Two 5′-ETS regions implicated in interactions with U3 snoRNA are required for small subunit rRNA maturation in Trypanosoma brucei

Early pre-rRNA processing events were examined in the ancient protozoan parasite Trypanosoma brucei and found to have both distinctive and conserved features. Two 5'-ETS cleavages occur: A' and the newly discovered A0. A' and A0 appear related to vertebrate and yeast primary pre-RNA cleavage sites, respectively. However, trypanosomatid primary rRNA transcripts can first be processed at the ITS1/5.8S boundary and 5'-ETS sequences then removed by consecutive cleavages at A', A0 and A1 at the 5'-ETS/SSU rRNA junction. 5'-ETS sequences previously crosslinked to U3 snoRNA were tested for their roles in rRNA processing using our new tagged rRNA system. Two distinct A'-adjacent sequence elements, which may pair with U3 hinge bases, were specifically required for SSU rRNA production, as was a downstream element. The latter element appears conserved with the yeast 5'-ETS U3 binding sequence, required for A0, A1 and A2 cleavages, in that they both share 10 bases complementary with U3 hinge sequences and lie upstream from A0 and A1 sites located in a potential stem-loop structure, The distinctive positioning of putative trypanosomatid U3 binding sites with respect to A' and A0 cleavages suggests that different U3-dependent mechanisms may direct each processing event.