Processing of precursor rRNA in Euglena gracilis: identification of intermediates in the pathway to a highly fragmented large subunit rRNA

We have identified and characterized the stable steady-state intermediates that appear during formation of the cytoplasmic rRNA in Euglena gracilis. A 10.2 kb RNA is the precursor to both the small subunit (SSU) rRNA and 14 discrete fragments that comprise the large subunit (LSU) rRNA. The SSU rRNA is produced via two intermediates of 4.4 kb and 3.2 kb, whereas the LSU rRNA is generated by way of two RNA species of 5.8 kb and 5.3 kb. A number of unique intermediates are associated with a novel processing pathway by which the 14 mature fragments of the LSU rRNA are produced. Analysis of transcripts mapping within ITS1, the internal transcribed spacer separating the SSU and LSU rRNA coding regions, revealed that the LSU1 (=5.8S) rRNA is heterogeneous at its 5'-end, with a major cluster of primer extension products terminating approx. 4-5 nucleotides upstream from the predominant, mature 5'-end and a second, low-level extension product appearing further upstream within ITS1. The results reported here define the pre-rRNA processing pathway in E. gracilis and provide the basis for further studies of the mechanism of excision of the novel ITSs in this system.