Fine‐tuning levels of heterologous gene expression in plants by orthogonal variation of the untranslated regions of a nonreplicating transient expression system

A transient expression system based on a deleted version of Cowpea mosaic virus (CPMV) RNA‐2, termed CPMV‐HT, in which the sequence to be expressed is positioned between a modified 5′ UTR and the 3′ UTR has been successfully used for the plant‐based expression of a wide range of proteins, including heteromultimeric complexes. While previous work has demonstrated that alterations to the sequence of the 5′ UTR can dramatically influence expression levels, the role of the 3′ UTR in enhancing expression has not been determined. In this work, we have examined the effect of different mutations in the 3′UTR of CPMV RNA‐2 on expression levels using the reporter protein GFP encoded by the expression vector, pEAQexpress‐HT‐GFP. The results showed that the presence of a 3′ UTR in the CPMV‐HT system is important for achieving maximal expression levels. Removal of the entire 3′ UTR reduced expression to approximately 30% of that obtained in its presence. It was found that the Y‐shaped secondary structure formed by nucleotides 125–165 of the 3′ UTR plays a key role in its function; mutations that disrupt this Y‐shaped structure have an effect equivalent to the deletion of the entire 3′ UTR. Our results suggest that the Y‐shaped secondary structure acts by enhancing mRNA accumulation rather than by having a direct effect on RNA translation. The work described in this paper shows that the 5′ and 3′ UTRs in CPMV‐HT act orthogonally and that mutations introduced into them allow fine modulation of protein expression levels.     

Albumin 3′untranslated region facilitates increased recombinant protein production from Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are used for recombinant protein production in the pharmaceutical industry but there is a need to improve expression levels. In the present work experiments were carried out to test the effectiveness of different 3′untranslated regions (3′UTRs) in promoting production of a naturally secreted luciferase. Seamless cloning was used to produce expression vectors in which Gaussia princeps luciferase coding sequences were linked to the human albumin, immunoglobulin or chymotrypsinogen 3′UTR. Stably transfected CHO cells expressing these constructs were selected. Luciferase activity in the culture medium was increased 2–3‐fold by replacing the endogenous 3′UTR with the albumin 3′UTR and decreased by replacement with immunoglobulin or chymotrypsinogen 3′UTR. Replacement of the native 3′UTR with the albumin 3′UTR led to a 10‐fold increase in luciferase mRNA levels. Deletion analysis of the albumin 3′UTR showed that loss of nucleotides 1–50, which removed an AU‐rich complex stem loop region, caused significant reductions in both luciferase protein expression and luciferase mRNA levels. The results suggest that recombinant protein expression and yield could be improved by the careful selection of appropriate 3′UTR sequences.     

Predictive models for the accumulation of a fluorescent marker protein in tobacco leaves according to the promoter/5′UTR combination

The promoter and 5′‐untranslated region (5′UTR) play a key role in determining the efficiency of recombinant protein expression in plants. Comparative experiments are used to identify suitable elements but these are usually tested in transgenic plants or in transformed protoplasts/suspension cells, so their relevance in whole‐plant transient expression systems is unclear given the greater heterogeneity in expression levels among different leaves. Furthermore, little is known about the impact of promoter/5′UTR interactions on protein accumulation. We therefore established a predictive model using a design of experiments (DoE) approach to compare the strong double‐enhanced Cauliflower mosaic virus 35S promoter (CaMV 35SS) and the weaker Agrobacterium tumefaciens Ti‐plasmid nos promoter in whole tobacco plants transiently expressing the fluorescent marker protein DsRed. The promoters were combined with one of three 5′UTRs (one of which was tested with and without an additional protein targeting motif) and the accumulation of DsRed was measured following different post‐agroinfiltration incubation periods in all leaves and at different leaf positions. The model predictions were quantitative, allowing the rapid identification of promoter/5′UTR combinations stimulating the highest and quickest accumulation of the marker protein in all leaves. The model also suggested that increasing the incubation time from 5 to 8 days would reduce batch‐to‐batch variability in protein yields. We used the model to identify promoter/5′UTR pairs that resulted in the least spatiotemporal variation in expression levels. These ideal pairs are suitable for the simultaneous, balanced production of several proteins in whole plants by transient expression. Biotechnol. Bioeng. 2013; 110: 471–482. © 2012 Wiley Periodicals, Inc.     

Cooperation between the chloroplast psbA 5′‐untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region

Chloroplast mRNA translation is regulated by the 5′‐untranslated region (5′‐UTR). Chloroplast 5′‐UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5′‐UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5′‐UTR with the E. coli phage T7 gene 10 5′‐UTR, a highly active 5′‐UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5′‐UTR with a cognate 5′‐coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5′‐UTR and its coding region is important for translational initiation.     

Novel RNA recognition motif domain in the cytoplasmic polyadenylation element binding protein 3

The family of cytoplasmic polyadenylation element binding proteins CPEB1, CPEB2, CPEB3, and CPEB4 binds to the 3′‐untranslated region (3′‐UTR) of mRNA, and plays significant roles in mRNA metabolism and translation regulation. They have a common domain organization, involving two consecutive RNA recognition motif (RRM) domains followed by a zinc finger domain in the C‐terminal region. We solved the solution structure of the first RRM domain (RRM1) of human CPEB3, which revealed that CPEB3 RRM1 exhibits structural features distinct from those of the canonical RRM domain. Our structural data provide important information about the RNA binding ability of CPEB3 RRM1. Proteins 2014; 82:2879–2886. © 2014 Wiley Periodicals, Inc.     

Synthesis and enzymatic deprotection of biodegradably protected dinucleoside-2',5'-monophosphates: 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl phosphoesters of 3'-O-(acyloxymethyl)adenylyl-2',5'-adenosines

As a first step towards a viable prodrug strategy for short oligoribonucleotides, such as 2–5A and its congeners, adenylyl‐2′,5′‐adenosines bearing a 3‐(acetyloxy)‐2,2‐bis(ethoxycarbonyl)propyl group at the phosphate moiety, and an (acetyloxy)methyl‐ or a (pivaloyloxy)methyl‐protected 3′‐OH group of the 2′‐linked nucleoside have been prepared. The enzyme‐triggered removal of these protecting groups by hog liver carboxyesterase at pH 7.5 and 37° has been studied. The (acetyloxy)methyl group turned out to be too labile for the 3′‐O‐protection, being removed faster than the phosphate‐protecting group, which results in 2′,5′‐ to 3′,5′‐isomerization of the internucleosidic phosphoester linkage. In addition, the starting material was unexpectedly converted to the 5′‐O‐acetylated derivative. (Pivaloyloxy)methyl group appears more appropriate for the purpose. The fully deprotected 2′,5′‐ApA was accumulated as a main product, although, even in this case, the isomerization of the starting material takes place.