RNase R mutants elucidate the catalysis of structured RNA: RNA-binding domains select the RNAs targeted for degradation
Résumé
The RNase II super family is an ubiquitous family of exoribonucleases essential for RNA metabolism. RNase II and RNase R degrade RNA in the 3' to 5' direction in a processive and sequence independent manner. However, while RNase R is capable of degrading highly structured RNAs, the RNase II activity is impaired by the presence of secondary structures. RNase II and RNase R share structural properties and have a similar modular domain organization. The eukaryotic RNase II homologue, Rrp44/Dis3, is the catalytic subunit of the exosome, one of the most important protein complexes involved in the maintenance of the correct levels of cellular RNAs. In this work we constructed truncated RNase II and RNase R proteins and point mutants and characterized them regarding their exoribonucleolytic activity and RNA-binding ability. We report that Asp-280 is crucial for RNase R activity without affecting RNA binding. When Tyr-324 was changed to alanine the final product changed from 2 to 5nts showing that this residue is responsible for setting the end-product. We have shown that the RNB domain of RNase II has catalytic activity. The most striking result is that the RNase R RNB domain per se degrades double stranded substrates even in the absence of a 3'-overhang. Moreover, we demonstrate for the first time that the substrate recognition of RNase R depends on the RNA binding domains that target the degradation of RNAs that are “tagged” by a 3'-tail. These results can have important implications for the study of poly(A)-dependent RNA degradation mechanisms.
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