The Arabidopsis CYSTM α 5' UTR Increases Protein Production from Transgenes in Plants and Bacteria

Background: Translational regulation constitutes a critical layer of gene expression control in plants, yet the contribution of endogenous 5' untranslated regions (5' UTRs) to translational efficiency remains incompletely defined. While viral and synthetic leader sequences have been widely used to enhance protein production, comparatively few native plant 5' UTRs have been systematically characterised. The objective of this study was to identify and functionally evaluate endogenous plant 5' UTR elements that promote translation through post-transcriptional mechanisms. Methods: A 79-nucleotide fragment (CYSTM α) derived from the 5' UTR of Arabidopsis thaliana CYSTM1 (AT1G05340) was cloned upstream of reporter genes and assessed using dual-luciferase assays in transient expression systems (Nicotiana benthamiana and A. thaliana) and in stable transgenic Arabidopsis lines. Translational activity was further evaluated in monocot wheat germ extract and in Escherichia coli. Transcript abundance was quantified by qRT-PCR. Publicly available ribosome profiling and m⁶A datasets were analysed to assess translational efficiency and RNA modification status. Results: In N. benthamiana and A. thaliana, CYSTM α increases reporter protein production 3-7 fold relative to the control and 30-130% above the benchmark Tobacco Mosaic Virus (TMV) Ω leader, without altering mRNA abundance. The CYSTM α sequence also enhances luciferase translation in monocot wheat germ extract and elevates translation 5-fold in E. coli. CYSTM α contains three motifs that may promote translation, namely three CAA repeats that are associated with translation initiation, an AMAYAA motif that is associated with eIF3 binding, and two N6-adenosine DRACH sites that are associated with cap-independent translation. Additionally, ribosome profiling revealed high translational efficiency (TE = 3.25) of native CYSTM1. Conclusions: CYSTM α represents a compact endogenous 5' UTR element that enhances translation across multiple experimental systems. These findings expand the repertoire of plant-derived translational enhancers and provide insight into sequence features associated with efficient mRNA translation in plants.