An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves

Rosenthal, Sun Hee; Diamos, Andrew G.; Mason, Hugh S.

doi:10.1007/s11103-018-0708-y

Cited by 25 publications

(56 citation statements)

References 61 publications

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“…These constructs were delivered to N. benthamiana leaves by agroinfiltration and evaluated for GFP production. To minimize leaf‐to‐leaf variation, each leaf was also infiltrated with a GFP vector containing the intronless tobacco extensin terminator (EU) as an internal control, which we previously found to be a potent enhancer of gene expression (Diamos et al ., ; Rosenthal et al ., ).…”

Section: Resultsmentioning

confidence: 97%

“…DsRed shares no sequence homology with GFP. For single terminators, the extensin terminator provided the highest level of gene expression (Figure ), in agreement with our data with GUS or Norwalk virus capsid protein (Rosenthal et al ., ). Other terminators performed similarly as when paired with GFP; however, some small differences were observed, such as the increased activity of NbHSP and 35S with DsRed compared with NOS (Figure ).…”

Section: Resultsmentioning

confidence: 97%

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Chimeric 3’ flanking regions strongly enhance gene expression in plants

Diamos

Mason

2018

Plant Biotechnology Journal

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115

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SummaryPlants represent a promising platform for the highly scalable production of recombinant proteins. Previously, we identified the tobacco extensin terminator lacking its intron as an element that reduced transcript read‐through and improved recombinant protein production in a plant‐based system. In this study, we systematically compared nonreplicating plant expression vectors containing over 20 commonly used or newly identified terminators from diverse sources. We found that eight gene terminators enhance reporter gene expression significantly more than the commonly used 35S and NOS terminators. The intronless extensin terminator provided a 13.6‐fold increase compared with the NOS terminator. Combining terminators in tandem produced large synergistic effects, with many combinations providing a >25‐fold increase in expression. Addition of the tobacco Rb7 or TM6 matrix attachment region (MAR) strongly enhanced protein production when added to most terminators, with the Rb7 MAR providing the greatest enhancement. Using deletion analysis, the full activity of the 1193 bp Rb7 MAR was found to require only a 463‐bp region at its 3’ end. Combined terminators and MAR together provided a >60‐fold increase compared with the NOS terminator alone. These combinations were then placed in a replicating geminiviral vector, providing a total of >150‐fold enhancement over the original NOS vector, corresponding to an estimated yield of 3–5 g recombinant protein per kg leaf fresh weight or around 50% of the leaf total soluble protein. These results demonstrate the importance of 3’ flanking regions in optimizing gene expression and show great potential for 3’ flanking regions to improve DNA‐based recombinant protein production systems.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 97%

Chimeric 3’ flanking regions strongly enhance gene expression in plants

Diamos

Mason

2018

Plant Biotechnology Journal

Self Cite

115

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“…While the NOS terminator is a common feature of many T-DNA vectors, several studies have shown that transgene expression can be considerably enhanced either when the NOS terminator is combined with a second terminator or when it is replaced by the terminator of plant endogenous genes [41][42][43]. For example, replacement of the NOS terminator with an extensin terminator was shown to reduce read-through transcript and improve expression of transgenes [44]. Altogether, these data suggest that mutations in the HPR1 and TEX1 genes may more prominently affect mRNA 3'processing for genes having unusual or weak polyadenylation signals, such as found in the NOS terminator.…”

Section: Plos Geneticsmentioning

confidence: 99%

The transcription and export complex THO/TREX contributes to transcription termination in plants

et al. 2020

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Transcription termination has important regulatory functions, impacting mRNA stability, localization and translation potential. Failure to appropriately terminate transcription can also lead to read-through transcription and the synthesis of antisense RNAs which can have profound impact on gene expression. The Transcription-Export (THO/TREX) protein complex plays an important role in coupling transcription with splicing and export of mRNA. However, little is known about the role of the THO/TREX complex in the control of transcription termination. In this work, we show that two proteins of the THO/TREX complex, namely TREX COMPONENT 1 (TEX1 or THO3) and HYPER RECOMBINATION1 (HPR1 or THO1) contribute to the correct transcription termination at several loci in Arabidopsis thaliana. We first demonstrate this by showing defective termination in tex1 and hpr1 mutants at the nopaline synthase (NOS) terminator present in a T-DNA inserted between exon 1 and 3 of the PHO1 locus in the pho1-7 mutant. Read-through transcription beyond the NOS terminator and splicing-out of the T-DNA resulted in the generation of a near full-length PHO1 mRNA (minus exon 2) in the tex1 pho1-7 and hpr1 pho1-7 double mutants, with enhanced production of a truncated PHO1 protein that retained phosphate export activity. Consequently, the strong reduction of shoot growth associated with the severe phosphate deficiency of the pho1-7 mutant was alleviated in the tex1 pho1-7 and hpr1 pho1-7 double mutants. Additionally, we show that RNA termination defects in tex1 and hpr1 mutants leads to 3'UTR extensions in several endogenous genes. These results demonstrate that THO/ TREX complex contributes to the regulation of transcription termination.

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“…While the NOS terminator is a common feature of many T-DNA vectors, several studies have shown that transgene expression can be considerably enhanced either when the NOS terminator is combined with a second terminator or when it is replaced by the terminator of plant endogenous genes [41–43]. For example, replacement of the NOS terminator with an extensin terminator was shown to reduce read-through transcript and improve expression of transgenes [44]. Altogether, these data suggest that mutations in the HPR1 and TEX1 genes may more prominently affect mRNA 3’processing for genes having unusual or weak polyadenylation signals, such as found in the NOS terminator.…”

Section: Discussionmentioning

confidence: 99%

The transcription and export complex THO/TREX contributes to transcription termination in plants

Khan

Deforges

Reis

et al. 2020

Preprint

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AbstractTranscription termination has important regulatory functions, impacting mRNA stability, localization and translation potential. Failure to appropriately terminate transcription can also lead to read-through transcription and the synthesis of antisense RNAs which can have profound impact on gene expression. The Transcription-Export (THO/TREX) protein complex plays an important role in coupling transcription with splicing and export of mRNA. However, little is known about the role of the THO/TREX complex in the control of transcription termination. In this work, we show that two proteins of the THO/TREX complex, namely TREX COMPONENT 1 (TEX1 or THO3) and HYPER RECOMBINATION1 (HPR1 or THO1) contribute to the correct transcription termination at several loci in Arabidopsis thaliana. We first demonstrate this by showing defective termination in tex1 and hpr1 mutants at the nopaline synthase (NOS) terminator present in a T-DNA inserted between exon 1 and 3 of the PHO1 locus in the pho1-7 mutant. Read-through transcription beyond the NOS terminator and splicing-out of the T-DNA resulted in the generation of a near full-length PHO1 mRNA (minus exon 2) in the tex1 pho1-7 and hpr1 pho1-7 double mutants, with enhanced production of a truncated PHO1 protein that retained phosphate export activity. Consequently, the strong reduction of shoot growth associated with the severe phosphate deficiency of the pho1-7 mutant was alleviated in the tex1 pho1-7 and hpr1 pho1-7 double mutants. Additionally, we show that RNA termination defects in tex1 and hpr1 mutants leads to 3’UTR extensions in several plant genes. These results demonstrate that THO/TREX complex contributes to the regulation of transcription termination.Author summaryProduction of messenger RNAs (mRNAs) involves numerous steps including initiation of transcription, elongation, splicing, termination, as well as export out of the nucleus. All these steps are highly coordinated and failure in any steps has a profound impact on the level and identity of mRNAs produced. The THO/TREX protein complex is associated with nascent RNAs and contributes to several mRNA biogenesis steps, including splicing and export. However, the contribution of the THO/TREX complex to mRNA termination was poorly defined. We have identified a role for two components of the THO/TREX complex, namely the proteins TEX1 and HPR1, in the control of transcription termination in the plant Arabidopsis thaliana. We show that the tex1 and hpr1 mutants have defects in terminating mRNA at the nopaline synthase (NOS) terminator found in T-DNA insertion mutants leading to the transcriptional read-through pass the NOS terminator. We also show that tex1 and hpr1 mutants have defects in mRNA termination at several endogenous genes, leading to the production of 3’UTR extensions. Together, these results highlight a role for the THO/TREX complex in mRNA termination.

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An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves

Cited by 25 publications

References 61 publications

Chimeric 3’ flanking regions strongly enhance gene expression in plants

Chimeric 3’ flanking regions strongly enhance gene expression in plants

The transcription and export complex THO/TREX contributes to transcription termination in plants

The transcription and export complex THO/TREX contributes to transcription termination in plants

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