Distinct regions of U3 SnoRNA interact at two sites within the 5′ external transcribed spacer of pre-rRNAs in Trypanosoma brucei cells

U3 snoRNA is required for early pre-rRNA processing events that include cleavage of the 5' external transcribed spacer (5'ETS) and 18S rRNA maturation. Herein, psoralen RNA crosslinking has been used to indicate novel in vivo interactions between the minimally-sized Trypanosoma brucei U3 snoRNA and pre-rRNAs. Two discrete U3 crosslinks were mapped to 5'ETS sequences, then individually isolated by hybrid selection following digestion of pre-rRNAs. Crosslink positions within these U3-site1 and U3-site2 complexes were resolved by RNaseH digestion and primer extension analyses. Hinge bases of U3 contacted site1 bases U140 and U142 just 3' of the processed primary site, This is the first experimental evidence of a U3 RNA interaction adjacent to a major 5'ETS cleavage site and supports a critical role for U3 in its processing. Highly conserved box A bases contacted site2 base U945, 187 nt upstream of 18S-like rRNA sequences. Site2 sequences are not required for primary processing, thus, a U3 interaction here might have roles in subsequent downstream processing events. These results clearly demonstrated that distinct U3 snoRNA sequences crosslinked different regions of the 5'ETS and support a model for U3 as a multifunctional snoRNA.