Cargando…

Identification of novel proteins associated with yeast snR30 small nucleolar RNA

H/ACA small nucleolar RNPs (snoRNPs) that guide pseudouridylation reactions are comprised of one small nucleolar RNA (snoRNA) and four common proteins (Cbf5, Gar1, Nhp2 and Nop10). Unlike other H/ACA snoRNPs, snR30 is essential for the early processing reactions that lead to the production of 18S ri...

Descripción completa

Detalles Bibliográficos
Autores principales: Lemay, Vincent, Hossain, Ahmed, Osheim, Yvonne N., Beyer, Ann L., Dragon, François
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3239182/
https://www.ncbi.nlm.nih.gov/pubmed/21893585
http://dx.doi.org/10.1093/nar/gkr659
_version_ 1782219137803616256
author Lemay, Vincent
Hossain, Ahmed
Osheim, Yvonne N.
Beyer, Ann L.
Dragon, François
author_facet Lemay, Vincent
Hossain, Ahmed
Osheim, Yvonne N.
Beyer, Ann L.
Dragon, François
author_sort Lemay, Vincent
collection PubMed
description H/ACA small nucleolar RNPs (snoRNPs) that guide pseudouridylation reactions are comprised of one small nucleolar RNA (snoRNA) and four common proteins (Cbf5, Gar1, Nhp2 and Nop10). Unlike other H/ACA snoRNPs, snR30 is essential for the early processing reactions that lead to the production of 18S ribosomal RNA in the yeast Saccharomyces cerevisiae. To determine whether snR30 RNP contains specific proteins that contribute to its unique functional properties, we devised an affinity purification strategy using TAP-tagged Gar1 and an RNA aptamer inserted in snR30 snoRNA to selectively purify the RNP. Northern blotting and pCp labeling experiments showed that S1-tagged snR30 snoRNA can be selectively purified with streptavidin beads. Protein analysis revealed that aptamer-tagged snR30 RNA was associated with the four H/ACA proteins and a number of additional proteins: Nop6, ribosomal proteins S9 and S18 and histones H2B and H4. Using antibodies raised against Nop6 we show that endogenous Nop6 localizes to the nucleolus and that it cosediments with snR30 snoRNA in sucrose density gradients. We demonstrate through primer extension experiments that snR30 snoRNA is required for cleavages at site A0, A1 and A2, and that the absence of Nop6 decreases the efficiency of cleavage at site A2. Finally, electron microscopy analyses of chromatin spreads from cells depleted of snR30 snoRNA show that it is required for SSU processome assembly.
format Online
Article
Text
id pubmed-3239182
institution National Center for Biotechnology Information
language English
publishDate 2011
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-32391822011-12-16 Identification of novel proteins associated with yeast snR30 small nucleolar RNA Lemay, Vincent Hossain, Ahmed Osheim, Yvonne N. Beyer, Ann L. Dragon, François Nucleic Acids Res Molecular Biology H/ACA small nucleolar RNPs (snoRNPs) that guide pseudouridylation reactions are comprised of one small nucleolar RNA (snoRNA) and four common proteins (Cbf5, Gar1, Nhp2 and Nop10). Unlike other H/ACA snoRNPs, snR30 is essential for the early processing reactions that lead to the production of 18S ribosomal RNA in the yeast Saccharomyces cerevisiae. To determine whether snR30 RNP contains specific proteins that contribute to its unique functional properties, we devised an affinity purification strategy using TAP-tagged Gar1 and an RNA aptamer inserted in snR30 snoRNA to selectively purify the RNP. Northern blotting and pCp labeling experiments showed that S1-tagged snR30 snoRNA can be selectively purified with streptavidin beads. Protein analysis revealed that aptamer-tagged snR30 RNA was associated with the four H/ACA proteins and a number of additional proteins: Nop6, ribosomal proteins S9 and S18 and histones H2B and H4. Using antibodies raised against Nop6 we show that endogenous Nop6 localizes to the nucleolus and that it cosediments with snR30 snoRNA in sucrose density gradients. We demonstrate through primer extension experiments that snR30 snoRNA is required for cleavages at site A0, A1 and A2, and that the absence of Nop6 decreases the efficiency of cleavage at site A2. Finally, electron microscopy analyses of chromatin spreads from cells depleted of snR30 snoRNA show that it is required for SSU processome assembly. Oxford University Press 2011-12 2011-09-05 /pmc/articles/PMC3239182/ /pubmed/21893585 http://dx.doi.org/10.1093/nar/gkr659 Text en © The Author(s) 2011. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Molecular Biology
Lemay, Vincent
Hossain, Ahmed
Osheim, Yvonne N.
Beyer, Ann L.
Dragon, François
Identification of novel proteins associated with yeast snR30 small nucleolar RNA
title Identification of novel proteins associated with yeast snR30 small nucleolar RNA
title_full Identification of novel proteins associated with yeast snR30 small nucleolar RNA
title_fullStr Identification of novel proteins associated with yeast snR30 small nucleolar RNA
title_full_unstemmed Identification of novel proteins associated with yeast snR30 small nucleolar RNA
title_short Identification of novel proteins associated with yeast snR30 small nucleolar RNA
title_sort identification of novel proteins associated with yeast snr30 small nucleolar rna
topic Molecular Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3239182/
https://www.ncbi.nlm.nih.gov/pubmed/21893585
http://dx.doi.org/10.1093/nar/gkr659
work_keys_str_mv AT lemayvincent identificationofnovelproteinsassociatedwithyeastsnr30smallnucleolarrna
AT hossainahmed identificationofnovelproteinsassociatedwithyeastsnr30smallnucleolarrna
AT osheimyvonnen identificationofnovelproteinsassociatedwithyeastsnr30smallnucleolarrna
AT beyerannl identificationofnovelproteinsassociatedwithyeastsnr30smallnucleolarrna
AT dragonfrancois identificationofnovelproteinsassociatedwithyeastsnr30smallnucleolarrna