NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

Abstract

Bacterial RNases process RNAs until only short oligomers (2–5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2–4 nucleotides in length, all of which were processed from their 5′ terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.

Structural and functional investigation of the DHH/DHHA1 family proteins in Deinococcus radiodurans

“…The results revealed that DrRecJ-like efficiently hydrolyzes ssRNA to 1 nt, with the highest activity observed in the presence of Mn 2+ as a cofactor (Figure 3A ). This observation aligns with findings for homologs from other bacteria ( 12 , 14 , 38–40 ). Additionally, Ni 2+ , Co 2+ and Mg 2+ proved to be effective metal cofactors, while Zn 2+ and Ca 2+ exhibited inhibitory effects.…”

“…Unlike BsNrnA, which degrades 2–5 nucleotide long RNA oligomers from the 3′ end, and longer RNA substrates from the 5′ end ( 41 ), our data indicate that DrNrnA cleaves both nanoRNA and longer oligos in the 3′-5′ direction. On the contrary, it was reported that NrnA-like proteins from Thermus thermophiles HB8, Enterococcus faecalis and Streptococcus pyogenes prefer to digest substrate in a 5′-3′ direction ( 38 , 45 ). Further research is required to test whether those non-conservative amino acid variances mentioned above may result in distinct substrate sequence and length preferences, and cleavage directionality of these proteins.…”

“…Interestingly, studies on the activity of NrnA-like proteins in different bacteria often yield very different conclusions, especially regarding the optimal bases, optimal substrate length and the confirmation of cleavage directionality ( 44 ). For example, B. subtilis NrnA (BsNrnA) possesses a slight preference for 5′ purine residues ( 38 ), while DrNrnA prefers digesting pyrimidine residues. NrnAs from M. tuberculosis and Mycoplasma pneumoniae M129 are known to preferentially digest ssRNA substrates of 2 and 5 nt in length, respectively ( 17 ), while DrNrnA prefers digesting 2–3 nucleotide long RNA.…”

Widespread PRC barrel proteins play critical roles in archaeal cell division

“…Notably, PRC barrel proteins are often found in the vicinity of genes encoding translation systems components or RNA processing enzymes. Specifically, cdpB1 is encoded next to nrnA , a 5’-3’ exonuclease involved in processing of short RNA substrates 38 , and HVO_1964 is encoded divergently to infB , translation initiation factor 2. These two genes are located in conserved neighborhoods in haloarchaea (Supplementary Table 2).…”