These analyses showed that expression of the chosen fusion proteins had no general cytotoxic effects as consequence of a potential increase in cytoplasmic RNAse activities. Moreover, indicating their incorporation into yeast ribosomes. Consistent with this, 80S ribosomal components co-purified in a specific and efficient manner with the MNase-HA tagged fusion proteins. Extracts were prepared of cells of these strains grown in medium containing glucose as carbon source to express solely the MNase fusions of the respective ribosomal proteins. Buffers used for extract preparation contained EGTA to quench excess of free calcium ions possibly released through breakage of subcellular compartments. Use of EGTA instead of EDTA for this purpose should preserve the concentration of free magnesium ions important for structure and function of many RNPs. Exogenous calcium was then added and extracts were incubated at 22uC. Total RNA was extracted from aliquots taken before or after addition of calcium and was analyzed by gel electrophoresis followed by ethidium bromide staining or Northern blotting. As seen in Figs. 1 and 2, lanes 1–5, only minor degradation of rRNA was detected in cellular extracts prepared from a strain expressing no MNase fusion protein in Fig. 2A, lane 5). By contrast, ethidium bromide staining revealed substantial calcium induced Silmitasertib fragmentation of rRNA in extracts from strains expressing MNase fusions of ribosomal proteins. The rRNA fragmentation patterns clearly differed for each of these strains. Fragmentation of 18S rRNA was seen in extracts of strains expressing MNase fusions of the small ribosomal subunit protein rpS13. Fragmentation of 25S rRNA was detected in extracts of strains expressing MNase fusions of large ribosomal subunit proteins rpL5 and rpL35. In addition to that, 5.8S rRNA was fragmented after addition of calcium to extracts of strains expressing MNase fusions of rpL35. Omission of calcium during extract incubation efficiently inhibited the observed rRNA fragmentation. Addition of recombinant MNase to an extract of yeast cells expressing no MNase fusion proteins resulted in a distinct rRNA fragmentation pattern consistent with various preferred MNase cleavage sites in rRNA distributed all over the 80S ribosome. The specific rRNA fragmentation pattern observed in the extracts expressing MNase fusion proteins strongly suggested that calcium dependent nuclease activity of MNase was tethered to local ribosomal environments through its fusion with the respective ribosomal protein. To correlate known ribosomal protein binding sites and calcium induced local RNA cleavage events in the in vivo formed recombinant ribosomes we aimed to characterize in more detail the sites of major nuclease actions in extracts of strains expressing MNase fusions of rpL5 and rpL35. Inspection of atomic resolution three dimensional structure models of yeast ribosomes revealed that the major cuts of MNase-rpL5 were localized in the central protrusion next to the rpL5 binding site. Cuts of MNase-rpL35 were localized on the opposite side of the ribosome clustered around the rpL35 binding site around the exit tunnel. Measurements on the basis of the atomic coordinates provided in indicated that the two major MNase-rpL5 cuts were about 1.5 and 4 nm, respectively, away from the rpL5 amino terminus. The MNase-rpL35 proximal cut in 5.8S rRNA was about 1 nm away from the rpL35 amino terminus while the two distal cuts in the 25S rRNA were between 5 and 5.5 nm away. We reasoned that the nucleolytic actions of MNAse fusion proteins detected in these experiments were defined by a few major determinants.