Reconstitution of the complete pathway of ITS2 processing at the pre-ribosome. Issue 1 (December 2017) - Fromm, Lisa
Abstract Removal of internal transcribed spacer 2 (ITS2) from pre-ribosomal RNA is essential to make functional ribosomes. This complicated processing reaction begins with a single endonucleolytic cleavage followed by exonucleolytic trimming at both new cleavage sites to generate mature 5.8S and 25S rRNA. We reconstituted the 7S→5.8S processing branch within ITS2 using purified exosome and its nuclear cofactors. We find that both Rrp44's ribonuclease activities are required for initial RNA shortening followed by hand over to the exonuclease Rrp6. During the in vitro reaction, ITS2-associated factors dissociate and the underlying 'foot' structure of the pre-60S particle is dismantled. 7S pre-rRNA processing is independent of 5S RNP rotation, but 26S→25S trimming is a precondition for subsequent 7S→5.8S processing. To complete the in vitro assay, we reconstituted the entire cycle of ITS2 removal with a total of 18 purified factors, catalysed by the integrated activities of the two participating RNA-processing machines, the Las1 complex and nuclear exosome. Excision of internal transcribed spacer 2 (ITS2) within eukaryotic pre-ribosomal RNA is essential for ribosome function. Here, the authors reconstitute the entire cycle of ITS2 processing in vitro using purified components, providing insights into the cleavage process and demonstrating that 26S pre-rRNA processing necessarily precedes 7S pre-rRNA processing.