Genome-Wide Analysis of C/D and H/ACA-Like Small Nucleolar RNAs in Leishmania major Indicates Conservation among Trypanosomatids in the Repertoire and in Their rRNA Targets

Abstract

Small nucleolar RNAs (snoRNAs) are a large group of noncoding RNAs that exist in eukaryotes and archaea and guide modifications such as 2-O-ribose methylations and pseudouridylation on rRNAs and snRNAs. Recently, we described a genome-wide screening approach with Trypanosoma brucei that revealed over 90 guide RNAs. In this study, we extended this approach to analyze the repertoire of the closely related human pathogen Leishmania major. We describe 23 clusters that encode 62 C/Ds that can potentially guide 79 methylations and 37 H/ACA-like RNAs that can potentially guide 30 pseudouridylation reactions. Like T. brucei, Leishmania also contains many modifications and guide RNAs relative to its genome size. This study describes 10 H/ACAs and 14 C/Ds that were not found in T. brucei. Mapping of 2-O-methylations in rRNA regions rich in modifications suggests the existence of trypanosomatid-specific modifications conserved in T. brucei and Leishmania. Structural features of C/D snoRNAs, such as copy number, conservation of boxes, K turns, and intragenic and extragenic base pairing, were examined to elucidate the great variation in snoRNA abundance. This study highlights the power of comparative genomics for determining conserved features of noncoding RNAs.In eukaryotes (2, 9, 17, 42) as well as in archaea (10, 29), the two major rRNA modifications, 2Ј-O-methylation (Nm) and pseudouridylation, are guided by small RNAs, which in eukaryotes are small nucleolar RNAs (snoRNAs). The 2Ј-Omethylations are guided by C/D box snoRNAs, which are named after short motifs known as the C box (RUGAUGA [R designates purines]) and the D box (CUGA). These boxes, together with the short sequences near the 5Ј and 3Ј ends of the RNA, which have the potential to form a K-turn structure, are essential for processing, localization, and stabilization of these molecules (7,19,40,44). The K-turn motif is the binding site for the 15.5-kDa/Snu13 snoRNP protein (26). Such motifs exist in C/D snoRNAs, which guide modification, and also in U3 snoRNP (26) and U4 snRNA (28). Most of the guide RNAs carry internal boxes related to the C and D boxes, known as CЈ and DЈ boxes. The recognition of the target by the guide RNA is based on complementarity of 10 to 21 nucleotides (nt) between these two molecules, located upstream of the D and DЈ sequences. The methylation site is situated 5 nt upstream from the D and DЈ boxes, within the domain of interaction between the snoRNA and the substrate (9, 18).

Elucidating the Role of C/D snoRNA in rRNA Processing and Modification inTrypanosoma brucei

“…We recently made a similar attempt to understand this phenomenon using 20 Leishmania major snoRNAs (31). Transcriptional regulation cannot explain the major differences that exist among levels of snoRNAs encoded by the same cluster.…”

“…A machine-learning algorithm was used to predict the level of snoRNA with 85% accuracy. Systematic mapping of Nm sites on rRNA identified an additional 47 Nms beyond the 84 Nms previously identified, suggesting that more C/D snoRNAs than identified in the published repertoire are expected to exist in the genome (31,35). Mapping of Nms in the two life stages of the parasite, the procyclic and bloodstream forms, revealed hypermethylation in the bloodstream form at certain positions, suggesting that this modification may help the parasite adapt to the higher temperature during cycling from the insect to the mammalian host.…”

Families of H/ACA ncRNA molecules in Trypanosomatids

“…Seed sequences. The repertoire of 34 H/ACA snoRNA sequences was extracted from Liang et al 12 in T. brucei as well as the 12 H/ACA molecules from T. brucei from Myslyuk et al 22 Similar data for 37 H/ACA sequences in L. major were taken from Liang et al 13 and 2 H/ACA sequences from Myslyuk et al 22 Trypanosmatid genomes. T. brucei (v4), 32 T. cruzi (v4) 33 and L. major (v5) 34 Homology searching.…”

“…1A). [11][12][13] Because of these changes in some publications these molecules are referred to as "H/ACA-like". In this manuscript, as Trypanosome H/ACAs operate in a similar fashion to their double-stem counterparts, we will refer to them simply as H/ACA.…”

“…Initially, 34 H/ACA snoRNA molecules, that guide 32 pseudouridylations, were identified in Trypanosoma brucei 12 and 37 H/ ACA snoRNAs that can potentially guide 30 pseudouridylations were identified in Leishmania major. 13 Recently, Psiscan, a computational tool for genome-wide detection of H/ACA molecules in Trypansomatids was developed. 22 This study identified 12 novel H/ACA molecules from Trypanosoma brucei and two from Leishmania major.…”