Interestingly, interaction between RNase R and the small ribosomal subunit protein S12, encoded by the rpsL gene, has recently been proposed, leading credence Selleckchem Thiazovivin to our conclusions [19]. After reaching its maximum, RNase R signal intensity decreased along the gradient,

but it could still be detected in the fraction corresponding to the 50S subunit and until the peak of the 70S ribosome (Figure  3A,B). The weaker detection of RNase R in the 50S subunit can be explained by the interaction of this enzyme with DeaD (also known as CsdA). DeaD is a helicase involved in the biogenesis of the 50S ribosomal subunit and its deletion leads to the dysfunction in biogenesis of this ribosomal subunit [20]. Figure 3 RNase R interacts with the small ribosomal subunit. Cellular extracts were separated on sucrose gradients. Position of ribosomal subunits, ribosomes and polysomes ARRY-438162 along the gradient were monitored by UV 280 absorbance (UV280). Amount of RNase R in each fraction of

the gradient was monitored using western blot. Amount of proteins along the gradient was monitored by Ponceau stain. (A) 10-30% sucrose gradient. Polysomes were separated from exponentially and cold shocked cells. (B) 5-20% sucrose gradients. Polysomes were separated from exponentially and cold shocked cells. Difference in subunits migration between the gradients is due to longer centrifugation time of cold shock sample. (C) 5-20% sucrose gradients. Polysome from cold shocked cells were separated, part of the sample was treated with EDTA which results in ribosomal subunits separation. The BCKDHB treatment find more changes pattern of RNase R in the gradient indicating its interaction

with ribosomes. Sample treatment with EDTA, which results in ribosome disruption and subunit separation, causes a change in the RNase R signal pattern, indicating that the position of RNase R in the gradient was due to an interaction with the ribosomes (Figure  3C). RNase R deletion does not impact ribosome formation Our results show that RNase R in vivo interacts with the ribosomes. Data from independent studies suggest that RNase R is involved in the ribosome quality control [9, 10], so interaction with the ribosomes can be important for this function. Overexpression of RNase R rescues phenotype of DeaD helicase deletion at low temperatures. One of the phenotypes of DeaD deletion is the dysfunction in biogenesis of 50S ribosomal subunit [5, 21]. The suppressing role of RNase R suggests that it may also be involved in the ribosome biogenesis. If RNase R is important for ribosome biogenesis, deletion of this enzyme may cause changes in ribosome number or accumulation of deficient ribosome species. To check such a possibility, the sucrose polysome profile of an RNase R deletion strain was compared to those obtained with the wild type cells.