Abstract
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 (Ψs) 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 Ψs by small RNA Ψ-seq. The analysis revealed 42 Ψs 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 Ψ 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 Ψs 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 Ψ strengthens U4/U6 RNA–RNA and U2B"/U2A’ proteins-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 may be related to their ability to cycle between their two hosts that differ in temperature.
Highlights
The most abundant modifications on ribosomal RNA are 2 -O-methylation (Nm) and pseudouridinylation ( )
To enrich for small RNAs, whole-cell extracts were prepared from both T. brucei procyclic form (PCF) and bloodstream form (BSF) parasites and were depleted of ribosomes, resulting in a post-ribosomal supernatant (PRS) enriched in small RNAs (Figure 1A)
Primer extension verified the existence of s such as 47 and 100 in U2 small nuclear RNAs (snRNAs) (Figure 1Ci); 47, 51, 54, 64 and 66 in U4 snRNA (Figure 1Cii) and 33 and 46 in U6 snRNA (Supplementary Figure S2C) which were detected in
Summary
The most abundant modifications on ribosomal RNA (rRNA) are 2 -O-methylation (Nm) and pseudouridinylation ( ). Whereas the modifications on rRNAs are performed in the nucleolus, guiding of modifications on spliceosomal U1, U2, U4 and U5 snRNAs as well as snRNP assembly takes place in the Cajal bodies (CB). These modifications are guided by small Cajal-body RNA species (scaRNA) that are bound by WDR79 protein [8]. SnRNA modifications were thought to be constitutive, but recent studies revealed that pseudouridylation can be induced by environmental signals at novel sites [9]. One such example are the two s that are induced under stress on U2 snRNA, compromising splicing [9,10]
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