title: Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3'-end-seq.
abstract: Despite the many advantages of Caenorhabditis elegans, biochemical approaches to study tissue-specific gene expression in post-embryonic stages are challenging. Here, we report a novel experimental approach for efficient determination of tissue-specific transcriptomes involving the rapid release and purification of nuclei from major tissues of post-embryonic animals by fluorescence-activated nuclei sorting (FANS), followed by deep sequencing of linearly amplified 3'-end regions of transcripts (3'-end-seq). We employed these approaches to compile the transcriptome of the developed C. elegans intestine and used this to analyse tissue-specific cleavage and polyadenylation. In agreement with intestinal-specific gene expression, highly expressed genes have enriched GATA-elements in their promoter regions and their functional properties are associated with processes that are characteristic for the intestine. We systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including more than 3000 sites that have previously not been identified. The detailed analysis of the 3'-ends of the nuclear mRNA revealed widespread alternative polyA site use (APA) in intestinally expressed genes. Importantly, we found that intestinal polyA sites that undergo APA tend to have U-rich and/or A-rich upstream auxiliary elements that may contribute to the regulation of 3'-end formation in the intestine.
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Simon
authors.initials: S
authors.lastname: Haenni
authors.forename: Zhe
authors.initials: Z
authors.lastname: Ji
authors.forename: Mainul
authors.initials: M
authors.lastname: Hoque
authors.forename: Nigel
authors.initials: N
authors.lastname: Rust
authors.forename: Helen
authors.initials: H
authors.lastname: Sharpe
authors.forename: Ralf
authors.initials: R
authors.lastname: Eberhard
authors.forename: Cathy
authors.initials: C
authors.lastname: Browne
authors.forename: Michael O
authors.initials: MO
authors.lastname: Hengartner
authors.forename: Jane
authors.initials: J
authors.lastname: Mellor
authors.forename: Bin
authors.initials: B
authors.lastname: Tian
authors.forename: André
authors.initials: A
authors.lastname: Furger
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000235:genetics; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D007413:Intestinal Mucosa / Q000378:metabolism; D026723:Polyadenylation; D017423:Sequence Analysis, RNA; D059467:Transcriptome