Dataset Information

Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering.

ABSTRACT: Chinese hamster ovary (CHO) cells are the predominant cell factory for the production of recombinant therapeutic proteins. Nevertheless, the lack in publicly available sequence information is severely limiting advances in CHO cell biology, including the exploration of microRNAs (miRNA) as tools for CHO cell characterization and engineering. In an effort to identify and annotate both conserved and novel CHO miRNAs in the absence of a Chinese hamster genome, we deep-sequenced small RNA fractions of 6 biotechnologically relevant cell lines and mapped the resulting reads to an artificial reference sequence consisting of all known miRNA hairpins. Read alignment patterns and read count ratios of 5' and 3' mature miRNAs were obtained and used for an independent classification into miR/miR* and 5p/3p miRNA pairs and discrimination of miRNAs from other non-coding RNAs, resulting in the annotation of 387 mature CHO miRNAs. The quantitative content of next-generation sequencing data was analyzed and confirmed using qPCR, to find that miRNAs are markers of cell status. Finally, cDNA sequencing of 26 validated targets of miR-17-92 suggests conserved functions for miRNAs in CHO cells, which together with the now publicly available sequence information sets the stage for developing novel RNAi tools for CHO cell engineering.

SUBMITTER: Hackl M

PROVIDER: S-EPMC3119918 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering.

Hackl Matthias M Jakobi Tobias T Blom Jochen J Doppmeier Daniel D Brinkrolf Karina K Szczepanowski Rafael R Bernhart Stephan H SH Höner Zu Siederdissen Christian C Bort Juan A Hernandez JA Wieser Matthias M Kunert Renate R Jeffs Simon S Hofacker Ivo L IL Goesmann Alexander A Pühler Alfred A Borth Nicole N Grillari Johannes J

Journal of biotechnology 20110330 1-2

Chinese hamster ovary (CHO) cells are the predominant cell factory for the production of recombinant therapeutic proteins. Nevertheless, the lack in publicly available sequence information is severely limiting advances in CHO cell biology, including the exploration of microRNAs (miRNA) as tools for CHO cell characterization and engineering. In an effort to identify and annotate both conserved and novel CHO miRNAs in the absence of a Chinese hamster genome, we deep-sequenced small RNA fractions o ...[more]

PMID: 21392545

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Project description:BACKGROUND: MicroRNA are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts through targeting of a microRNA-protein complex by base-pairing of the microRNA sequence to cognate recognition sequences in the 3' untranslated region (UTR) of the mRNA. Target identification for a given microRNA sequence is generally accomplished by informatics analysis of predicted mRNA sequences present in the genome or in databases of transcript sequence for the tissue of interest. However, gene models for porcine skeletal muscle transcripts in current databases, specifically complete sequence of the 3' UTR, are inadequate for this exercise. METHODOLOGY/PRINCIPAL FINDINGS: To provide data necessary to identify gene targets for microRNA in porcine skeletal muscle, normalized cDNA libraries were sequenced using Roche 454 GS-FLX pyrosequencing and de novo assembly of transcripts enriched in the 3' UTR was performed using the MIRA sequence assembly program. Over 725 million bases of sequence were generated, which assembled into 18,202 contigs. Sequence reads were mapped to a 3' UTR database containing porcine sequences. The 3' UTR that mapped to the database were examined to predict targets for previously identified microRNA that had been separately sequenced from the same porcine muscle sample used to generate the cDNA libraries. For genes with microRNA-targeted 3' UTR, KEGG pathways were computationally determined in order to identify potential functional effects of these microRNA-targeted transcripts. CONCLUSIONS: Through next-generation sequencing of transcripts expressed in skeletal muscle, mapping reads to a 3' UTR database, and prediction of microRNA target sites in the 3' UTR, our results identified genes expressed in porcine skeletal muscle and predicted the microRNA that target these genes. Additionally, identification of pathways regulated by these microRNA-targeted genes provides us with a set of genes that can be further evaluated for their potential role in skeletal muscle development and growth.

Dataset Information

Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering.

Publications

Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering.

Similar Datasets

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