Abstract

To keep mRNA homeostasis, the RNA degradation, quality control and silencing systems should act in balance in plants. Degradation of normal mRNA starts with deadenylation, then deadenylated transcripts are degraded by the SKI-exosome 3′-5′ and/or XRN4 5′-3′ exonucleases. RNA quality control systems identify and decay different aberrant transcripts. RNA silencing degrades double-stranded transcripts and homologous mRNAs. It also targets aberrant and silencing prone transcripts. The SKI-exosome is essential for mRNA homeostasis, it functions in normal mRNA degradation and different RNA quality control systems, and in its absence silencing targets normal transcripts. It is highly conserved in eukaryotes, thus recent reports that the plant SKI-exosome is associated with RST1 and RIPR proteins and that, they are required for SKI-exosome–mediated decay of silencing prone transcripts were unexpected. To clarify whether RST1 and RIPR are essential for all SKI-exosome functions or only for the elimination of silencing prone transcripts, degradation of different reporter transcripts was studied in RST1 and RIPR inactivated Nicotiana benthamiana plants. As RST1 and RIPR, like the SKI-exosome, were essential for Non-stop and No-go decay quality control systems, and for RNA silencing- and minimum ORF-mediated decay, we propose that RST1 and RIPR are essential components of plant SKI-exosome supercomplex.

Highlights

  • To generate Tobacco Rattle Virus (TRV)-P-RST1 and TRVP-RIPR Virusinduced gene silencing (VIGS) vectors, 487 and 413 nt long PCR fragments were amplified with Nb Rst1 VIGS EcoRI F / Nb Rst1 VIGS EcoRI R and Nb Ripr VIGS EcoRI F / Nb Ripr VIGS EcoRI R primer pairs from Nicotiana benthamiana cDNA, and the EcoRI cleaved fragments were cloned into TRV-PDS vector

  • We found that the level of the 5′ cleavage products of the minimum uORF (MinuORF) reporter mRNA was higher in the P + RST1 and P + RIPR test as well as in the P + SKI2 positive control VIGS plants than in the PDS silenced negative control plants

  • We have found that SKI2 is required for the degradation of Nonstop decay (NSD) target transcripts and for the elimination of 5′ cleavage fragments generated by No-go decay (NGD), miRNA-programmed RISC (miRISC) or viral siRNA programmed RISC (vsiRISC) (Szádeczky-Kardoss et al 2018a, b)

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Summary

Introduction

Long A-stretches can block elongation and the stalled ribosomes activate NGD (Szádeczky-Kardoss et al 2018b) It cleaves the transcript upstream of the A-stretch, and the 3′ and 5′cleavage fragments are degraded by the XRN4 and the SKI-exosome, respectively. RST1 and RIPR cooperate with the SKI-exosome system to degrade silencing amplification prone mRNAs. It was hypothesized that that the RST1-RIPR complex is involved in other SKI-exosome activities including NGD and NSD quality control systems (Lange et al 2019). As endonuclease cleaved transcripts are sensitive to RNA silencing mediated rqc-siRNA generation, we wanted to study the cis requirement of NGD-mediated cleavage and tried to experimentally test the hypothesis that the RST1 and RIPR proteins play a role in NGD and NSD quality controls. As we found that RST1 and RIPR are involved in all tested cytoplasmic SKI-exosome activities, we propose that these proteins are essential for the function of the SKI-exosome supercomplex in plants

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