Breaking the silence: tethering a translational enhancer to improve transgene expression

Abstract

Genetic transformation has become a routine technique in plant biology: transgenes are widely used as tools in fundamental research, as expression systems for high-value proteins and—despite advances in CRISPR-based gene editing—remain the method of choice to introduce traits absent from a species' breeding pool. However, these applications are frequently hampered by gene silencing, which can lead to the decline or even complete loss of transgene expression over successive generations. This effect has been attributed to two major processes: transcriptional gene silencing (TGS) via methylation of the transgene DNA, and post-transcriptional gene silencing (PTGS) mediated by the RNA interference (RNAi) pathway (Molnar et al., 2011). PTGS involves the formation of short-interfering RNAs (siRNAs) of 21–25 nucleotides that are complementary to the transgene's mRNA. These siRNAs are loaded into RNA-induced silencing complexes, guiding them to their target mRNA for degradation or translational inhibition. PTGS typically precedes TGS, and continuous production of siRNAs can trigger activation of RNA-directed DNA methylation of a transgene's promoter regions (Matzke & Mosher, 2014).

Keith Slotkin and his lab investigate gene silencing mechanisms in plants, and as part of this broader effort, also explore strategies to improve transgene expression. They recently leveraged the RNA-binding protein BRUNO-LIKE 1 (BRN1) to establish an in vivo protein–mRNA tethering system (Cuerda-Gil et al., 2022). BRN1 binds a seven-nucleotide recognition sequence in the 3′ UTR of the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) transcript and thereby interferes with its translation (Kim et al., 2013). A truncated BRN1 RNA-binding domain (BD), while still able to bind the SOC1 3′ UTR, does not repress translation and can be fused to other proteins to influence the fate of the tethered mRNA. For example, fusion of a deadenylase protein to BD triggered SOC1 transcript deadenylation and subsequent degradation, while fusion of the conserved 40S ribosomal subunit RIBOSOMAL PROTEIN S6 (RPS6) increased translation efficiency (Cuerda-Gil et al., 2022).

Senior Research Scientist Yu-Hung Hung, first author of the highlighted paper, extended this approach to test whether the BD-RPS6 tethering system can be used to improve expression of transgenes. As a target Hung and colleagues chose Cas9, a widely used transgene that generates a quantifiable output. They generated Cas9 expression constructs with different BRN1 binding site configurations at the 3′ end: no (0xBS), one (1xBS) or four (4xBS) BRN1 binding sites, or the full SOC1 3′ UTR (Figure 1A). These constructs were expressed together with a guide RNA targeting the ALCOHOL DEHYDROGENASE 1 (ADH1) gene and transformed into Arabidopsis plants with or without the BD-RPS6 tethering system.

To determine the effect of RPS6 tethering on transgene expression, the Cas9 protein was quantified using ELISA and Western blotting. Although expression varied substantially across lines, constructs with BRN1 binding sites consistently showed higher Cas9 protein levels than the 0xBS controls. This effect occurred only in the presence of the BD-RPS6 system, confirming that enhanced protein synthesis relied on RPS6 tethering. Increased Cas9 abundance translated to higher mutation rates at the ADH1 locus, an effect that was assessed indirectly via an allyl alcohol survival assay. When treated with allyl alcohol, wild-type seeds die because functional ADH1 converts allyl alcohol into toxic allyl aldehyde; this reaction is abolished in adh1 mutants (Jacobs et al., 1988). In agreement with the increased Cas9 levels, lines with 1xBS, 4xBS or the full-length SOC1 3′ UTR had higher survival rates than 0xBS lines, and all surviving seedlings were either homozygous or biallelic for mutations in ADH1.

To explore the link between RPS6 tethering and gene silencing, the authors measured siRNA accumulation and promoter methylation in Cas9 T1 plants. Lines with 1xBS, 4xBS, or the SOC1 3′ UTR produced fewer siRNAs than the 0xBS control, while none showed promoter methylation above background. This suggests that Cas9 silencing in T1 plants occurs predominantly via PTGS and likely contributes to the observed differences in Cas9 levels.

The team next assessed whether enhanced Cas9 expression persisted across generations. Compared with T1 plants, all T4 plants showed reduced Cas9 levels, consistent with progressive silencing of the transgene. But while high variability in the 4xBS lines precluded firm conclusions, the 1xBS and SOC1 3′ UTR lines consistently maintained higher Cas9 expression than the 0xBS controls, suggesting that the BD-RPS6 system reduces transgenerational silencing. The T5 progeny of the 0xBS and 1xBS lines was subsequently tested for transgene methylation: the authors observed high variability across individual plants, with the 1xBS lines showing a moderate but significant reduction in relative methylation compared with the 0xBS controls. These findings suggest that lower Cas9 levels are, at least in part, due to changes in TGS.

Overall, Hung et al. demonstrate that tethering with RPS6 can effectively shift mRNAs from RNAi-mediated silencing toward translation, substantially improving transgene performance. Hung emphasizes that future iterations of the BD-RPS6 system may further optimize expression by adjusting the number and position of BRN1 binding sites, or by adding alternative translational enhancers. Beyond boosting expression and mitigating silencing, this versatile tethering system can be used to manipulate RNA fate, interrogate RNA–protein interactions, or assess protein function in an mRNA-specific context.