Fragmentation Of Ribosomal 23S RRNA In Cyanobacteria

In most prokaryotes such as E. coli,23 S rRNA within the ribosomal large subunit is a single molecule.While recent studies showed that, in some species (such as Salmonella) the 23 S rRNA in ribosome is not an intact sequence, instead it consists of two or more pieces. The origin of this phenomenon and its effects on cell activities remain unclear. When analyzing the total RNA isolated from two cyanobacterial species(Anabaena sp. PCC 7120 and Synechocystis sp. PCC 6803), we noticed that a) the intensity ratio of 23 S rRNA and 16S rRNA was significantly lower than 2; b) in addition to the bands corresponging to the full-size 23 S rRNA,16S rRNA and 5S rRNA, another two clear bands, with their sizes close to that of the intact 23 S rRNA, were shown. We speculated that these two bands were fragments generated from 23 S rRNA.Thus the phenomenon and the underlying mechanism was investigated in this study. Using 23 S rRNA specific fluorescent probes, it was proved that the additional bands are indeed the pieces of 23 S rRNA. Subsequently, we examined the total RNA samples from several bacteria (including cyanobacterial and non-cyanobacterial species) by electrophoresis, and found that all cyanobacterial RNA samples exhibited very similar patterns (i.e., having the bands of fragmented 23 S rRNA), while the RNA samples of E. coli and Bacillus subtilis did not have such patterns. It indicates that fragmentation of 23 S rRNA has special physiological significance for cyanobacteria. By Ligation-Dependent RT-PCR, we successfully cloned the 5’and 3’ends of the intact 23 S rRNAs and their cleaved products from Anabaena sp. PCC 7120 and Synechocystis sp. PCC 6803. The results show that the cleavage sites in 23S rRNA are identical in both cyanobacteria. Further sequence analysis revealed that the nucleatides around the cleavage site are highly conserved in all sequenced cyanobacteria, implying that 23 S rRNA fragmentation is a universal phenomenon in cyanobacteria. We further found that fragmented 23 S rRNA species are present in each of 50S,70S and polysome of ribosomes isolated by sucrose density gradient centrifuge, suggesting that fragmentation occurs in functional ribosomes. In order to determine the ribonuclease responsible for the cleavage of 23 S rRNA, we tested the activities of two predicted endoribonucles, RNase E and RNase J, on the isolated intact 70S ribosome and several synthetic cleavage-site containing sub-regions of 23 S RNA. However, none of the enzymes could cleave the substrate RNAs at expected sites. It is spectulated that cyanobacterial 23S rRNA fragmentation is a process under strict regulation and involved in many unkonwn factors in vivo.