A dedicated diribonuclease resolves a key bottleneck for the terminal step of RNA degradation

Abstract

Degradation of RNA polymers, an ubiquitous process in all cells, is catalyzed by specific subsets of endo- and exoribonucleases that together recycle RNA fragments into nucleotide monophosphate. In γ-proteobacteria, 3-‘5’ exoribonucleases comprise up to eight distinct enzymes. Among them, Oligoribonuclease (Orn) is unique as its activity is required for clearing short RNA fragments, which is important for cellular fitness. However, the molecular basis of Orn’s unique cellular function remained unclear. Here, we show that Orn exhibits exquisite substrate preference for diribonucleotides. Crystal structures of substrate-bound Orn reveal an active site optimized for diribonucleotides. While other cellular RNases process oligoribonucleotides down to diribonucleotide entities, Orn is the one and only diribonucleotidase that completes the terminal step of RNA degradation. Together, our studies indicate RNA degradation as a step-wise process with a dedicated enzyme for the clearance of a specific intermediate pool, diribonucleotides, that affects cellular physiology and viability.

Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria

“…To gain molecular insight into the mechanism and specificity of nucleotide degradation by REXO2, we crystallized the human protein comprising residues 39-216, which retains catalytic activity (Figures S1F-S1I), and determined its structure to 2.0 Å resolution (Table 1). As previously reported (Chu et al, 2019;Kim et al, 2019), the protein crystallizes as a dimer and has an RNase-H-like fold that is composed of five central b strands surrounded by nine a helices on opposite sides (Figures 2A and S2C). Each monomer harbors an active site formed by the conserved acidic DEDD residues D47, E49, D147, and D199 that are positioned for nucleotide binding (Figure 2A), and mutation of any one of the conserved DEDD residues to alanine severely impaired the catalytic activity of the enzyme ( Figures 2B-2D), consistent with their key role in catalysis.…”

“…ExoG releases dinucleotides as reaction products and is active upon RNA (Szymanski et al, 2017;Wu et al, 2019) and so could potentially fulfill this role. Interestingly, a very recent study of E. coli Orn has similarly revealed a stark preference for diribonucleotide substrates (Kim et al, 2019), suggesting that oligoribonucleases have a more specialized and evolutionarily conserved role than previously understood. A second potential source of nanoRNAs in mitochondria are cycles of abortive transcription by POLRMT.…”

“…This process requires the conserved and essential oligoribonuclease Orn (Ghosh and Deutscher, 1999;Niyogi and Datta, 1975). Orn is a 3 0 -5 0 exonuclease of the DEDDh family that releases mononucleotides as reaction products (Datta and Niyogi, 1975) and was recently found to show a strong preference for dinucleotide substrates (Kim et al, 2019). The loss of Orn results in large-scale alterations in gene expression that have been attributed to the ability of nano-RNAs to mediate transcription priming (Druzhinin et al, 2015;Goldman et al, 2011;Vvedenskaya et al, 2012).…”

Draft Genome Sequence of Pseudomonas aeruginosa Strain PA14-UM

“… Pseudomonas aeruginosa is an opportunistic pathogen that is associated with significant global morbidity and mortality among hospitalized burn and cystic fibrosis patients, as well as immunocompromised individuals ( 1 ). P. aeruginosa PA14, originally isolated by the Ausubel laboratory ( 2 ), was acquired by the laboratory of Steven Lory and passed on to the laboratory of Vincent Lee, where it has been named PA14-UM to indicate the version of PA14 that resides at the University of Maryland ( 3 – 10 ). PA14 is a commonly used strain for studies of P. aeruginosa and represents a lineage that is distinct from another commonly used reference isolate, PAO1.…”

NrnA is a linear dinucleotide phosphodiesterase with limited function in cyclic dinucleotide metabolism inListeria monocytogenes

“…A subset of nanoRNases, including Orn, NrnA, NrnB, and NrnC can readily degrade pGpG (14). Indeed, Orn has a strong preference for linear dinucleotides in bacterial cells over longer substrates (15). Accordingly, a P. aeruginosa orn mutant, which is severely diminished for pGpG degradation, also accumulates c-di-GMP (11,12).…”