PPubMed31 Jan 2005Cited 90×

Identification of ciliated sensory neuron-expressed genes in <i> Caenorhabditis elegans </i> using targeted pull-down of poly(A) tails

Hirofumi Kunitomo, Hiroko Uesugi, Yuji Kohara, Yuichi Iino

Abstract

An mRNA-tagging method was used to selectively isolate mRNA from a small number of cells for subsequent cDNA microarray analysis. The approach was used to identify genes specifically expressed in ciliated sensory neurons of Caenorhabditis elegans.

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A targeted mRNA-tagging method isolated and identified genes specifically expressed in C. elegans ciliated sensory neurons, validating the approach with microarray enrichment.

What’s new

i.Developed an mRNA‑tagging protocol using FLAG‑PAB‑1 to isolate poly(A) RNA from defined neuron types
ii.Performed cDNA microarray analysis to rank genes enriched in che‑2‑expressing ciliated sensory neurons
iii.Validated enrichment by showing 13 of 15 tested genes (87%) were expressed in sensory neurons
iv.Identified nematode‑specific genes and protein motifs enriched among top‑ranked genes
v.Linked C. elegans homologs of human nephrocystin disease genes to sensory neuron expression

How they did it

The authors expressed FLAG‑tagged poly(A)‑binding protein (PAB‑1) under a che‑2 promoter to bind polyadenylated RNAs in ciliated sensory neurons. After formaldehyde cross‑linking, FLAG‑PAB‑1‑RNA complexes were immunoprecipitated, purified, and labeled for hybridization to a 8,348‑spot cDNA microarray. Gene enrichment was assessed by the Cy5/Cy3 intensity ratio between che‑2 and acr‑5 samples.

What they found

Microarray contained 8,348 cDNA spots representing 7,088 C. elegans genes
20 known sensory‑neuron genes and 5 motor‑neuron genes were present on the array, with sensory genes ranking highest
13 of 15 genes (87%) tested by reporter analysis were expressed in sensory neurons
X‑box‑containing promoter genes showed highly uneven enrichment, confirming specificity
Nephrocystin homologs ranked at positions 15 and 25, suggesting a link to sensory neuron function

Caveats

✕Enrichment of motor‑neuron genes was less efficient, with weak RT‑PCR bands observed
✕PAB‑1 may bind some transcripts with higher affinity, causing bias in recovered RNAs
✕Using a cDNA microarray covered only ~40% of predicted genes, missing many sensory‑neuron genes
✕Potential loss of transcripts with low poly(A) tail accessibility during immunoprecipitation

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Identification of ciliated sensory neuron-expressed genes in <i> Caenorhabditis elegans </i> using targeted pull-down of poly(A) tails

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