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Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [sRNA-Seq]

ABSTRACT: Improvement and research of plants depends on the long-term expression of transgenes. However, the durability of transgene expression is routinely hampered by silencing pathways that start as the post-transcriptional process of transcript cleavage by RNA interference (RNAi). To avoid transgene silencing, we aimed to inhibit the sorting of transgene mRNAs into RNAi. We manipulated a well-studied protein / RNA-binding module from Arabidopsis into a transgene transcript, where the transcript is now bound by an engineered RNA-binding protein that preferentially sorts the RNA into translation, rather than RNAi. We used the Cas9 transcript as a proof-of-principle and demonstrated higher Cas9 protein production and editing rates. In addition, transgenes with the engineered protein / RNA-binding module had improved long-term durability of transgene expression, as after four generations these plants had higher Cas9 protein accumulation and lower levels of DNA methylation, a hallmark of transgene silencing. Our engineered system represents a successful manipulation of post-transcriptional RNA sorting for improved transgene performance, and could be applied to any transgene transcript.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE287530 | GEO | 2025/06/02

REPOSITORIES: GEO

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Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [BSAS]

Project description:Improvement and research of plants depends on the long-term expression of transgenes. However, the durability of transgene expression is routinely hampered by silencing pathways that start as the post-transcriptional process of transcript cleavage by RNA interference (RNAi). To avoid transgene silencing, we aimed to inhibit the sorting of transgene mRNAs into RNAi. We manipulated a well-studied protein / RNA-binding module from Arabidopsis into a transgene transcript, where the transcript is now bound by an engineered RNA-binding protein that preferentially sorts the RNA into translation, rather than RNAi. We used the Cas9 transcript as a proof-of-principle and demonstrated higher Cas9 protein production and editing rates. In addition, transgenes with the engineered protein / RNA-binding module had improved long-term durability of transgene expression, as after four generations these plants had higher Cas9 protein accumulation and lower levels of DNA methylation, a hallmark of transgene silencing. Our engineered system represents a successful manipulation of post-transcriptional RNA sorting for improved transgene performance, and could be applied to any transgene transcript.

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Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [BSAS]

Project description:Engineering an RNA-protein binding module for higher transgene protein production and improved long-term durability [BSAS]

| PRJNA1213155 | ENA

mutant axel-4 seedlings

Project description:A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a ?-glucuronidase (GUS) reporter gene and a hygromycin phosphotransferase (HPH)?selectable marker gene. This screen yielded several mutants, and five of the mutations (axe1-1 to axe1-5) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3-like histone deacetylase gene, referred to as HDA6. The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity. A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Keywords: replicate_design

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