Trypanosoma brucei 5 ' ETS A '-cleavage is directed by 3 '-adjacent sequences, but not two U3 snoRNA-binding elements, which are all required for subsequent pre-small subunit rRNA processing events

Trypanosoma brucei pre-rRNA processing commences by cleavage near the 5' en d of 5.8 S sequences. The 5' external transcribed spacer (FETS) is removed from pre-small subunit (SSU) rRNAs by sequential cleavages at internal A' a nd AO sites, and Al at the 5' end of SSU rRNA. The A' and AO sites position ally resemble the U3 small nucleolar RNA-dependent, primary pre-rRNA cleava ges of vertebrates and yeast, respectively. Uniquely in T. brucei, two U3-c rosslinkable FETS sites are essential for SSU rRNA production: site1b is no vel in its 3' location to the A' site, and site3 lies upstream of AO in a p osition analogous to the yeast U3-binding site. Here, in vivo analysis of m utated 5'ETS sequences shows that sequences 5' to the A' site are not neede d for A' cleavage or SSU rRNA production. A' cleavage is linked to, but is not sufficient to trigger, downstream pre-SSU rRNA processing events. These events require an intact 11 nt sequence, 3'-adjacent to A', which directs efficient and accurate A' cleavage. Neither the A' nearby site1b nor the si te3 U3-binding elements affect A processing, yet each is required for AO an d A' cleavage, and SSU rRNA production. The same U3 3' hinge bases evidentl y bind a core element, UGUu/gGGU, within site1a and site3; the U3-site1b in teraction is less reliant on base-pairing than the U3-site3 interaction. As yeast U3 5' hinge bases pair to FETS sequences, it is clear that distinct U3 hinge regions can interact at both novel and related FETS sites to promo te 3'-proximal FETS processing events in diverse organisms. The T. brucei d ata fit a model wherein processing factors assemble at the 5'ETS site1a to affect A' cleavage and stabilize a U3-site1b complex, which may work in con cert with the downstream U3-site3 complex to assist processing events leadi ng to ribosomal SSU production. (C) 2001 Academic Press.