The key role of terminators on the expression and post‐transcriptional gene silencing of transgenes. (26th July 2020)

• Record Type: Journal Article
• Authors: F. de Felippes, Felipe; McHale, Marcus; Doran, Rachel L.; Roden, Sally; Eamens, Andrew L.; Finnegan, E. Jean; Waterhouse, Peter M.
• Is Part Of: Plant journal. Volume 104:Number 1(2020)
• Page Start: 96
• Page End: 112
• Publication Date: 2020-07-26
• Subjects: PTGS -- siRNAs -- silencing -- transcription termination -- transitivity -- HSP terminator; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.14907
• Languages: English
• ISSNs: 0960-7412

Abstract

Summary: Transgenes have become essential to modern biology, being an important tool in functional genomic studies and also in the development of biotechnological products. One of the major challenges in the generation of transgenic lines concerns the expression of transgenes, which, compared to endogenes, are particularly susceptible to silencing mediated by small RNAs (sRNAs). Several reasons have been put forward to explain why transgenes often trigger the production of sRNAs, such as the high level of expression induced by commonly used strong constitutive promoters, the lack of introns, and features resembling viral and other exogenous sequences. However, the relative contributions of the different genomic elements with respect to protecting genes from the silencing machinery and their molecular mechanisms remain unclear. Here, we present the results of a mutagenesis screen conceived to identify features involved in the protection of endogenes against becoming a template for the production of sRNAs. Interestingly, all of the recovered mutants had alterations in genes with proposed function in transcription termination, suggesting a central role of terminators in this process. Indeed, using a GFP reporter system, we show that, among different genetic elements tested, the terminator sequence had the greatest effect on transgene‐derived sRNA accumulation and that a well‐defined poly(A) site might be especially important. Finally, we describe an unexpected mechanism, where transgenes containing certain intron/terminator combinations lead to an increase in the production of sRNAs, which appears to interfere with splicing. Significance Statement: Transgenes are key elements to modern biology but, compared to endogenes, are particularly susceptible to silencing mediated by small RNAs (sRNAs). In the present study, we show that, among different genetic elements tested, terminators are the main feature protecting mRNAs from silencing and we also identify molecular mechanisms associated with the use of distinct 3′ regulatory regions potentially leading to sRNA generation.