Structural Basis for the Bidirectional Activity of Bacillus nanoRNase NrnA

Abstract

NanoRNAs are RNA fragments 2 to 5 nucleotides in length that are generated as byproducts of RNA degradation and abortive transcription initiation. Cells have specialized enzymes to degrade nanoRNAs, such as the DHH phosphoesterase family member NanoRNase A (NrnA). This enzyme was originally identified as a 3′ → 5′ exonuclease, but we show here that NrnA is bidirectional, degrading 2–5 nucleotide long RNA oligomers from the 3′ end, and longer RNA substrates from the 5′ end. The crystal structure of Bacillus subtilis NrnA reveals a dynamic bi-lobal architecture, with the catalytic N-terminal DHH domain linked to the substrate binding C-terminal DHHA1 domain via an extended linker. Whereas this arrangement is similar to the structure of RecJ, a 5′ → 3′ DHH family DNase and other DHH family nanoRNases, Bacillus NrnA has gained an extended substrate-binding patch that we posit is responsible for its 3′ → 5′ activity.

The trimeric Hef-associated nuclease HAN is a 3′→5′ exonuclease and is probably involved in DNA repair

“…Although the amino acid similarity among DHH superfamily members is less than 20%, a Dali search revealed that their three-dimensional structures are notably similar. The topology of PfuHAN_DND is similar to the structures of several DHH superfamily members (Figure 4D , and Supplementary Figure S9 ), including bacterial RecJs ( 44–46 ), GAN ( 47 ), NrnA ( 48 ), and PPase ( 49 ). Each of them has an N-terminal DHH and a C-terminal DHHA1/2 domain, suggesting they might have evolved from a common ancestor.…”

“…Unlike BsuNrnA, which forms a dimer via interactions between two DHHA1 domains ( 48 ), PfuHAN forms a symmetric trimer through both DHH and DHHA1 domains, as well as the helix linker α13 (Figure 7A and B ). The interaction strength of each subunit is primarily dictated by hydrogen bonds and salt bridges.…”

“…In contrast to bacterial RecJ, bacterial NrnA can hydrolyse short ssRNA (nanoRNA), pAp, and c-di-GMP, but not ssDNA ( 48 , 54 ). In the structures of NrnA, the split alpha helix connecting the DHH and DHHA1 domains further separates the two domains and forms a wide and short binding patch that supports the hydrolysis of short nanoRNA, c-di-GMP and pAp ( 48 , 54 ). Therefore, it might be plausible to identify the residues in bacterial RecJ that sterically repulse the ribose by comparing differences in the substrate binding patches of HAN, NrnA, and RecJ.…”

“…Bacterial RecJ is a 5′-exonuclease ( 55 ), and archaeal HAN is a 3′-exonuclease ( 8 , 51 ). In contrast, NrnA is a bidirectional exonuclease ( 48 ), while archaeal GAN has a more complicated hydrolytic polarity: 5′-exonuclease specific for ssDNA and 3′-exonuclease for ssRNA ( 15 , 16 , 56 , 57 ). The 5′-phosphate-binding pocket, which consists of the polar residues R109, R280, S371, and R373, is present above the DraRecJ active site and determines the 5′-3′ polarity of RecJ for ssDNA ( 46 ).…”

“…The structure of PfuHAN_DND does not have this specific pocket for binding the 3′-nucleotide; thus, it might determine the hydrolysis polarity based on the full binding patch for single-stranded substrates (Figure 6D ). The conserved residues R262 and R264 in BsuNrnA are key residues for determining the 3′-5′ digestion of nanoRNA ( 48 ). NrnA also hydrolyses the cyclic dinucleotides c-di-AMP and c-di-GMP and does not require a specific pocket for determining hydrolysis polarity ( 48 , 54 ).…”