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Transient expression of intron-containing transgenes generates non-spliced aberrant pre-mRNAs that are processed into siRNAs

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In this study, we show that aberrant pre-mRNAs from non-spliced and non-polyadenylated intron-containing transgenes are channelled to the RNA silencing pathway.

In plants, improperly processed transcripts are called aberrant RNAs (ab-RNAs) and are eliminated by either RNA silencing or RNA decay mechanisms. Ab-RNAs transcribed from intronless genes are copied by RNA-directed RNA polymerases (RDRs) into double-stranded RNAs which are subsequently cleaved by DICER-LIKE endonucleases into small RNAs (sRNAs). In contrast, ab-RNAs from intron-containing genes are suggested to be channelled post-splicing to exonucleolytic degradation. Yet, it is not clear how non-spliced aberrant pre-mRNAs are eliminated. We reasoned that transient expression of agroinfiltrated intron-containing transgenes in Nicotiana benthamiana would allow us to study the steady-state levels of non-spliced pre-mRNAs. SRNA deep sequencing of the agroinfiltrated transgenes revealed the presence of sRNAs mapping to the entire non-spliced pre-mRNA suggesting that RDRs (most likely RDR6) processed aberrant non-spliced pre-mRNAs. Primary and secondary sRNAs with lengths of 18–25 nucleotides (nt) were detected, with the most prominent sRNA size class of 22 nt. SRNAs also mapped to the terminator sequence, indicating that RDR substrates also comprised read-through transcripts devoid of polyadenylation tail. Importantly, the occurring sRNAs efficiently targeted cognate mRNA for degradation but failed to cleave the non-spliced pre-mRNA, corroborating the notion that sRNAs are not triggering RNA cleavage in the nucleus.

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Acknowledgements

We are thankful to Dr. Guenther Buchholz for providing the plasmid pBAM-dTOM and Mario Braun for providing the plant codon-optimized dTOMATO sequence. This project was supported by the German Research Foundation (DFG; Grant WA 1019/14-1).

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Authors and Affiliations

  1. RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, 67435, Neustadt, Germany

    Athanasios Dalakouras, Anja Lauter, Alexandra Bassler, Gabi Krczal & Michael Wassenegger

  2. Centre for Organismal Studies (COS) Heidelberg, University of Heidelberg, 69120, Heidelberg, Germany

    Michael Wassenegger

  3. Institute of Plant Breeding and Genetic Resources ELGO-DEMETER, 57001, Thessaloniki, Greece

    Athanasios Dalakouras

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  1. Athanasios Dalakouras
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  2. Anja Lauter
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  3. Alexandra Bassler
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  4. Gabi Krczal
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  5. Michael Wassenegger
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Correspondence to Athanasios Dalakouras or Michael Wassenegger.

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Dalakouras, A., Lauter, A., Bassler, A. et al. Transient expression of intron-containing transgenes generates non-spliced aberrant pre-mRNAs that are processed into siRNAs. Planta 249, 457–468 (2019). https://doi.org/10.1007/s00425-018-3015-6

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