Endonucleolytic processing plays a critical role in the maturation of ribosomal RNA in .

Ribosomal RNA (rRNA) processing and maturation are fundamentally important for ribosome biogenesis, but the mechanisms in archaea, the third form of life, remains largely elusive. This study aimed to investigate the rRNA maturation process in , a representative archaeon lacking known 3'-5' exonucleases. Through cleavage site identification and enzymatic assays, the splicing endonuclease EndA was determined to process the bulge-helix-bulge (BHB) motifs in 16S and 23S rRNA precursors. After splicing, the circular processing intermediates were formed and this was confirmed by quantitative RT-PCR and Northern blot. Ribonuclease assay revealed a specific cleavage at a 10-nt A/U-rich motif at the mature 5' end of pre-16S rRNA, which linearized circular pre-16S rRNA intermediate. Further 3'-RACE and ribonuclease assays determined that the endonuclease Nob1 cleaved the 3' extension of pre-16S rRNA, and so generated the mature 3' end. Circularized RT-PCR (cRT-PCR) and 5'-RACE identified two cleavage sites near helix 1 at the 5' end of 23S rRNA, indicating that an RNA structure-based endonucleolytic processing linearized the circular pre-23S rRNA intermediate. In the maturation of pre-5S rRNA, multiple endonucleolytic processing sites were determined at the 10-nt A/U-rich motif in the leader and trailer sequence. This study demonstrates that endonucleolytic processing, particularly at the 10-nt A/U-rich motifs play an essential role in the pre-rRNA maturation of . , indicating diverse pathways of rRNA maturation in archaeal species.