Hello, my name is Sebastian Gruenberg and I am a Research Scientist in the RNA division here at New England Biolabs. I’m excited to present one of our recent publications, titled “E. coli RNase I exhibits a strong Ca2+-dependent inherent double-stranded RNase activity”.
RNase I is a ribonuclease that has been studied since the early 60s and has been described as a metal-independent and non-sequence specific endoribonuclease that targets single stranded RNA. These features make it a favorite tool in molecular biology to remove ssRNA from reactions. Inspired by the observation that some other RNases showed increased affinity for structured RNAs in the presence of Ca2+ and other metal ions, we decided to take a closer look at RNase I and characterize the effect of metal ions on its activity.
As shown previously, RNase I showed very low cleavage activity and affinity towards double stranded RNA in the absence of Ca2+. However, when we added Ca2+, the enzyme cleaved dsRNA with 90-fold higher efficiency.
As shown previously, RNase I showed very low cleavage activity and affinity towards double stranded RNA in the absence of Ca2+. However, when we added Ca2+, the enzyme cleaved dsRNA with 90-fold higher efficiency. Interestingly, Ca2+ did not affect RNase I activity on ssRNA, suggesting that Ca2+ has no effect on the enzyme’s targeting of ssRNA substrates.
Another enzymatic activity of RNase I that we described for the first time is a hybridase activity. In the presence of Ca2+, RNase I will degrade the RNA strand of an RNA:DNA hybrid, leaving the DNA strand untouched.
It was quite surprising to us that these Ca2+ dependent activities had not been reported, considering how much research was done on – and with – RNase I over the decades.
When we closely studied the crystal structure of RNase I that had been published in 2008, we noticed that it contained a Ca2+ binding site with a bound Ca2+. So we wondered if this Ca2+ bound state was the native state, and if the ion – and with it the enzymatic properties that depend on it – might simply get lost during standard protein purifications?
When we purified RNase I without EDTA, which is a metal chelating agent that is commonly used in protein purifications to inhibit metal dependent nucleases and proteases, the enzyme was able to cleave dsRNA effectively even in the absence of Ca2+.
This and other experiments in the paper strongly suggest that native RNase I in fact contains a bound Ca2+ that is required for efficient cleavage of dsRNA.
RNase I’s function in the cell has been described as a scavenger enzyme that recycles unwanted RNA into monophosphates. Obviously, the ability to efficiently cleave dsRNA is desirable for the recycling of highly structured RNA that could not be processed by purely ssRNA cleaving RNases.
What we learned from this work is that there is a good chance that there might be activities hidden even in enzymes we’ve worked with for many years, probably obscured by purification methods or reaction conditions.
And these activities could lead the way to new applications and technologies. In the case of RNase I, adding Ca2+ to a reaction will allow for a much more efficient removal of structured RNA from DNA or protein preparations and Ribonuclease protection assays.
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