Degradation of nanoRNA is performed by multiple redundant RNases in Bacillus subtilis

Abstract

Escherichia coli possesses only one essential oligoribonuclease (Orn), an enzyme that can degrade oligoribonucleotides of five residues and shorter in length (nanoRNA). Firmicutes including Bacillus subtilis do not have an Orn homolog. We had previously identified YtqI (NrnA) as functional analog of Orn in B. subtilis. Screening a genomic library from B. subtilis for genes that can complement a conditional orn mutant, we identify here YngD (NrnB) as a second nanoRNase in B. subtilis. Like NrnA, NrnB is a member of the DHH/DHHA1 protein family of phosphoesterases. NrnB degrades nanoRNA 5-mers in vitro similarily to Orn. Low expression levels of NrnB are sufficient for orn complementation. YhaM, a known RNase present in B. subtilis, degrades nanoRNA efficiently in vitro but requires high levels of expression for only partial complementation of the orn(-) strain. A triple mutant (nrnA(-), nrnB(-), yhaM(-)) in B. subtilis is viable and shows almost no impairment in growth. Lastly, RNase J1 seems also to have some 5'-to-3' exoribonuclease activity on nanoRNA and thus can potentially finish degradation of RNA. We conclude that, unlike in E. coli, degradation of nanoRNA is performed in a redundant fashion in B. subtilis.

Figure 1.

Complementation of the conditional orn mutant by expression of NrnB but not the NrnB-DHH mutant. Transformants of strain UM341 with pBAD18 (vector control), pUM408 (arabinose-inducible orn), pUM414 (arabinose-inducible nrnB) or pUM443 (arabinose-inducible NrnB-DHH mutant) were spread on LB plates containing 0.2% arabinose in the absence of anhydrotetracycline (Atc).

Figure 2.

NrnB-catalyzed degradation of nanoRNA. Shown is the separation of reaction products on 22% PAA gels (A, B, E, F). The reverse migration can be accounted for by the fact that cyanine dyes have a lower net negative charge than nucleic acids: thus, removing nucleotides will reduce the charge relative to the mass of the oligonucleotide and cause it to shift up instead of down. Panels (C) and (D) show quantifications of reaction products and intermediates of degradation of 5′Cy5-CCCCC3′ and 5′Cy5-AAAAA3′, respectively. 50 μl reactions contained 0.25 μg NrnB and 6.0 μM RNA (5′Cy5-CCCCC3′ for A and C, 5′Cy5-AAAAA3′ for B and D, 5′Cy5-CCC3′ for E, and 5′Cy5-AAA3′ for F). The minus indicates controls lacking enzyme. (C and D) Closed circles: 5-mers, open circles: 4-mers, closed triangles: 3-mers, open triangles: 2-mers, squares: 1-mers. (G) Comparison of initial rates of degradation of 5′Cy5-AAAAA3′, 5A; 5′Cy5-CCCCC3′, 5C; 5′Cy5-AAA3′, 3A; and 5′Cy5-CCC3′, 3C. Specific activities measured as disappearance of substrate were normalized according to the activity on 5′Cy5-AAAAA3′, which was set to be 1.

Figure 3.

Activity of NrnB-DHH on nanoRNA (5′Cy5-CCCCC3′). Fifty-microliter reactions contained 5 μg NrnB-DHH and 6 μM RNA 5-mer (5′Cy5-CCCCC3′).

Figure 4.

Activity of NrnB on RNA 24-mers. Reactions containing 5′ ³³P-labeled RNA 24-mers (5′CACACACACACACACACACACACA3′) and 0.5 μg NrnB or no enzyme (minus) were incubated at 37°C. M, decade marker; H, alkaline hydrolysis control.

Figure 5.

Lacking or partial complementation of the conditional orn mutant by expression of YhaM from different constructs differing in copy number. Transformants of strain UM341 with pBAD18 (vector control), vector; pUM408 (arabinose-inducible orn), orn; pUM413 (arabinose-inducible yhaM in pBAD18, a low-copy (lc) number vector), yhaMlc; or pFM1 (yhaM in pGEMT-Easy, a high-copy (hc) number vector), yhaMhc were spread on LB plates containing 0.2% arabinose in the absence of anhydrotetracycline (Atc).

Figure 6.

Comparison of YhaM activity on RNA and DNA substrates. Shown is the separation of reaction products on 22% PAA gels. 50 μl reactions contained (A) 20 μg YhaM and 5.4 μM RNA oligo (5′Cy5-CCCCC3′) or (B) 4 μg YhaM and 5.4 μM DNA oligo (5′Cy5-CCCCC3′).

Figure 7.

Degradation of nanoRNA 5-mers by RNase J1. Thirty-microliter reactions contained 3 μg RNase J1 or 5 μg RNase J1 mutant protein (RNase J1 H46A, H46A mut) and 5 μM RNA oligo (5′Cy5-AAAAA3′, Figure 7A; or 5′Cy5-CCCCC3′, Figure 7B). The minus indicates controls lacking enzyme.