Advertisement

The Nucleolus pp 231-241 | Cite as

Deep Sequencing Analysis of Nucleolar Small RNAs: RNA Isolation and Library Preparation

  • Baoyan Bai
  • Marikki LaihoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1455)

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.

Key words

Nucleolus Small RNA purification Library preparation Deep sequencing 

Notes

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.

References

  1. 1.
    Pederson T (2011) The nucleolus. Cold Spring Harb Perspect Biol 3:pii:a000638.28Google Scholar
  2. 2.
    Fatica A, Tollervey D (2002) Making ribosomes. Curr Opin Cell Biol 14:313–318CrossRefPubMedGoogle Scholar
  3. 3.
    Boisvert FM, van Koningsbruggen S, Navascués J, Lamond AI (2007) The multifunctional nucleolus. Nat Rev Mol Cell Biol 8:574–585CrossRefPubMedGoogle Scholar
  4. 4.
    Haag JR, Pikaard CS (2007) RNA polymerase I: a multifunctional molecular machine. Cell 131:1224–1225CrossRefPubMedGoogle Scholar
  5. 5.
    Russell J, Zomerdijk JC (2006) The RNA polymerase I transcription machinery. Biochem Soc Symp 73:203–216CrossRefPubMedGoogle Scholar
  6. 6.
    Turowski TW, Tollervey D (2015) Cotranscriptional events in eukaryotic ribosome synthesis. Wiley Interdiscip Rev RNA 6:129–139CrossRefPubMedGoogle Scholar
  7. 7.
    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 3:397–414CrossRefPubMedGoogle Scholar
  8. 8.
    Bai B, Laiho M (2012) Efficient sequential recovery of nucleolar macromolecular components. Proteomics 12:3044–3048CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Bai B, Laiho M (2015) Sequential recovery of macromolecular components of the nucleolus. Methods Mol Biol 1228:43–51CrossRefPubMedGoogle Scholar
  10. 10.
    Tissot C (2008) Analysis of miRNA content in total RNA preparations using the Agilent 2100 bioanalyzer. Agilent Technologies, Palo Alto, CAGoogle Scholar
  11. 11.
    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 4:441–449CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Bai B, Yegnasubramanian S, Wheelan SJ, Laiho M (2014) RNA-Seq of the nucleolus reveals abundant SNORD44-derived small RNAs. PLoS One 9:e107519CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, and Centre for Cancer BiomedicineUniversity of OsloOsloNorway
  2. 2.Department of Radiation Oncology and Molecular Radiation SciencesJohns Hopkins University School of MedicineBaltimoreUSA
  3. 3.Sidney Kimmel Comprehensive Cancer CenterJohns Hopkins University School of MedicineBaltimoreUSA

Personalised recommendations