E. coli RNase I exhibits a strong Ca2+-dependent inherent double-stranded RNase activity

Abstract

Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca2+)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca2+-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca2+ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca2+ in the modulation of RNase I activity. Mutation of a previously overlooked Ca2+ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca2+, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca2+-dependency of RNase I may be useful as a tool in applied molecular biology.

Figure 1.

Calcium significantly stimulates RNase I activity on dsRNA. (A) 33mer dsRNA (0.1 μM) substrate with 2 nt 3′ overhangs was incubated at 37°C for 15 min without (NE) or with various concentrations of RNase I as indicated above the lanes (from 6.7 μM to 6.7 × 10⁻⁴ μM) in either the absence (left panel) or presence (right panel) of 4 mM CaCl₂. Asterisks indicate the RNase I concentrations used for the subsequent MS analysis. Red diamonds mark primary cleavage products. (B) Quantification of the relative amount of uncleaved RNA substrate ± CaCl₂. (C) MS results showing the location of the cleavage (location of the bar above the sequence) and the relative abundance of the resulting fragment (height of the bar) for reactions containing 67 nM RNase I without Ca²⁺ (top) and 6.7 nM RNase I with Ca²⁺ (bottom). FL, full-length oligonucleotide (uncleaved substrate). (D) 6.7 × 10⁻³ μM (left panel) or 1.34 × 10⁻³ μM (right panel) of RNase I were incubated in the absence or presence of Ca²⁺, respectively, and various concentrations of dsRNA as indicated. Reactions were quenched at the indicated timepoints and the amount of digested full-length dsRNA over time (circles) was plotted. Solid traces are exponential curve fits to the data.

Figure 2.

Calcium is not required for RNase I activity on ssRNA. Reactions in (A) contained a 33mer ssRNA (0.1 μM) substrate with either no enzyme (NE) or various concentrations of RNase I (from 0.67 to 1.34 × 10⁻⁴ μM) for 15 minutes at 37°C. The relative amount of uncleaved RNA is shown in (B). (C) 6.7 × 10⁻⁴ μM of RNase I were incubated with various concentrations of ssRNA from 0.67 to 3.35 μM. Reactions were quenched at the indicated timepoints and the amount of digested full length ssRNA over time (circles) was plotted. Solid (no Ca²⁺) and dashed (plus Ca²⁺) traces are exponential curve fits to the data.

Figure 3.

RNase I exhibits hybridase activity. Calcium induced activation of RNase I against 0.1 μM of a double stranded 31mer hybrid substrate containing a DNA top and an RNA bottom strand as shown above the gel. (A) 0.67 μM to 1.34 × 10⁻³ μM of RNase I were incubated with the substrate in the absence (lanes 1–5) or presence of 4 mM CaCl₂ (lanes 8–12). Lanes 6 and 7 show the separate RNA and DNA strands, respectively. (B) Quantification of the uncleaved RNA.

Figure 4.

Effect of metal ions on RNase I activity against dsRNA. (A) To test whether other divalent cations exhibit a similar stimulatory effect on dsRNA cleavage by RNase I, 0.1 μM dsRNA substrate was incubated with 0.067 μM RNase I in the presence of 0.5 mM EDTA and either 1 mM or 4 mM CaCl₂, MgCl₂, MnCl₂ or ZnCl₂. (B) Broader range titration of CaCl₂ and MnCl₂ from 0.5 to 10 mM. Asterisks indicate no-enzyme control reactions containing 4 mM of the respective metal ion.

Figure 5.

RNase I purified in the absence of EDTA exhibits an inherent strong dsRNase activity. (A) The crystal structure of RNase I (2PQX; Rodriguez et al., 2008) contained a previously overlooked Ca²⁺ binding site (colored in grey, coordinating Ca²⁺ represented as magenta sphere) that is located approximately 18 Å away from the enzyme's active site (colored in light pink). 0.1 μM dsRNA was incubated with various concentrations of RNase I as indicated above the lanes (from 6.7 to 6.7 × 10⁻⁴ μM) in the absence (B) or presence (C) of 4 mM CaCl₂. No DTT or EDTA was present in the reactions shown in (B) and (C). The asterisks indicate the RNase I dilutions selected for MS analysis. The uncleaved RNA was quantified and plotted in (D). (E) MS results showing the location of the cleavage and the relative abundance of the resulting fragment for reactions containing 6.7 nM RNase I without (top) and with Ca²⁺ (bottom). FL, full-length oligonucleotide (uncleaved substrate).

Figure 6.

EDTA removes RNase I bound Ca²⁺ leading to the inhibition of its dsRNase activity. (A) 0.1 μM dsRNA and various concentrations of RNase I purified in the presence (top panel) or absence (bottom panel) of DTT and EDTA (–DTT/–EDTA) were incubated without or with 0.5 mM EDTA in the reaction, as indicated below the gels. Reactions in lanes 10–18 contained 4 mM CaCl₂. The quantification of the uncleaved RNA substrate is shown in (B). NE, no enzyme control.

Figure 7.

Activity of RNase I D241L and D241E mutants. RNase I cleavage of synthetic dsRNA or ssRNA using dilution series of RNase I mutant enzymes. (A) Structure (wt) and homology models (D241L/E) of the Ca²⁺ coordinating site on RNase I. Either a dsRNA substrate (B, F) or a ssRNA substrate (D) was cleaved by the indicated amount of RNase I_D241L (B, D) or RNase I_D241E (F) in the absence or presence of Ca²⁺. The concentration for each enzyme is indicated at the top of each lane. (C, E, G) show the respective quantifications of the uncleaved RNA substrates in (B, D, F). NE, no enzyme control.