on the basis of their TPM score from the list of all expressed VRs detected by nanoCAGE. DNA molecular weight Marker VI is used as DNA ladder (Roche Applied Science). (B) The TSS of Vmn2r69 identified by nanoCAGE was validated by RT-PCR with a forward primer designed in proximity of the TSSs and a reverse primer designed on the first exon. The sequence of the RT-PCR product is shown uploaded in the UCSC Genome Browser along with the nanoCAGE data.
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Figure 2: Validation of nanoCAGE data by RT-PCR confirms the expression in the MOE of V1Rs, V2Rs, and key components of the pheromone transduction pathway. (A) RT-PCR validation was carried out starting from the same total RNA sample of the MOE used for the nanoCAGE workflow. V1Rs and V2Rs to be validated were chosen by interest or on the basis of their TPM score from the list of all expressed VRs detected by nanoCAGE. DNA molecular weight Marker VI is used as DNA ladder (Roche Applied Science). (B) The TSS of Vmn2r69 identified by nanoCAGE was validated by RT-PCR with a forward primer designed in proximity of the TSSs and a reverse primer designed on the first exon. The sequence of the RT-PCR product is shown uploaded in the UCSC Genome Browser along with the nanoCAGE data.

Mentions: We validated the nanoCAGE data of selected genes by performing a standard RT-PCR starting from the same total RNA sample used for the synthesis of the nanoCAGE libraries. We amplified transcripts for the V2Rs family (Vmn2r29, Vmn2r69, and Vmn2r95), the V1Rs family (Vmn1r51, Vmn1r50, Vmn1r10) and the components of the vomeronasal transduction pathway (Gαo, Gαi2, and Trpc2); Omp was chosen as a positive control for its high expression in the MOE (Figure 2A). Because of the high level of intra-cluster homology, RT-PCRs for Vmn2r29 and Vmn2r95 amplified additional V2Rs including Vmn2r30, Vmn2r31, and Vmn2r42 on chromosome 7 as well as Vmn2r104 and Vmn2r107 on chromosome 17. We also validated the expression of Vmn2r26, the only V2Rs that has been experimentally proven to bind a MHC class I peptide in the VNO (Leinders-Zufall et al., 2009) and for which nanoCAGE identified a sharp TSS mapped 105 bases upstream of the annotated Refseq gene. Cloning and sequencing confirmed the identity of all validated transcripts.


NanoCAGE analysis of the mouse olfactory epithelium identifies the expression of vomeronasal receptors and of proximal LINE elements.

Pascarella G, Lazarevic D, Plessy C, Bertin N, Akalin A, Vlachouli C, Simone R, Faulkner GJ, Zucchelli S, Kawai J, Daub CO, Hayashizaki Y, Lenhard B, Carninci P, Gustincich S - Front Cell Neurosci (2014)

Validation of nanoCAGE data by RT-PCR confirms the expression in the MOE of V1Rs, V2Rs, and key components of the pheromone transduction pathway. (A) RT-PCR validation was carried out starting from the same total RNA sample of the MOE used for the nanoCAGE workflow. V1Rs and V2Rs to be validated were chosen by interest or on the basis of their TPM score from the list of all expressed VRs detected by nanoCAGE. DNA molecular weight Marker VI is used as DNA ladder (Roche Applied Science). (B) The TSS of Vmn2r69 identified by nanoCAGE was validated by RT-PCR with a forward primer designed in proximity of the TSSs and a reverse primer designed on the first exon. The sequence of the RT-PCR product is shown uploaded in the UCSC Genome Browser along with the nanoCAGE data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927265&req=5

Figure 2: Validation of nanoCAGE data by RT-PCR confirms the expression in the MOE of V1Rs, V2Rs, and key components of the pheromone transduction pathway. (A) RT-PCR validation was carried out starting from the same total RNA sample of the MOE used for the nanoCAGE workflow. V1Rs and V2Rs to be validated were chosen by interest or on the basis of their TPM score from the list of all expressed VRs detected by nanoCAGE. DNA molecular weight Marker VI is used as DNA ladder (Roche Applied Science). (B) The TSS of Vmn2r69 identified by nanoCAGE was validated by RT-PCR with a forward primer designed in proximity of the TSSs and a reverse primer designed on the first exon. The sequence of the RT-PCR product is shown uploaded in the UCSC Genome Browser along with the nanoCAGE data.
Mentions: We validated the nanoCAGE data of selected genes by performing a standard RT-PCR starting from the same total RNA sample used for the synthesis of the nanoCAGE libraries. We amplified transcripts for the V2Rs family (Vmn2r29, Vmn2r69, and Vmn2r95), the V1Rs family (Vmn1r51, Vmn1r50, Vmn1r10) and the components of the vomeronasal transduction pathway (Gαo, Gαi2, and Trpc2); Omp was chosen as a positive control for its high expression in the MOE (Figure 2A). Because of the high level of intra-cluster homology, RT-PCRs for Vmn2r29 and Vmn2r95 amplified additional V2Rs including Vmn2r30, Vmn2r31, and Vmn2r42 on chromosome 7 as well as Vmn2r104 and Vmn2r107 on chromosome 17. We also validated the expression of Vmn2r26, the only V2Rs that has been experimentally proven to bind a MHC class I peptide in the VNO (Leinders-Zufall et al., 2009) and for which nanoCAGE identified a sharp TSS mapped 105 bases upstream of the annotated Refseq gene. Cloning and sequencing confirmed the identity of all validated transcripts.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Area of Neuroscience, International School for Advanced Studies (SISSA) Trieste, Italy ; RIKEN Yokohama Institute, Center for Life Science Technologies, Division of Genomic Technologies Tsurumi-ku, Yokohama, Japan.

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.

No MeSH data available.


Related in: MedlinePlus