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' end of 5.8 S sequences. The 5' external transcribed spacer (5'ETS) is removed from pre-small subunit (SSU) rRNAs by sequential cleavages at internal A' and A0 sites, and A1 at the 5' end of SSU rRNA. The A' and A0 sites positionally resemble the U3 small nucleolar RNA-dependent, primary pre-rRNA cleavages of vertebrates and yeast, respectively. Uniquely in T. brucei, two U3-crosslinkable 5'ETS sites are essential for SSU rRNA production: site1b is novel in its 3' location to the A' site, and site3 lies upstream of A0 in a position analogous to the yeast U3-binding site. Here, in vivo analysis of mutated 5'ETS sequences shows that sequences 5' to the A' site are not needed 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 site3 U3-binding elements affect A' processing, yet each is required for A0 and A1 cleavage, and SSU rRNA production. The same U3 3' hinge bases evidently bind a core element, UGUu/gGGU, within site1a and site3; the U3-site1b interaction is less reliant on base-pairing than the U3-site3 interaction. As yeast U3 5' hinge bases pair to 5'ETS sequences, it is clear that distinct U3 hinge regions can interact at both novel and related 5'ETS sites to promote 3'-proximal 5'ETS processing events in diverse organisms. The T. brucei data 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 concert with the downstream U3-site3 complex to assist processing events leading to ribosomal SSU production.