Abstract
Selection of mammalian high-producer cell lines remains a major challenge for the biopharmaceutical manufacturing industry. Ribosomal RNA (rRNA) genes encode the major component of the ribosome but many rRNA gene copies are not transcribed [1]–[5] due to epigenetic silencing by the nucleolar remodelling complex (NoRC) [6], which may limit the cell's full production capacity. Here we show that the knockdown of TIP5, a subunit of NoRC, decreases the number of silent rRNA genes, upregulates rRNA transcription, enhances ribosome synthesis and increases production of recombinant proteins. However, general enhancement of rRNA transcription rate did not stimulate protein synthesis. Our data demonstrates that the number of transcriptionally competent rRNA genes limits efficient ribosome synthesis. Epigenetic engineering of ribosomal RNA genes offers new possibilities for improving biopharmaceutical manufacturing and provides novel insights into the complex regulatory network which governs the translation machinery in normal cellular processes as well as in pathological conditions like cancer.
Highlights
On the way from DNA to product translation is a major bottleneck which may limit the specific productivity of mammalian production cell lines
Ribosome assembly occurs within the nucleolus and requires coordinated expression of four ribosomal RNA (rRNA) (45S pre-rRNA, which is subsequently processed into 18S, 5.8S, 28S and 5S rRNA) and about 80 ribosomal proteins (r-proteins) [8]. 45S pre-rRNA is transcribed in the nucleolus by polymerase I (Pol I), 5S RNA is transcribed by Pol III at the nucleolar periphery and imported into the nucleolus [9] and r-proteins are transcribed by Pol II
With the aim of engineering cells for increased synthesis of recombinant proteins, we determined whether a decrease in the number of silent rRNA genes enhances 45S pre-rRNA synthesis and, as consequence, stimulates ribosome biogenesis and increases the number of translation-competent ribosomes
Summary
On the way from DNA to product translation is a major bottleneck which may limit the specific productivity of mammalian production cell lines. Cells can upregulate the rate of protein synthesis either by increasing the translational efficiency of existing ribosomes or by increasing the capacity of translation through the production of new ribosomes (ribosome biogenesis) [7]. With about 80% of total nuclear transcription being dedicated to the synthesis of ribosomal RNA (rRNA), ribosome biogenesis is one of the major metabolic activity of mammalian cells. 45S pre-rRNA is transcribed in the nucleolus by polymerase I (Pol I), 5S RNA is transcribed by Pol III at the nucleolar periphery and imported into the nucleolus [9] and r-proteins are transcribed by Pol II. Ribosome biogenesis requires orchestration of transcription by different polymerases operating in different compartments. In mammalian cells, these processes are largely unknown
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