Abstract
The nucleolus is a subcellular compartment with a key essential function in ribosome biogenesis. The nucleolus is rich in noncoding RNAs, mostly the ribosomal RNAs and small nucleolar RNAs. Surprisingly, also several miRNAs have been detected in the nucleolus, raising the question as to whether other small RNA species are present and functional in the nucleolus. We have developed a strategy for stepwise enrichment of nucleolar small RNAs from the total nucleolar RNA extracts and subsequent construction of nucleolar small RNA libraries which are suitable for deep sequencing. Our method successfully isolates the small RNA population from total RNAs and monitors the RNA quality in each step to ensure that small RNAs recovered represent the actual small RNA population in the nucleolus and not degradation products from larger RNAs. We have further applied this approach to characterize the distribution of small RNAs in different cellular compartments.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pederson T (2011) The nucleolus. Cold Spring Harb Perspect Biol 3:pii:a000638.28
Fatica A, Tollervey D (2002) Making ribosomes. Curr Opin Cell Biol 14:313–318
Boisvert FM, van Koningsbruggen S, Navascués J, Lamond AI (2007) The multifunctional nucleolus. Nat Rev Mol Cell Biol 8:574–585
Haag JR, Pikaard CS (2007) RNA polymerase I: a multifunctional molecular machine. Cell 131:1224–1225
Russell J, Zomerdijk JC (2006) The RNA polymerase I transcription machinery. Biochem Soc Symp 73:203–216
Turowski TW, Tollervey D (2015) Cotranscriptional events in eukaryotic ribosome synthesis. Wiley Interdiscip Rev RNA 6:129–139
Watkins NJ, Bohnsack MT (2012) The box C/D and H/ACA snoRNPs: key players in the modification, processing and the dynamic folding of ribosomal RNA. Wiley Interdiscip Rev RNA 3:397–414
Bai B, Laiho M (2012) Efficient sequential recovery of nucleolar macromolecular components. Proteomics 12:3044–3048
Bai B, Laiho M (2015) Sequential recovery of macromolecular components of the nucleolus. Methods Mol Biol 1228:43–51
Tissot C (2008) Analysis of miRNA content in total RNA preparations using the Agilent 2100 bioanalyzer. Agilent Technologies, Palo Alto, CA
Bai B, Liu H, Laiho M (2014) Small RNA expression and deep sequencing analyses of the nucleolus reveal the presence of nucleolus-associated microRNAs. FEBS Open Bio 4:441–449
Bai B, Yegnasubramanian S, Wheelan SJ, Laiho M (2014) RNA-Seq of the nucleolus reveals abundant SNORD44-derived small RNAs. PLoS One 9:e107519
Acknowledgments
The original work leading to the development of this protocol has been supported by NIH P30 CA006973 and Johns Hopkins University start-up funds.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Bai, B., Laiho, M. (2016). Deep Sequencing Analysis of Nucleolar Small RNAs: RNA Isolation and Library Preparation. In: Németh, A. (eds) The Nucleolus. Methods in Molecular Biology, vol 1455. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3792-9_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3792-9_18
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3790-5
Online ISBN: 978-1-4939-3792-9
eBook Packages: Springer Protocols