Abstract
The RNA3 species of the beet necrotic yellow vein virus (BNYVV), a multipartite positive-stranded RNA phytovirus, contains the ‘core’ nucleotide sequence required for its systemic movement in Beta macrocarpa. Within this ‘core’ sequence resides a conserved “coremin” motif of 20 nucleotides that is absolutely essential for long-distance movement. RNA3 undergoes processing steps to yield a noncoding RNA3 (ncRNA3) possessing “coremin” at its 5′ end, a mandatory element for ncRNA3 accumulation. Expression of wild-type (wt) or mutated RNA3 in Saccharomyces cerevisiae allows for the accumulation of ncRNA3 species. Screening of S. cerevisiae ribonuclease mutants identified the 5′-to-3′ exoribonuclease Xrn1 as a key enzyme in RNA3 processing that was recapitulated both in vitro and in insect cell extracts. Xrn1 stalled on ncRNA3-containing RNA substrates in these decay assays in a similar fashion as the flavivirus Xrn1-resistant structure (sfRNA). Substitution of the BNYVV-RNA3 ‘core’ sequence by the sfRNA sequence led to the accumulation of an ncRNA species in yeast in vitro but not in planta and no viral long distance occurred. Interestingly, XRN4 knockdown reduced BNYVV RNA accumulation suggesting a dual role for the ribonuclease in the viral cycle.
Highlights
In eukaryotic cells, the RNA decay machinery plays a major role in determining both the quantity and quality of gene expression
We previously showed that cellular proteins were implicated in beet necrotic yellow vein virus (BNYVV)-RNA3 processing, leading to noncoding RNA3 (ncRNA3) accumulation in a heterologous, non-viral wheat germ extract system [33]
To further explore ncRNA3 biogenesis, Saccharomyces cerevisiae FY4 strains were transformed with plasmid constructs expressing either RNA3 deleted of its 50 UTR (Figure S1A), wt RNA3, or RNA3E, a mutated form of RNA3 (Figure 1A and Figure S1A) in which the “coremin”
Summary
The RNA decay machinery plays a major role in determining both the quantity and quality of gene expression. Nuclear RNA surveillance complexes remove aberrant and unprocessed transcripts, while the cytoplasmic RNA decay machinery coordinates with translation to determine the half-life of mRNAs [1]. Viral transcripts are significantly impacted by these RNA decay pathways. Viruses 2018, 10, 137 and stabilize their messengers or genome components. These include cleavage of decay-promoting factors, such as AUF1, by picornaviruses [3], recruitment of the mRNA stabilizing protein HuR by alphaviruses [4], or repression of the decay machinery itself (for reviews see [5,6,7])
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