Coordinated Ribosomal ITS2 RNA Processing by the Las1 Complex Integrating Endonuclease, Polynucleotide Kinase, and Exonuclease Activities

Highlights

• Las1-Grc3-Rat1-Rai1 complex processes ITS2 pre-rRNA on pre-ribosomes

• Las1 is the endonuclease cleaving 27SB pre-rRNA at site C₂

• Las1 generates 2′,3′ cyclic phosphate on 7S and 5′ OH on 26S pre-rRNA

• Grc3 phosphorylates 26S rRNA for efficient processing by Rat1-Rai1

Summary

The rapidly evolving internal transcribed spacer 2 (ITS2) in the pre-ribosomal RNA is one of the most commonly applied phylogenetic markers at species and genus level. Yet, during ribosome biogenesis ITS2 is removed in all eukaryotes by a common, but still unknown, mechanism. Here we describe the existence of an RNA processome, assembled from four conserved subunits, Las1-Grc3-Rat1-Rai1, that carries all the necessary RNA processing enzymes to mediate coordinated ITS2 rRNA removal. Las1 is the long-sought-after endonuclease cleaving 27SB pre-rRNA at site C₂ to yield a 5′-OH end at the 26S pre-rRNA and 2′,3′ cyclic phosphate at the 3′ end of 7S pre-rRNA. Subsequently, polynucleotide kinase Grc3 catalyzes ATP-dependent 5′-OH phosphorylation of 26S pre-rRNA, which in turn enables Rat1-Rai1 exonuclease to generate 25S′ pre-rRNA. ITS2 processing is reminiscent of tRNA splicing, but instead of subsequent tRNA ligation, the Las1 complex carries along an exonuclease tool to degrade the ITS2 rRNA.