Prof. Michaeli Shulamit

Nucleic Acids Research, 2019 1–15

doi: 10.1093/nar/gkz477

Pseudouridines on Trypanosoma brucei spliceosomal small nuclear RNAs and their implication for RNA and protein interactions

K. Shanmugha Rajan1, Tirza Doniger 1, Smadar Cohen-Chalamish1, Dana Chen1,

Oz Semo1, Saurav Aryal1, Efrat Glick Saar2, Vaibhav Chikne1, Doron Gerber 1, Ron Unger1, Christian Tschudi3 and Shulamit Michaeli 1,*

1The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute,

Bar-Ilan University, Ramat-Gan 52900, Israel, 2Sheba Medical Center, Tel-HaShomer 5265601, Israel and

3Departmentof Epidemiology and Microbial Diseases, Yale School of Public Health, New Haven, CT 06536, USA

Received February 21, 2019; Revised May 11, 2019; Editorial Decision May 15, 2019


The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an insect and a mammalian host. Here, we investigated the presence of pseudouridines (!s) on the spliceosomal small nuclear RNAs (snRNAs), which may enable growth at the very different temperatures characterizing the two hosts. To this end, we performed the first high-throughput mapping of spliceosomal snRNA !s by small RNA !-seq. The analysis revealed 42 !s on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding ! on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA !s are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth at elevated temperatures. We further demonstrate that ! strengthens U4/U6 RNA–RNA and U2B’/U2A’-U2 snRNA interaction at elevated temperatures. The existence of single hairpin RNAs that modify both the

spliceosome and ribosome RNAs is unique for these parasites, and maybe related to their ability to cycle between their two hosts that differ in temperature.