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

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.

Developmentally Regulated Novel Non-coding Anti-sense Regulators of mRNA Translation in Trypanosoma brucei

“…Total cell lysate was prepared, RNPs were extracted with 300mM KCl, and the ribosomes were removed by ultracentrifugation. RNA from the PRS was used to prepare RNA-seq libraries from both PCF and BSF, as previously described ( Chikne et al., 2016 ; Rajan et al., 2019a , 2019b , 2020 ). The libraries contained the entire population of ncRNAs (U snRNA, snoRNAs, 7SL RNA, Vault RNA, and tRNAs, Figure 1 B) but also ∼62 relatively abundant RNAs with unknown function ( Data S1 , S2 , and S3 ).…”

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Figure 1 TBsRNAs Are Developmentally Regulated (A) Enrichment of ncRNAs from T. brucei cell extracts. Whole cell extracts from 2 × 10 9 cells were prepared and depleted of ribosomes, as described in ( Chikne et al., 2016 ; Rajan et al., 2019a , 2019b ). RNA was extracted from PRS and total cell lysate (T), separated on a 10% denaturing gel, and stained with ethidium bromide.

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“…As a first step toward exploring the function of the novel ncRNAs, their association with other RNPs was examined. Two major types of RNPs greater than 80S were described in trypanosomes: polyribosomes and the processome involved in rRNA processing ( Rajan et al., 2019a , 2019b ). To this end, whole cell extracts were fractionated on 15–45% sucrose gradients, and the RNA was subjected to Northern analysis with gene-specific probes.…”

“…Trypanosomes possess all the conventional eukaryotic small RNA types such as U snRNAs ( Liang et al., 2003 ; Rajan et al., 2019a , 2019b ), snoRNAs ( Chikne et al, 2016 , 2019 ; Michaeli et al., 2012 ; Rajan et al., 2020 ), telomerase RNA ( Gupta et al., 2013a ; Sandhu et al., 2013 ), 7SL RNA ( Michaeli, 2014 ), and Vault RNA ( Kolev et al., 2019 ). Surprisingly, trypanosomes possess almost double the number of snoRNAs compared to yeast, which have a similar genome-size ( Rajan et al., 2019a , 2019b ). Pseudouridines guided by H/ACA snoRNAs, are developmentally regulated, with higher levels in the BSF, enabling the parasite to cope with the temperature shift while cycling between the hosts ( Chikne et al., 2016 ).…”

Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

“…Classical approaches for RT-silent pseudouridine (Ψ) detection are based on the use of soluble carbodiimide ( N -Cyclohexyl- N ’-(2-morpholinoethyl)carbodiimide metho- p -toluenesulfonate, usually abbreviated as CMCT) [ 65 ] and they were developed in the 1990s for low-throughput Ψ mapping. Several groups successfully coupled this protocol to NGS almost simultaneously and independently [ 66 , 67 , 68 , 69 , 70 , 71 , 72 ]. A promising variant of the method used a “clickable” CMCT derivative for the subsequent enrichment of derivatized, pseudouridine-containing RNA [ 73 , 74 ].…”