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

• Verfasst von: Dalakouras, Athanasios [VerfasserIn]
• Verfasst von: Wassenegger, Michael [VerfasserIn]
• Titel: Transient expression of intron-containing transgenes generates non-spliced aberrant pre-mRNAs that are processed into siRNAs
• Verf.angabe: Athanasios Dalakouras, Anja Lauter, Alexandra Bassler, Gabi Krczal, Michael Wassenegger
• Jahr: 2019
• Umfang: 12 S.
• Fussnoten: Published online: 24 September 2018 ; Gesehen am 22.07.2019
• Titel Quelle: Enthalten in: Planta
• Ort Quelle: Berlin : Springer, 1925
• Jahr Quelle: 2019
• Band/Heft Quelle: 249(2019), 2, Seite 457-468
• ISSN Quelle: 1432-2048
• DOI: doi:10.1007/s00425-018-3015-6
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: DCLs
• Sach-SW: Intron
• Sach-SW: Nicotiana benthamiana
• Sach-SW: RDRs
• Sach-SW: RNA silencing
• Sach-SW: Small RNAs
• Sach-SW: Splicing
• Sach-SW: Transgene
• K10plus-PPN: 1669555208

Abstract

Main conclusionIn 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.