Contribution of 5′- and 3′-untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes

Abstract

Expression of chloroplast genes is primarily regulated posttranscriptionally, and a number of RNA elements, found in either the 5'- or 3'-untranslated regions (UTRs) of plastid mRNAs, that impact gene expression have been identified. Complex regulatory and feedback mechanisms influence both translation and protein accumulation, making assignment of roles for specific RNA elements difficult. To identify specific contributions made by various UTRs on translation of plastid mRNAs, we used a heterologous gfp reporter gene that is fused combinatorially to chloroplast 5'- and 3'-UTRs. In general, the 5'-UTR, including the promoter, of the plastid atpA and psbD genes produced the highest levels of chimeric mRNA and protein accumulation, while the 5'-UTR of the rbcL and psbA genes produced less mRNA and protein. Varying the 3'-UTR had little impact on mRNA and protein accumulation, as long as a 3'-UTR was present. Overall, accumulation of chimeric mRNAs was proportional to protein accumulation, with a few notable exceptions. Light-regulated translation continues to operate in chimeric mRNAs containing the 5'-UTR of either the psbA or psbD mRNAs, despite translation of these two chimeric mRNAs at very different efficiencies, suggesting that translational efficiency and light-regulated translation are separate events. Translation of some chimeric mRNAs was much more efficient than others, suggesting that interactions between the untranslated and coding sequences can dramatically impact translational efficiency.

A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (HGAD65)

“…Franklin et al [44] showed an 80-fold increase in expression levels of GFP (green fluorescent protein) in C. reinhardtii chloroplasts when a synthetic GFP gene with increased AT content (66% AT content in synthetic GFP in comparison to native GFP with 60% AT content) was used. Furthermore, as the 5'-UTR region of chloroplast mRNAs has a profound effect on the translational efficiency of C. reinhardtii chloroplast genes [45,46], the use of different 5'-UTRs of chloroplast genes such as the 5'-UTR of the plastid psbA gene (D1 protein of photosystem II), combined with a strong promoter, could serve as another approach to enhance hGAD65 expression in C. reinhardtii cells. One additional strategy to improve hGAD65 expression may be the use of different host strains of C. reinhardtii.…”

- From Biodiesel and Bioethanol to Liquid Hydrocarbon Fuels: New Hydrotreating and Advanced Microbial Technologies

“…Microalgae have recently attracted attention as potential lowcost platform for the production of a broad range of commercial products including biofuels, nutraceuticals, therapeutics, industrial chemicals and animal feeds [1][2][3][4][5][6][7][8][9][10][11]; and genome engineering will enable and enhance algae-produced bio-products [1,5,6,[12][13][14][15][16][17][18][19]. However, while much has been written about the potential of transgenic microalgae, little of that potential has yet to be commercialized.…”

The microalgaChlamydomonas reinhardtiias a platform for the production of human protein therapeutics

“…16 The promoter regions are responsible for initiating transcription, while 5' UTRs function to regulate both mRNA stability as well as translation. 19 The 3' UTRs appear to influence mRNA processing and stability, [20][21][22][23] but may also interact with 5' UTRs to influence translation. 19,24 In general, the regulation of chloroplast gene expression is primarily controlled at the level of translation.…”

“…19 The 3' UTRs appear to influence mRNA processing and stability, [20][21][22][23] but may also interact with 5' UTRs to influence translation. 19,24 In general, the regulation of chloroplast gene expression is primarily controlled at the level of translation. [25][26][27] Three of the human therapeutic proteins; domain 14 of fibronectin (14FN3), VEGF and HMGB1 expressed well in each of the three vectors (table 1).…”