Non−coding RNAs in CHO cells and their contribution to genome stability and process−relevant phenotypes

Background.

Over the last few years genome and transcriptome information for Chinese Hamster ovary cells has become publicly available (see Brinkrolf 2013, Lewis 2013, Rupp 2014, Xu 2011 above). Our group contributed significantly to the knowledge on expressed microRNAs (Hackl et al., 2011 and 2012a) and piRNAs (Gerstl et al., 2013), has developed relevant protocols for evaluation of their phenotypic effects in CHO cells (Hernandez Bort et al., 2012; Jadhav et al., 2012 and 2014; Hackl et al. 2014; Klanert et al., 2014), and outlined approaches for the use of these small regulators of phenotype as engineering targets (Jadhav et al., 2013; Hackl et al., 2012b). piRNAs for instance are described as preventing translocation of transposons in sperm cells, however we have observed that they are differentially expressed in a variety of CHO cell types and culture states, indicating that their function in this cell line is not yet entirely understood. In addition, cells express many more categories of non-coding RNAs (Hangauer et al., 2013), whose mechanism, regulation and impact on recombinant protein production may be of high relevance to applications of CHO cells for this purpose. Such ncRNAs are involved in global and gene-specific control of transcription and silencing (Bergmann and Spector, 2014; Marques et al., 2013; Karta and Subramanian, 2014), in mRNA splicing (Cech and Steitz, 2014), and in the processing of the protein translational machinery including tRNAs and rRNAs (Martin et al., 2013) as well as in regulation of metabolism (Kornfeld and Brüning, 2014). All of these biological functions are of prime importance in the context of recombinant protein production in CHO cells.

Aims and methods.

The aim of this project is to characterise the regulatory network of ncRNAs in the context of transcription and translation as well as control of chromatin structure and genome stability in recombinant CHO cell lines. With several genome sequences of CHO cell lines and the Chinese Hamster reference genome available, the first approach will be the identification and annotation of all known non-coding RNAs in these genomes, followed by generation of RNA-seq data to identify those that are expressed in different cell lines and under defined culture conditions. The gained information will be included in the current effort of the scientific community to generate a high-quality annotated reference genome for the Chinese Hamster. Based on the RNA-seq data, characteristic expression patterns that correlate to defined phenotypes will be established and verified by overexpression or knockout strategies.

Precise understanding of the molecular mechanisms used by ncRNAs to impact transcription and translation as well as chromatin structure could have many applications: as ncRNA are typically involved in rapid responses to stress, their expression patterns could serve as predictive reporters for process optimisation; their involvement in protein synthesis could be used for selection of cells with the capacity to handle high protein production loads; and finally their expression could be engineered to optimise cell factories, although their complex and interlinked network will require a detailed understanding of their function and differential responses to allow for that in the future. As ncRNAs are also involved in epigenetic regulation, the results of this project may generate synergies and cross-fertilisation with the results from the project on genome and epigenome variation. 

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