Implication of the box C/D snoRNP assembly factor Rsa1p in U3 snoRNP assembly

Abstract

The U3 box C/D snoRNA is one key element of 90S pre-ribosome. It contains a 5΄ domain pairing with pre-rRNA and the U3B/C and U3C΄/D motifs for U3 packaging into a unique small nucleolar ribonucleoprotein particle (snoRNP). The RNA-binding protein Snu13/SNU13 nucleates on U3B/C the assembly of box C/D proteins Nop1p/FBL and Nop56p/NOP56, and the U3-specific protein Rrp9p/U3-55K. Snu13p/SNU13 has a much lower affinity for U3C΄/D but nevertheless forms on this motif an RNP with box C/D proteins Nop1p/FBL and Nop58p/NOP58. In this study, we characterized the influence of the RNP assembly protein Rsa1 in the early steps of U3 snoRNP biogenesis in yeast and we propose a refined model of U3 snoRNP biogenesis. While recombinant Snu13p enhances the binding of Rrp9p to U3B/C, we observed that Rsa1p has no effect on this activity but forms with Snu13p and Rrp9p a U3B/C pre-RNP. In contrast, we found that Rsa1p enhances Snu13p binding on U3C΄/D. RNA footprinting experiments indicate that this positive effect most likely occurs by direct contacts of Rsa1p with the U3 snoRNA 5΄ domain. In light of the recent U3 snoRNP cryo-EM structures, our data suggest that Rsa1p has a dual role by also preventing formation of a pre-mature functional U3 RNP.

Ribosome Biogenesis in Plants: From Functional 45S Ribosomal DNA Organization to Ribosome Assembly Factors

“…U3 is a C/D box small nucleolar RNA (U3 snoRNA) required for nucleolar processing (cleavage, not methylation) of pre-18S rRNA in all eukaryotes (Rothé et al, 2017). U3 snoRNA associates with proteins common to all C/D box snoRNAs, including Nop56, Nop58, Nop1/FIBRILLARIN, and Small nuclear protein13 (Snu13), and with the U3-specific proteins Rrp9/U3-55k/Sof1 (RRNA pro-cessing9/Suppressor of fibrillarin1), Mpp10p (M phase phospho-protein10), Lcp5p (Lethal conditional pap1 [poly(A) polymerase] allele), Imp3 and Imp4 (Interacting with Mpp10), and Rrp9/U3-55k to form U3 snoRNP (Rothé et al, 2017). Protein-based affinity purification of the U3 snoRNP complex identified the Utp proteins (U three) and demonstrated that U3 and Utp proteins are the main components of the 90S/SSU processome (Supplemental Data Set 2; Gallagher et al, 2004;Phipps et al, 2011).…”

Mapping targets for small nucleolar RNAs in yeast

“…What is the mRNA target of snR77? Could the authors comment on Rrp9 binding and U3 function/yeast viability taking into account their data and preceding data on in vitro and in vivo Rrp9 binding on U3 and U3 mutants 2 - 4 .snoRNA-snoRNA hybrids revealed potential interactions between mature snoRNAs and precursor forms of target snoRNAs. Figure 6 shows a preferred location in the 3’ sequence of the targeted precursor form.…”

“…What is the mRNA target of snR77? Could the authors comment on Rrp9 binding and U3 function/yeast viability taking into account their data and preceding data on in vitro and in vivo Rrp9 binding on U3 and U3 mutants 2 - 4 .…”

Caracterização da interação entre a subunidade do R2TP, Nop17, e da proteína de transferência de <i>clusters</i> de Fe/S, Dre2, em <i>Saccharomyces cerevisiae</i>

“…Boxes C e D interagem formando uma estrutura de torção conhecida como K-turn (stem-internal loop-stem structure) (FromontRacine et al, 2005;Thomson et al, 2013;Rothé et al, 2017). A maioria dos snoRNAs de box C/D contém cópias adicionais bem conservadas, denominadas box C´ e D´.…”

“…Além da montagem dos snoRNPs de box C/D e box H/ACA, o complexo R2TP-Hsp90 participa da montagem das RNA Polimerases I, II e III, PIKKs, e telomerase (Rothé et al, 2017). …”