Effect of transcription terminator usage on the establishment of transgene transcriptional gene silencing

Abstract

ObjectiveObtaining high and stable transgene expression is of vital importance for plant genetic engineering. A lot is known about the relationship between terminator efficiency and gene expression, but no studies have addressed the relationship between terminator usage and transgene expression stability or heritable gene silencing. In this paper, we aim to analyze if terminators are a determining factor in the establishment of promoter DNA methylation of plant transgenes.ResultsOur experiments comparing plants with a LUC reporter under the 35S CaMV promoter and good efficiency terminators (Thsp, T35S) show that the use of efficient terminator sequences does not avoid the accumulation of promoter DNA methylation and transgene silencing. However, Thsp lead to a higher reporter gene expression and lower promoter DNA methylation levels than T35S, supporting that terminator usage is indeed involved in the establishment of TGS by methylation of transgenes’ promoters. In the case of a terminatorless construct, the PTGS initiated by the improperly terminated mRNAs is not followed by the establishment of heritable silencing in the form of strong promoter DNA methylation, like in the case of TAS genes and reactivated TEs (for the transgene DNA methylation levels remained below the 20%).Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3649-2) contains supplementary material, which is available to authorized users.

Plant 3’ Regulatory Regions From mRNA-Encoding Genes and Their Uses to Modulate Expression

“…In addition, mutated PAS can generate readthrough events, with RNA Polymerase II (RNAPII) sequestering factors involved in initiation and stretching, with negative effects on expression, consistent with the findings by Wei et al (2017). Peŕez-Gonzaĺez and Caro (2018), also demonstrated that the absence of a 3' regulatory region results in high methylation of the promoter through RdDM events, with negative effects on the expression of the target gene. It is likely that the use of 3' regulatory regions with weak polyadenylation signals may also promote RdDM.…”

“…Indeed, 35S has greater potential in regulating expression than NOS and OCS, both in monocot (rice) or dicot (tobacco) plants (Mitsuhara et al, 1996;Nagaya et al, 2010). However, although the number of 3' regulatory regions identified and validated in plants is still reduced, several studies have shown that plant 3' regulatory regions have a higher potential to increase expression compared to NOS, OCS or 35S 3' regulatory regions (Table 1) (Richter et al, 2000;Weeks et al, 2008;Yang et al, 2009;Nagaya et al, 2010;Hirai et al, 2011;Hiwasa-Tanase et al, 2011;Schaart et al, 2011;Kurokawa et al, 2013;Limkul et al, 2015;Diamos et al, 2016;Diamos and Mason, 2018;Yamamoto et al, 2018;Peŕez-Gonzaĺez and Caro, 2018;Rosenthal et al, 2018).…”

“…However, we believe 3' regulatory region is a more appropriate term, as it will be referred to here. This is because transcription termination is only one of the roles of the 3' regulatory regions, which, in many cases, can have profound effects on gene expression, as it will be discussed in this review (Menossi et al, 2003;Yang et al, 2009;Nagaya et al, 2010;Hirai et al, 2011;Hiwasa-Tanase et al, 2011;Matsui et al, 2014;Diamos and Mason, 2018;Peŕez-Gonzaĺez and Caro, 2018;Rosenthal et al, 2018;Yamamoto et al, 2018).…”

The Whys and Wherefores of Transitivity in Plants

“…During transcription, sequences in the terminator region are recognized by the 3′ end processing machinery resulting in the addition of a poly(A) tail to the nascent transcript and triggering the end of the polymerization process by the RNA polymerase II ( Mandel et al, 2007 ; Kumar et al, 2019 ; Thore and Fribourg, 2019 ). A growing number of studies have shown that different terminators can have a distinct influence in the efficiency of transgene expression ( Ingelbrecht et al, 1989 ; Nagaya et al, 2009 ; Yang et al, 2009 ; Hirai et al, 2011 ; Pérez-González and Caro, 2018 ; de Felippes et al, 2020 ). The improper formation of the mRNA 3′ end has also been implicated with enhanced silencing of transgenes.…”

The key role of terminators on the expression and post‐transcriptional gene silencing of transgenes

“…Similarly, splicing has been shown to affect gene stability and siRNA accumulation from transgenes (Christie et al ., 2011). Finally, the use of some terminators, such as the 3′ regulatory element of the HEAT SHOCK PROTEIN 18.2 gene from Arabidopsis thaliana , resulted in higher transgene expression and mRNA stability, as well as reduced predisposition to TGS (Nagaya et al ., 2009; Hirai et al ., 2011; Pérez‐González and Caro, 2018). Nevertheless, the contributions of these genetic components to transgene expression and stability, as well as the molecular mechanisms behind it, still remain largely unknown.…”