Optimization of mRNA design for protein expression in the crustacean Daphnia magna

The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 (EF1α-1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α-1 3′ UTR failed to induce fluorescence. To assess reporter expression, the length of the 3′ UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3′ end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3′ UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3′ UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3′ UTRs of Daphnia EF1α-1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia.     

MicroRNA-378 Regulates Adiponectin Expression in Adipose Tissue: A New Plausible Mechanism

Aims

Mechanisms regulating adiponectin expression have not been fully clarified. MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, are involved in biological processes, including obesity and insulin resistance. We evaluated whether the miRNA-378 pathway is involved in regulating adiponectin expression.

Methods and Results

First, we determined a putative target site for miRNA-378 in the 3 prime untranslated region (3''UTR) of the adiponectin gene by in silico analysis. The levels of adiponectin mRNA and protein were decreased in 3T3-L1 cells overexpressing the mimic of miRNA-378. Luminescence activity in HEK293T cells expressing a renilla-luciferase-adiponectin-3''UTR sequence was inhibited by overexpressing the mimic of miRNA-378, and the decrease was reversed by adding the inhibitor of miRNA-378. Moreover, we confirmed the inhibitory effects of the mimic were cancelled in a deleted mutant of the miR-378 3′-UTR binding site. Addition of tumor necrosis factor-α (TNFα) led a upregulation of miR-378 and downregulation of adiponectin at mRNA and protein levels in 3T3-L1 cells. Level of miR-378 was higher and mRNA level of adiponectin was lower in diabetic ob/ob mice than those of normal C57BL/6 mice and levels of miR378 and adiponectin were negatively well correlated (r = −0.624, p = 0.004).

Conclusions

We found that levels of miRNA-378 could modulate adiponectin expression via the 3''UTR sequence-binding site. Our findings warrant further investigations into the role of miRNAs in regulating the adiponectin expression.     

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.     

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.     

IRES-dependent translational control of Cbfa1/Runx2 expression

The P1 and P2 promoters of the Cbfa1/Runx2 gene produce Type I and II mRNAs with distinct complex 5'-untranslated regions, respectively designated UTR1 and UTR2. To evaluate whether the 5'-UTRs impart different translational efficiencies to the two isoforms, we created SV40 promoter-UTR-luciferase reporter (luc) constructs in which the translational potential of the 5'-UTR regions was assessed indirectly by measurement of luciferase activity in transfected cell lines in vitro. In MC3T3-E1 pre-osteoblasts, UTR2 was translated approximately twice as efficiently as the splice variants of UTR1, whereas translation of unspliced UTR1 was repressed. To determine if the UTRs conferred internal ribosome entry site (IRES)-dependent translation, we tested bicistronic SV40 promoter-Rluc-UTR-Fluc constructs in which Fluc is expressed only if the intercistronic UTR permits IRES-mediated translation. Transfection of bicistronic constructs into MC3T3-E1 osteoblasts demonstrated that both UTR2 and the spliced forms of UTR1 possess IRES activity. Similar to other cellular IRESs, activity increased with genotoxic stress induced by mitomycin C. In addition, we observed an osteoblastic maturation-dependent increase in IRES-mediated translation of both UTR2 and the spliced forms of UTR1. These findings suggest that Cbfa1 UTRs have IRES-dependent translational activities that may permit continued Cbfa1 expression under conditions that are not optimal for cap-dependent translation.     

Fine-tuning of fgf8a expression through alternative polyadenylation has a selective impact on Fgf-associated developmental processes

The formation of distinct 3′UTRs through alternative polyadenylation is a mechanism of gene expression regulation that has been implicated in many physiological and pathological processes. However, its functions in the context of vertebrate embryonic development have been largely unaddressed, in particular with a gene-specific focus. Here we show that the most abundant 3′UTR for the zebrafish fgf8a gene in the developing embryo mediates a strong translational repression, when compared to a more sparsely used alternative 3′UTR, which supports a higher translation efficiency. By inducing a shift in the selection efficiency of the associated polyadenylation sites, we show a temporally and spatially specific impact of fgf8a 3′UTR usage on embryogenesis, in particular at late stages during sensory system development. In addition, we identified a previously undescribed role for Fgf signalling in the initial stages of superficial retinal vascularization. These results reveal a critical functional importance of gene-specific alternative 3′UTRs in vertebrate embryonic development.