Small nucleolar RNAs controlling rRNA processing in Trypanosoma brucei.

Vaibhav Chikne,Hava Madmoni,Viplov K Biswas,Shulamit Michaeli,Kathryn Decker,Ron Unger,K Shanmugha Rajan,Sachin Kumar Gupta,Moran Shalev-Benami,Tirza Doniger,Smadar Cohen-Chalamish,Christian Tschudi,Elisabetta Ullu

doi:10.1093/nar/gky1287

Vaibhav Chikne, Hava Madmoni + Show 11 more

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https://doi.org/10.1093/nar/gky1287

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Abstract

In trypanosomes, in contrast to most eukaryotes, the large subunit (LSU) ribosomal RNA is fragmented into two large and four small ribosomal RNAs (srRNAs) pieces, and this additional processing likely requires trypanosome-specific factors. Here, we examined the role of 10 abundant small nucleolar RNAs (snoRNAs) involved in rRNA processing. We show that each snoRNA involved in LSU processing associates with factors engaged in either early or late biogenesis steps. Five of these snoRNAs interact with the intervening sequences of rRNA precursor, whereas the others only guide rRNA modifications. The function of the snoRNAs was explored by silencing snoRNAs. The data suggest that the LSU rRNA processing events do not correspond to the order of rRNA transcription, and that srRNAs 2, 4 and 6 which are part of LSU are processed before srRNA1. Interestingly, the 6 snoRNAs that affect srRNA1 processing guide modifications on rRNA positions that span locations from the protein exit tunnel to the srRNA1, suggesting that these modifications may serve as check-points preceding the liberation of srRNA1. This study identifies the highest number of snoRNAs so far described that are involved in rRNA processing and/or rRNA folding and highlights their function in the unique trypanosome rRNA maturation events.

Highlights

Ribosomal RNA processing in eukaryotes is a complex, multi-step process process that starts in the nucleolus, proceeds in the nucleoplasm and culminates in the cytoplasm [1,2]
To obtain a more comprehensive overview of the small RNome, and verify small nucleolar RNAs (snoRNAs) abundance using a different fractionation approach, RNA-seq was performed on RNAs enriched in the post-ribosomal supernatant (PRS), which should include the entire subset of snoRNAs (Figure 1A)
We found that the majority of abundant snoRNAs (RPKM > 15 000) that were previously described by FPLC sizing column RNA seq [44] are abundant in the PRS RNA libraries

Summary

Introduction

Ribosomal RNA (rRNA) processing in eukaryotes is a complex, multi-step process process that starts in the nucleolus, proceeds in the nucleoplasm and culminates in the cytoplasm [1,2]. The process begins with a pre-rRNA transcript that includes RNAs destined for both the small subunit (SSU; 18S) and large subunit (5.8S, LSU; 25S/28S in yeast/mammals, respectively) [1,2]. These elements are separated by long internally transcribed spacers (ITSs) that are processed during ribosome biogenesis [1,2]. It was demonstrated that C/D snoRNAs guide rRNA base acetylation by establishing extended bipartite complementarity around the cytosines targeted for acetylation, similar to the pseudouridylation pocket formation by the H/ACA snoRNPs [9]. Five major snoRNAs were reported to function in SSU processing; these include U3, U14 and U17 (snR30), which are conserved in both higher and lower eukaryotes, U22, which is specific to mammalian cells, and snR10 which is specific to yeast [10]

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