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.     

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.     

Distinct roles of the histone chaperones NAP1 and NRP and the chromatin‐remodeling factor INO80 in somatic homologous recombination in Arabidopsis thaliana

Homologous recombination (HR) of nuclear DNA occurs within the context of a highly complex chromatin structure. Despite extensive studies of HR in diverse organisms, mechanisms regulating HR within the chromatin context remain poorly elucidated. Here we investigate the role and interplay of the histone chaperones NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) and NAP1‐RELATED PROTEIN (NRP) and the ATP‐dependent chromatin‐remodeling factor INOSITOL AUXOTROPHY80 (INO80) in regulating somatic HR in Arabidopsis thaliana. We show that simultaneous knockout of the four AtNAP1 genes and the two NRP genes in the sextuple mutant m123456‐1 barely affects normal plant growth and development. Interestingly, compared with the respective AtNAP1 (m123‐1 and m1234‐1) or NRP (m56‐1) loss‐of‐function mutants, the sextuple mutant m123456‐1 displays an enhanced plant hypersensitivity to UV or bleomycin treatments. Using HR reporter constructs, we show that AtNAP1 and NRP act in parallel to synergistically promote somatic HR. Distinctively, the AtINO80 loss‐of‐function mutation (atino80‐5) is epistatic to m56‐1 in plant phenotype and telomere length but hypostatic to m56‐1 in HR determinacy. Further analyses show that expression of HR machinery genes and phosphorylation of H2A.X (γ‐H2A.X) are not impaired in the mutants. Collectively, our study indicates that NRP and AtNAP1 synergistically promote HR upstream of AtINO80‐mediated chromatin remodeling after the formation of γ‐H2A.X foci during DNA damage repair.