Bacillus subtilis ribonucleases J1 and J2 form a complex with altered enzyme behaviour

Abstract

SummaryRibonucleases J1 and J2 are recently discovered enzymes with dual 5Ј-to-3Ј exoribonucleolytic/ endoribonucleolytic activity that plays a key role in the maturation and degradation of Bacillus subtilis RNAs. RNase J1 is essential, while its paralogue RNase J2 is not. Up to now, it had generally been assumed that the two enzymes functioned independently. Here we present evidence that RNases J1 and J2 form a complex that is likely to be the predominant form of these enzymes in wild-type cells. While both RNase J1 and the RNase J1/J2 complex have robust 5Ј-to-3Ј exoribonuclease activity in vitro, RNase J2 has at least two orders of magnitude weaker exonuclease activity, providing a possible explanation for why RNase J1 is essential. The association of the two proteins also has an effect on the endoribonucleolytic properties of RNases J1 and J2. While the individual enzymes have similar endonucleolytic cleavage activities and specificities, as a complex they behave synergistically to alter cleavage site preference and to increase cleavage efficiency at specific sites. These observations dramatically change our perception of how these ribonucleases function and provide an interesting example of enzyme subfunctionalization after gene duplication.

What is the role of RNase J in mRNA turnover?

“…10 However, we failed to see a stable interaction between RNase J1, RNase J2 or the RNase J1/J2 complex and any other proteins in extensive yeast 2-hybrid and 3-hybrid screens in vivo. 9 We also failed to see an interaction between Flag-tagged RNase J1 and any other protein besides RNase J2 in pulldown experiments from B. subtilis whole cell extracts. The functional importance of such a complex, if it exists, remains to be seen.…”

“…That this only caused a two-fold decrease in growth rate suggests that, at about 2,500 molecules per cell, 9 RNase J1 is produced in significant excess over the cell's needs. A key question therefore is whether more transcripts would show increased stability under more stringent RNase J1 depletion conditions, where the cells exhibit a severe growth phenotype.…”

“…It is also clear that RNase J2 has very poor 5'-to-3' exoribonuclease activity compared to RNase J1. 9 Our current hypothesis is that this explains why RNase J1 is essential, i.e., that RNase J2 alone cannot functionally replace this property of RNase J1. A recent paper from the Stülke laboratory suggested that, in addition to interacting with each other, RNase J1 and J2 were part of a B. subtilis 'degradosome' complex containing two other ribonucleases, RNase Y and PNPase, phosphofructokinase and enolase.…”

“…When expressed together in the same cell, RNase J1/J2 preferentially form heteromers, suggesting that the homomeric complexes of the individual enzymes are not as stable. 9 Thus, although RNase J2 has little 5'-3' exoribonuclease activity itself, cells lacking RNase J2 may have less 5'-to-3' exoribonuclease activity due to the inherent instability of RNase J1 dimers or tetramers. This would account for the apparent differences in the importance of RNase J2 observed under conditions of mild and severe RNase J1 depletion and would place RNase J1 5'-3' exoribonuclease activity to the forefront.…”

“…4 We also now know that RNase J1 and J2 form a complex and that this is likely to be the predominant form of these two enzymes wild-type cells in vivo. 9 The purified complex is primarily a heterotetramer, with a tendency to dissociate to heterodimers. Interestingly, the RNase J1/ J2 complex has different endonucleolytic cleavage site preferences to the individual enzymes in vitro and this will have significant bearing on the design of future experiments with these two enzymes.…”