Functional intron-derived miRNAs and host-gene expression in plants

Umidjon Shapulatov, M.D. van Hoogdalem, M.E.L. Schreuder, H.J. Bouwmeester, Ibrokhim Y. Abdurakhmonov, A.R. van der Krol

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Background: Recently, putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene pre-mRNA and maturation of the miRNAs from the intron. Here, we demonstrated that such dual functionality is indeed possible, using as host gene the firefly luciferase gene with intron (ffgLUC), and different artificial intronic miRNAs (aimiRNA) placed within the intron of ffgLUC. Results: The miRNAs were based on the structure of the natural miR319a. Luciferase (LUC) activity in planta was used to evaluate a correct splicing of the ffgLUC mRNA. Different target sequences were inserted into the aimiRNA to monitor efficiency of silencing of different target mRNAs. After adjusting the insertion cloning strategy, the ffgLUCaimiR-319a gene showed dual functionality with correct splicing of ffgLUC and efficient silencing of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 transcription factor genes targeted in-trans by aimiR-319a or targeting the transgene ffLUC in-cis by an aimiR-LUC. Silencing of endogenous target genes by aimiRNA or amiRNA is efficient both in transient assays and stable transformants. A behave as strong phenotype the PHYTOCHROME B (PHYB) gene was also targeted by ffgLUCaimiR-PHYB. The lack of silencing of the PHYB target was most likely due to an insensitive target site within the PHYB mRNA which can potentially form a double stranded stem structure. Conclusion: The combination of an overexpression construct with an artificial intronic microRNA allows for a simultaneous dual function in plants. The concept therefore adds new options to engineering of plant traits that require multiple gene manipulations.
LanguageEnglish
Article number83
JournalPlant Methods
Volume14
DOIs
Publication statusPublished - 24 Sep 2018

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MicroRNAs
Introns
introns
Gene Expression
gene expression
Genes
genes
luciferase
Luciferases
Messenger RNA
Synthetic Genes
Firefly Luciferases
Plant Genes
RNA Precursors
Transgenes
Zea
Zea mays
Organism Cloning
Plantae
Transcription Factors

Cite this

Shapulatov, Umidjon ; van Hoogdalem, M.D. ; Schreuder, M.E.L. ; Bouwmeester, H.J. ; Abdurakhmonov, Ibrokhim Y. ; van der Krol, A.R. / Functional intron-derived miRNAs and host-gene expression in plants. In: Plant Methods. 2018 ; Vol. 14.
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title = "Functional intron-derived miRNAs and host-gene expression in plants",
abstract = "Background: Recently, putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene pre-mRNA and maturation of the miRNAs from the intron. Here, we demonstrated that such dual functionality is indeed possible, using as host gene the firefly luciferase gene with intron (ffgLUC), and different artificial intronic miRNAs (aimiRNA) placed within the intron of ffgLUC. Results: The miRNAs were based on the structure of the natural miR319a. Luciferase (LUC) activity in planta was used to evaluate a correct splicing of the ffgLUC mRNA. Different target sequences were inserted into the aimiRNA to monitor efficiency of silencing of different target mRNAs. After adjusting the insertion cloning strategy, the ffgLUCaimiR-319a gene showed dual functionality with correct splicing of ffgLUC and efficient silencing of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 transcription factor genes targeted in-trans by aimiR-319a or targeting the transgene ffLUC in-cis by an aimiR-LUC. Silencing of endogenous target genes by aimiRNA or amiRNA is efficient both in transient assays and stable transformants. A behave as strong phenotype the PHYTOCHROME B (PHYB) gene was also targeted by ffgLUCaimiR-PHYB. The lack of silencing of the PHYB target was most likely due to an insensitive target site within the PHYB mRNA which can potentially form a double stranded stem structure. Conclusion: The combination of an overexpression construct with an artificial intronic microRNA allows for a simultaneous dual function in plants. The concept therefore adds new options to engineering of plant traits that require multiple gene manipulations.",
author = "Umidjon Shapulatov and {van Hoogdalem}, M.D. and M.E.L. Schreuder and H.J. Bouwmeester and Abdurakhmonov, {Ibrokhim Y.} and {van der Krol}, A.R.",
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language = "English",
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Functional intron-derived miRNAs and host-gene expression in plants. / Shapulatov, Umidjon; van Hoogdalem, M.D.; Schreuder, M.E.L.; Bouwmeester, H.J.; Abdurakhmonov, Ibrokhim Y.; van der Krol, A.R.

In: Plant Methods, Vol. 14, 83, 24.09.2018.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Functional intron-derived miRNAs and host-gene expression in plants

AU - Shapulatov, Umidjon

AU - van Hoogdalem, M.D.

AU - Schreuder, M.E.L.

AU - Bouwmeester, H.J.

AU - Abdurakhmonov, Ibrokhim Y.

AU - van der Krol, A.R.

PY - 2018/9/24

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N2 - Background: Recently, putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene pre-mRNA and maturation of the miRNAs from the intron. Here, we demonstrated that such dual functionality is indeed possible, using as host gene the firefly luciferase gene with intron (ffgLUC), and different artificial intronic miRNAs (aimiRNA) placed within the intron of ffgLUC. Results: The miRNAs were based on the structure of the natural miR319a. Luciferase (LUC) activity in planta was used to evaluate a correct splicing of the ffgLUC mRNA. Different target sequences were inserted into the aimiRNA to monitor efficiency of silencing of different target mRNAs. After adjusting the insertion cloning strategy, the ffgLUCaimiR-319a gene showed dual functionality with correct splicing of ffgLUC and efficient silencing of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 transcription factor genes targeted in-trans by aimiR-319a or targeting the transgene ffLUC in-cis by an aimiR-LUC. Silencing of endogenous target genes by aimiRNA or amiRNA is efficient both in transient assays and stable transformants. A behave as strong phenotype the PHYTOCHROME B (PHYB) gene was also targeted by ffgLUCaimiR-PHYB. The lack of silencing of the PHYB target was most likely due to an insensitive target site within the PHYB mRNA which can potentially form a double stranded stem structure. Conclusion: The combination of an overexpression construct with an artificial intronic microRNA allows for a simultaneous dual function in plants. The concept therefore adds new options to engineering of plant traits that require multiple gene manipulations.

AB - Background: Recently, putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene pre-mRNA and maturation of the miRNAs from the intron. Here, we demonstrated that such dual functionality is indeed possible, using as host gene the firefly luciferase gene with intron (ffgLUC), and different artificial intronic miRNAs (aimiRNA) placed within the intron of ffgLUC. Results: The miRNAs were based on the structure of the natural miR319a. Luciferase (LUC) activity in planta was used to evaluate a correct splicing of the ffgLUC mRNA. Different target sequences were inserted into the aimiRNA to monitor efficiency of silencing of different target mRNAs. After adjusting the insertion cloning strategy, the ffgLUCaimiR-319a gene showed dual functionality with correct splicing of ffgLUC and efficient silencing of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 transcription factor genes targeted in-trans by aimiR-319a or targeting the transgene ffLUC in-cis by an aimiR-LUC. Silencing of endogenous target genes by aimiRNA or amiRNA is efficient both in transient assays and stable transformants. A behave as strong phenotype the PHYTOCHROME B (PHYB) gene was also targeted by ffgLUCaimiR-PHYB. The lack of silencing of the PHYB target was most likely due to an insensitive target site within the PHYB mRNA which can potentially form a double stranded stem structure. Conclusion: The combination of an overexpression construct with an artificial intronic microRNA allows for a simultaneous dual function in plants. The concept therefore adds new options to engineering of plant traits that require multiple gene manipulations.

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DO - 10.1186/s13007-018-0351-2

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VL - 14

JO - Plant Methods

T2 - Plant Methods

JF - Plant Methods

SN - 1746-4811

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