Identification of ciliated sensory neuron-expressed genes in Caenorhabditis elegans using targeted pull-down of poly(A) tails - PubMed (original) (raw)

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

Hirofumi Kunitomo et al. Genome Biol. 2005.

Abstract

It is not always easy to apply microarray technology to small numbers of cells because of the difficulty in selectively isolating mRNA from such cells. We report here the preparation of mRNA from ciliated sensory neurons of Caenorhabditis elegans using the mRNA-tagging method, in which poly(A) RNA was co-immunoprecipitated with an epitope-tagged poly(A)-binding protein specifically expressed in sensory neurons. Subsequent cDNA microarray analyses led to the identification of a panel of sensory neuron-expressed genes.

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Figures

Figure 1

Figure 1

Principle of the mRNA-tagging method. Step 1, FLAG-tagged poly(A)-binding protein (PABP) is expressed from a transgene using a cell-specific promoter. Step 2, PABP and poly(A)+ RNA are crosslinked in situ by formaldehyde. Step 3, poly(A)-RNA/FLAG-PABP complexes are purified by anti-FLAG affinity purification. Step 4, RNA-PABP crosslinks are reversed and RNA is isolated. Step 5, purified RNA is used for microarray analysis.

Figure 2

Figure 2

Quantification of tissue-specific transcripts in RNA prepared by mRNA tagging. The transcript indicated on the left of each row was amplified by RT-PCR using gene-specific primers. Poly(A)+RNA from wild-type (WT) animals was used as a template in lane 1. RNA prepared by mRNA tagging from che-2::PABP (JN501), acr-5::PABP (JN502) and myo-3::PABP (JN503) was used in lanes 2, 3 and 4, respectively.

Figure 3

Figure 3

Rank orders of che-2::PABP/acr-5::PABP values for specific genes in the microarray analyses. (a) Distribution of genes with known expression patterns. Genes known to be specifically expressed in sensory neurons, motor neurons, muscles or the intestine, respectively, were collected from WormBase (see Materials and methods) and the rank orders of their che-2::PABP/acr-5::PABP signal ratios were plotted. Vertical bars indicate the medians. Genes expressed in sensory neurons are specifically enriched in the che-2::PABP RNA preparations, while motor neuron- and intestine-expressed genes are enriched in the acr-5::PABP RNA preparations. Note that although only five genes were found as motor neuron-expressed genes, nine data points were plotted in (a), because multiple cDNA clones were present on the microarray for three of the genes (see Additional data file 2). (b) Distribution of genes with X-boxes in their promoter regions. Genes that carry one or more X-boxes in their promoter regions were collected from the genome database (see Materials and methods) and their rank orders of che-2::PABP/acr-5::PABP signal ratios were plotted. These genes, which are expected to be expressed in ciliated sensory neurons under the control of the DAF-19 transcription factor, are also enriched in the che-2::PABP RNA preparations.

Figure 4

Figure 4

Expression patterns of newly identified sensory neuron-expressed genes. The genes indicated were each fused to GFP in-frame, and the reporters introduced into wild-type animals. Overlaid images of the Nomarski and GFP fluorescence images of transgenic worms between larval stages 1 and 3 are shown. Gene expression is indicated by the green fluorescence. Scale bar, 50 μm. See Table 1 for the identity of the expressing cells.

Figure 5

Figure 5

Sensory neuron-specific genes are less likely to be classified into Gene Ontology categories and more likely to be worm-specific. (a) All genes on the microarray were ordered by descending che-2::PABP/acr-5::PABP value and the fraction of GO-annotated genes in each bin is indicated for a bin width of 50 rank orders. Only the top 1,500 genes are shown in (a)-(c). (b) The fraction of genes with homologs in C. briggsae, and not in humans, mice, flies, fission yeast or budding yeast (cutoff BLASTP score E = 1 × 10-20) in each bin is indicated as in (a). (c) The fraction of genes with homologs in both animals and yeasts, namely in humans, mice or flies and in fission yeast or budding yeast (cutoff BLASTP score E = 1 × 10-20) in each bin is indicated as in (a). In all panels, the red dotted line indicates the average of all the genes, and the blue dotted lines indicate the 95% confidence limits assuming a random binominal distribution.

Figure 6

Figure 6

Categories of genes enriched in the sensory neuron fraction. Genes were categorized according to the GO molecular function categories. (a) Categorization of all the genes on the microarray; (b) categorization of genes within the top 500 che-2::PABP/acr-5::PABP ranks. In both panels, the fraction of genes in each category in respect of all annotated genes is shown.*P < 0.05; **P < 0.01 (binominal distribution).

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