The RNA exosome provides eukaryotic cells with an essential 3'-5' exoribonucleolytic activity, which processes or eliminates many classes of RNAs. Its nine-subunit core (Exo9) is structurally related to prokaryotic phosphorolytic exoribonucleases. Yet, yeast and animal Exo9s have lost the primordial phosphorolytic capacity and rely instead on associated hydrolytic ribonucleases for catalytic activity. Here, we demonstrate that Arabidopsis Exo9 has retained a distributive phosphorolytic activity, which contributes to rRNA maturation processes, the hallmark of exosome function. High-density mapping of 3' extremities of rRNA maturation intermediates reveals the intricate interplay between three exoribonucleolytic activities coordinated by the plant exosome. Interestingly, the analysis of RRP41 protein diversity across eukaryotes suggests that Exo9's intrinsic activity operates throughout the green lineage, and possibly in some earlier-branching non-plant eukaryotes. Our results reveal a remarkable evolutionary variation of this essential RNA degradation machine in eukaryotes.

Flaviviruses such as Yellow fever, Dengue, West Nile, and Zika generate disease-linked viral noncoding RNAs called subgenomic flavivirus RNAs. Subgenomic flavivirus RNAs result when the 5'-3' progression of cellular exoribonuclease Xrn1 is blocked by RNA elements called Xrn1-resistant RNAs located within the viral genome's 3'-untranslated region that operate without protein co-factors. Here, we show that Xrn1-resistant RNAs can halt diverse exoribonucleases, revealing a mechanism in which they act as general mechanical blocks that 'brace' against an enzyme's surface, presenting an unfolding problem that confounds further enzyme progression. Further, we directly demonstrate that Xrn1-resistant RNAs exist in a diverse set of flaviviruses, including some specific to insects or with no known arthropod vector. These Xrn1-resistant RNAs comprise two secondary structural classes that mirror previously reported phylogenic analysis. Our discoveries have implications for the evolution of exoribonuclease resistance, the use of Xrn1-resistant RNAs in synthetic biology, and the development of new therapies.

RNA degradation is an essential process that allows bacteria to control gene expression and adapt to various environmental conditions. It is usually initiated by endoribonucleases (endoRNases), which produce intermediate fragments that are subsequently degraded by exoribonucleases (exoRNases). However, global studies of the coordinated action of these enzymes are lacking. Here, we compare the targetome of endoRNase Y with the targetomes of 3'-to-5' exoRNases from Streptococcus pyogenes, namely, PNPase, YhaM, and RNase R. We observe that RNase Y preferentially cleaves after guanosine, generating substrate RNAs for the 3'-to-5' exoRNases. We demonstrate that RNase Y processing is followed by trimming of the newly generated 3' ends by PNPase and YhaM. Conversely, the RNA 5' ends produced by RNase Y are rarely further trimmed. Our strategy enables the identification of processing events that are otherwise undetectable. Importantly, this approach allows investigation of the intricate interplay between endo- and exoRNases on a genome-wide scale.