The stem-loop region of the tobacco psbA 5′UTR is an important determinant of mRNA stability and translation efficiency

Abstract

Regulation of chloroplast gene expression involves networked and concerted interactions of nucleus-encoded factors with their target sites on untranslated regions (UTRs) of chloroplast transcripts. So far, only a few cis-acting elements within such 5'UTR sequences have been identified as functional determinants of mRNA stability and efficient translation in Chlamydomonas in vivo. In this study, we have used chloroplast transformation and site-directed mutagenesis to analyse the functions of the 5'UTRs of tobacco psbA and rbcL fused to the coding region of the reporter gene uidA. Various mutant versions of the psbA leader, as well as rbcL/psbA hybrid leader elements, were investigated. Our results showed a 1.5- to 3-fold decrease in uidA mRNA levels and a 1.5- to 6-fold reduction in uidA translation efficiency in all psbA 5'UTR stem-loop mutants generated by sequence deletions and base alterations. This indicates that the correct primary sequence and secondary structure of the psbA 5'UTR stem-loop are required for mRNA stabilisation and translation. The 5'-terminal segment of the rbcL 5'UTR did not enhance the stability or translational activity of chimeric uidA mRNA under the standard light-dark regime of 16 h light and 8 h dark. Stabilising effects were, however, observed when the cells were kept continuously in the dark. Possible reasons for the influence of the 5'UTR of the tobacco psbA on mRNA stability and translation efficiency are discussed.

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…Among more than 150 transgenes expressed in chloroplasts, more than 75% utilized psbA regulatory sequences (Jin and Daniell, 2015;Daniell et al, 2016aDaniell et al, , 2016b. In addition, three ribosome-binding regions in the 5ʹ UTR of psbA recruit ribosomes and efficiently form the translational initiation complex (Zou et al, 2003). Therefore, it is expected that improvement of translation elongation in heterologous genes should increase transgene expression.…”

Synthetic Lethality in the Tobacco Plastid Ribosome and Its Rescue at Elevated Growth Temperatures

“…For supertransformation, a modified Drps15 vector was built that contained the kanamycin resistance gene aphA-6 instead of the spectinomycin resistance gene aadA (Bateman and Purton, 2000;Huang et al, 2002;Fleischmann et al, 2011). The aphA-6 expression cassette was driven by the psbA promoter from Chlamydomonas reinhardtii and the rbcL 39 untranslated region from C. reinhardtii (Zou et al, 2003). The cassette was excised with SmaI and inserted into a unique HincII restriction site in the rps15 coding region (Fleischmann et al, 2011;Figure 1).…”

Expanding the ‘central dogma’: the regulatory role of nonprotein coding genes and implications for the genetic liability to schizophrenia

“…Rather, alternative codons may affect the tertiary structure of mRNA, thereby affecting mRNA degradation rate, mRNA half-life, and hence the rate of protein expression. 30 …”