Engineering Translation in Mammalian Cell Factories to Increase Protein Yield: The Unexpected Use of Long Non-Coding SINEUP RNAs

Zucchelli, S.; Patrucco, Laura; Persichetti, Francesca; Gustincich, Stefano; Cotella, Diego

doi:10.1016/j.csbj.2016.10.004

Cited by 35 publications

(25 citation statements)

References 114 publications

(127 reference statements)

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“…As shown earlier, lncRNAs are deemed to be an essential regulator in almost all biological processes. Moreover, a recent study reported that a family of antisense lncRNAs (SINEUPs) are able to increase translation of partially overlapping protein‐coding messenger RNAs (mRNAs) (Zucchelli et al ., ). Therefore, it is valuable to know whether lncRNAs relate to silk yield variation between domestic and wild silkworms.…”

Section: Introductionmentioning

confidence: 97%

Identification and comparison of long non‐coding RNAs in the silk gland between domestic and wild silkworms

et al. 2017

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Under long-term artificial selection, the domestic silkworm (Bombyx mori) has increased its silk yield tremendously in comparison with its wild progenitor, Bombyx mandarina. However, the molecular mechanism of silk yield increase is still unknown. Comparative analysis of long non-coding RNAs (lncRNAs) may provide some insights into understanding this phenotypic variation. In this study, using RNA sequencing technology data of silk gland in domestic and wild silkworms, we identified 599 lncRNAs in the silk gland of the silkworm. Compared with protein-coding genes, the silk gland lncRNA genes tend to have fewer exon numbers, shorter transcript length and lower GC-content. Moreover, we found that three lncRNA genes are significantly and differentially expressed between domestic and wild silkworms. The potential targets of two differentially expressed lncRNAs (DELs) (dw4sg_0040 and dw4sg_0483) and the expression-correlated genes with the two DELs are mainly enriched in the related processes of silk protein translation. This implies that these DELs may affect the phenotypic variation in silk yield between the domestic and wild silkworms through the post-transcriptional regulation of silk protein.

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Section: Introductionmentioning

confidence: 97%

Identification and comparison of long non‐coding RNAs in the silk gland between domestic and wild silkworms

et al. 2017

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“…As the majority of the lncRNAs reported in the literature are discussed and related to human or model organism systems, our work aimed at unveiling the role of lncRNAs in CHO under industrially relevant conditions to identify new targets for manipulation to sustain proliferation. Examples of successful cell engineering of lncRNAs to selectively enhance translation and product yield have already been reported in CHO, demonstrating the potential of manipulation of lncRNAs for enhancing industrial processes. Moreover, since it was reported up to 15% of the total ribosome occupancy can be occupied by a single recombinant mRNA, the intrinsic characteristics of lncRNAs place them as ideal candidates for cell line engineering of protein production cell factories, as they do not add any translational burden on top of the coding gene of interest…”

Section: Discussionmentioning

confidence: 99%

“…Despite the importance of lncRNAs in controlling cellular processes, and unlike small non‐coding RNAs (e.g., siRNAs, microRNAs), the impact(s) of lncRNA expression on CHO cell bioprocessing with regard to growth/proliferation and recombinant protein yields and quality has barely been explored, with only a small number of studies reported . However, these two studies show that manipulation of lncRNAs can impact upon recombinant protein production from CHO cells.…”

Section: Introductionmentioning

confidence: 99%

The Long Non‐Coding RNA Transcriptome Landscape in CHO Cells Under Batch and Fed‐Batch Conditions

Vito

Smales

2018

Biotechnology Journal

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The role of non-coding RNAs in determining growth, productivity, and recombinant product quality attributes in Chinese hamster ovary (CHO) cells has received much attention in recent years, exemplified by studies into microRNAs in particular. However, other classes of non-coding RNAs have received less attention. One such class are the non-coding RNAs known collectively as long non-coding RNAs (lncRNAs). The authors have undertaken the first landscape analysis of the lncRNA transcriptome in CHO using a mouse based microarray that also provided for the surveillance of the coding transcriptome. The authors report on those lncRNAs present in a model host CHO cell line under batch and fed-batch conditions on two different days and relate the expression of different lncRNAs to each other. The authors demonstrate that the mouse microarray is suitable for the detection and analysis of thousands of CHO lncRNAs and validated a number of these by qRT-PCR. The authors then further analyzed the data to identify those lncRNAs whose expression changed the most between growth and stationary phases of culture or between batch and fed-batch culture to identify potential lncRNA targets for further functional studies with regard to their role in controlling growth of CHO cells. The authors discuss the implications for the publication of this rich dataset and how this may be used by the community.

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“…LncRNAs are able to regulate manyfold cellular processes (Geisler & Coller, ; Wang & Chang, ) and became as well of interest for cell line engineering as a lncRNA with a complementary sequence to the NF‐kappa‐B inhibitor alpha (NFKBIA) was identified to enhance culture longevity and productivity when stably overexpressed (Tabuchi, ). Moreover, so called SINEUP lncRNAs are of great potential for cell line engineering as they can be designed to target a specific mRNA to increase translation (Zucchelli, Patrucco, Persichetti, Gustincich, & Cotella, ). However, the function of the vast majority of lncRNAs is still unknown complicating the analysis of potential pathways induced by these RNA molecules.…”

Section: Discussionmentioning

confidence: 99%

Stable miRNA overexpression in human CAP cells: Engineering alternative production systems for advanced manufacturing of biologics using miR‐136 and miR‐3074

Weis

Guth

Fischer

et al. 2018

Biotech & Bioengineering

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Chinese hamster ovary (CHO) cells still represent the major production host for therapeutic proteins. However, multiple limitations have been acknowledged leading to the search for alternative expression systems. CEVEC's amniocyte production (CAP) cells are human production cells demonstrated to enable efficient overexpression of recombinant proteins with human glycosylation pattern. However, CAP cells have not yet undergone any engineering approaches to optimize process parameters for a cheaper and more sustainable production of biopharmaceuticals. Thus, we assessed the possibility to enhance CAP cell production capacity via cell engineering using miRNA technology. Based on a previous high-content miRNA screen in CHO-SEAP cells, selected pro-productive miRNAs including, miR-99b-3p, 30a-5p, 329-3p, 483-3p, 370-3p, 219-1-3p, 3074-5p, 136-3p, 30e-5p, 1a-3p, and 484-5p, were shown to act pro-productive and product independent upon transient transfection in CAP and CHO antibody expressing cell lines. Stable expression of miRNAs established seven CAP cell pools with an overexpression of the pro-productive miRNA strand. Subsequent small-scale screening as well as upscaling batch experiments identified miR-136 and miR-3074 to significantly increase final mAb concentration in CAP-mAb cells. Transcriptomic changes analyzed by microarrays identified several lncRNAs as well as growth and apoptosis-related miRNAs to be differentially regulated in CAP-mAb-miR-136 and -miR-3074. This study presents the first engineering approach to optimize the alternative human expression system of CAP-cells.

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Engineering Translation in Mammalian Cell Factories to Increase Protein Yield: The Unexpected Use of Long Non-Coding SINEUP RNAs

Cited by 35 publications

References 114 publications

Identification and comparison of long non‐coding RNAs in the silk gland between domestic and wild silkworms

Identification and comparison of long non‐coding RNAs in the silk gland between domestic and wild silkworms

The Long Non‐Coding RNA Transcriptome Landscape in CHO Cells Under Batch and Fed‐Batch Conditions

Stable miRNA overexpression in human CAP cells: Engineering alternative production systems for advanced manufacturing of biologics using miR‐136 and miR‐3074

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