miR-CATCH identifies biologically active miRNA regulators of the pro-survival gene XIAP, in Chinese hamster ovary cells

Genetic engineering of mammalian cells, in particular Chinese hamster ovary (CHO) cells, is of critical interest to the biopharmaceutical industry as a means to further boost the yields of therapeutic proteins. Complimentary to already in place advanced bioprocesses, stable overexpression of the pro-survival X-linked inhibitor of apoptosis (XIAP) is one example of the successful manipulation of CHO cell genetics resulting in prolonged culture survival, ultimately increasing recombinant protein productivity. However, saturation or burdening of the cells translational machinery can occur in instances of forced expression of a trans-gene thereby achieving the anticipated cellular phenotype without the associated improvement in productivity. Ribosomal footprint sequencing has demonstrated that ~15% of an IgG-producing CHO cell translatome is occupied by the Neomycin selection marker. microRNAs (miRNAs) have the ability to fine tune endogenous gene expression thereby achieving elevated gene levels without the excess that could negatively impact global gene expression. Additionally, not only does a single miRNA have the capacity to regulate multiple mRNA transcripts simultaneously but individual mRNAs can be regulated by a multitude of miRNAs at the post-transcriptional level. This can facilitate the maximal translation of an endogenous gene without surpassing the superphysiological threshold associated with diminished productivity. The promiscuous nature of miRNA represented by the variety of binding patterns associated with mRNA targeting limits the predictability of high confidence miRNA regulators of attractive engineering candidates. This results in a lengthy list of falsely predicted in-silico miRNA regulators for a single gene. We explored the identification of direct miRNA regulators of the pro-survival endogenous XIAP gene in CHO-K1 cells by using a miR-CATCH1 protocol. A biotin-tagged antisense DNA oligonucleotide was designed for an exposed predicted secondary structure loop of endogenous CHO XIAP. This mRNA anchor resulted in the pulldown of XIAP and all associated RNA/protein complexes thereby enriching for all bound miRNAs. Two miRNAs were chosen out of the 14 miRNAs identified for further validation, miR-124-3p and miR-19b-3p. Transient transfection of mimics for both resulted in the diminished translation of endogenous CHO XIAP protein whereas their inhibition increased XIAP protein levels (Fig. 1). Please click Additional Files below to see the full abstract

Improved yield of rhEPO in CHO cells with synthetic 5′ UTR

The impact of local structure on mRNA translation is not well-defined pertaining to the 5′ UTR. Reports suggest structural remodelling of the 5′ UTR can significantly influence mRNA translation both in cis and trans however a new layer of complexity has been applied to this model with the now known reversible post-transcriptional chemical modification of RNA. N6-methyladenosine (m6A) is the most abundant internal base modification in mammalian mRNA. It has been reported that mRNAs harbouring m6A motifs in their 5′ UTR have improved translation efficiency. The present study evaluated the addition of putative m6A motifs to the 5′ UTR of a model recombinant human therapeutic glycoprotein, Erythropoietin (EPO), in a direct comparison with an A to T mutant and a no adenosine control. The m6A construct yielded significantly improved EPO titer in transient batch culture over no adenosine and m6T controls by 2.84 and 2.61-fold respectively. This study highlights that refinement of transgene RNA elements can yield significant improvements to protein titer.Science Foundation IrelandUpdate issue date during checkdate report - A

Improved yield of rhEPO in CHO cells with synthetic 5′ UTR

The impact of local structure on mRNA translation is not well-defined pertaining to the 5′ UTR. Reports suggest structural remodelling of the 5′ UTR can significantly influence mRNA translation both in cis and trans however a new layer of complexity has been applied to this model with the now known reversible post-transcriptional chemical modification of RNA. N6-methyladenosine (m6A) is the most abundant internal base modification in mammalian mRNA. It has been reported that mRNAs harbouring m6A motifs in their 5′ UTR have improved translation efficiency. The present study evaluated the addition of putative m6A motifs to the 5′ UTR of a model recombinant human therapeutic glycoprotein, Erythropoietin (EPO), in a direct comparison with an A to T mutant and a no adenosine control. The m6A construct yielded significantly improved EPO titer in transient batch culture over no adenosine and m6T controls by 2.84 and 2.61-fold respectively. This study highlights that refinement of transgene RNA elements can yield significant improvements to protein titer.Science Foundation IrelandUpdate issue date during checkdate report - A

CRISPR/Cas9 mediated targeting of microRNA-24 improves the bioprocess phenotype of Chinese Hamster Ovary cells

Chinese Hamster Ovary (CHO) cells are the prominent cell line used in biopharmaceutical production. Although optimisation efforts have led to a vast increase in productivity, CHO cells yield less than other expression systems like yeast or bacteria. To improve yields and find beneficial bioprocess phenotypes, genetic engineering plays an essential role in recent research. The miR-23 cluster with its genomic paralogues (miR-23a and miR-23b) was first identified as differentially expressed during temperature shift, suggesting its role in proliferation and productivity. The common approach to deplete miRNAs is the use of a sponge decoy which, requires the introduction of reporter genes. As an alternative this work aims to knockdown miRNA expression using the recently developed CRISPR/Cas9 system which does not require a reporter transcript. This system consists of two main components: the single guide RNA (sgRNA) and an endonuclease (Cas9) which induces double strand breaks (DSBs). These DSBs can result in insertion or deletion (indels) of base pairs which can disrupt miRNA function and processing. A CHO-K1 cell line stably expressing an IgG was used for knockdown experiments. SgRNAs were designed to target the seed region of miR-24-3p and stable mixed populations were generated. It was shown that miRNA expression for miR-24-3p as well as miR-24-5p was significantly reduced in mixed populations. Please click Additional Files below to see the full abstract

Versatile dual reporter gene systems for investigating stop codon readthrough in plants.

Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA) in an mRNA. However, many viruses (and some cellular mRNAs) contain "stop" codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a "readthrough", or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context.We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP.We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a 'leaky' UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV) readthrough context

A proteomic profiling dataset of recombinant Chinese hamster ovary cells showing enhanced cellular growth following miR-378 depletion

The proteomic data presented in this article provide supporting information to the related research article ''Depletion of endogenous miRNA-378-3p increases peak cell density of CHO DP12 cells and is correlated with elevated levels of Ubiquitin Carboxyl-Terminal Hydrolase 14'' (Costello et al., in press) [1]. Control and microRNA-378 depleted CHO DP12 cells were profiled using label-free quantitative proteomic profiling. CHO DP12 cells were collected on day 4 and 8 of batch culture, subcellular proteomic enrichment was performed, and subsequent fractions were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Here we provide the complete proteomic dataset of proteins significantly differentially expressed by greater than 1.25-fold change in abundance between control and miR-378 depleted CHO DP12 cells, and the lists of all identified proteins for each condition

Detection of stop codon readthrough in stably transformed <i>Arabidopsis</i> plants.

<p>(A) Reporter gene constructs introduced into the <i>Arabidopsis</i> nuclear genome. The strong constitutive p045 promoter (open arrow) was used to drive expression of <i>NAN-GUS</i> dual reporter constructs containing either the wild-type TMV leaky stop codon region (p045:TMV-TAG) or the NRStop region (p045:TMV-NRStop) in stably transformed plants. (B) Histochemical analysis of progeny of single insertion transgenic <i>Arabidopsis</i> lines. Each panel contains six seedlings, 3 stained for NAN activity (left half) and 3 stained for GUS activity (right half). NAN activity stains blue, GUS activity magenta. Seedlings expressing a p045:GUS control construct show, as expected, strong GUS activity but no NAN activity (top panel). Seedlings expressing the p045:TMV-NRStop construct show strong NAN activity but negligible GUS activity (bottom panel), while seedlings expressing p045:TMV-TAG show strong NAN activity and low, but significant GUS activity (middle panel). (C) GUS/NAN activity ratios of seedlings of three individual transgenic lines expressing the p045:TMV-TAG (lines 7, 10, 11) or the p045:TMV-NRStop (lines 8, 11, 12) construct. NAN and GUS activity was quantified in total protein extracts from two pools of twelve seedlings of each line.</p

<i>In planta</i> measurement of stop codon readthrough using a NAN-GUS dual reporter vector.

<p>(A) Structure of the dual reporter gene cassette in the vector pOF. The <i>NAN</i> gene lacks a stop codon and a multiple cloning site separates the <i>NAN</i> and <i>GUS</i> orfs, which are translationally out-of-frame. CGT (bold and underlined) represents the 3^rd codon of the <i>GUS</i> gene. Test sequences were inserted between the <i>Bam</i>H I and <i>Sac</i> I restriction sites (underlined). (B) Diagrammatic representation of the TMV genome. The methyltransferase/helicase (MT/HEL) subunit of the viral RNA-dependent RNA polymerase is synthesized when translation terminates at a leaky UAG codon (underlined, with flanking sequence context). Stop codon readthrough extends the C-terminus of MT/HEL, adding the 57 kDa polymerase (POL) domain. Translation of the 183 kDa MT/HEL/POL protein terminates at a non-readthrough stop codon (NRStop) (underlined, with flanking sequence context). (C) Viral test sequences cloned as oligonucleotide linkers between the <i>NAN</i> and <i>GUS</i> orfs in pOF using the <i>Bam</i>H I and <i>Sac</i> I sites. Cloning sites and stop codons are underlined. Test sequences comprised leaky stop codon regions from the replicase orf of BNYVV, TRV and CarMV; the wild-type TMV readthrough region (TMV-TAG) and mutants with altered nucleotides (shown in red) at various positions flanking the leaky stop codon (TMV-5M, -I, -CtoG, -M); TMV mutants with alternate stop codons (TMV-TAA, TMV-TGA); and the region flanking the non-readthrough stop codon in the TMV replicase orf (TMV-NRStop). (D) Stop codon readthrough efficiencies of the various test sequences were calculated as described in the text. TMV-NRStop was used as a readthrough negative control.</p