Abstract
Assembly of eukaryotic ribosomes is an elaborate biosynthetic process that begins in the nucleolus and requires hundreds of cellular factors. Analysis of rRNA processing has been instrumental for studying the mechanisms of ribosome biogenesis and effects of stress conditions on the molecular milieu of the nucleolus. Here, we describe the quantitative analysis of the steady-state levels of rRNA precursors, applicable to studies in mammalian cells and other organisms. We include protocols for gel electrophoresis and northern blotting of rRNA precursors using procedures optimized for the large size of these RNAs. We also describe the ratio analysis of multiple precursors, a technique that facilitates the accurate assessment of changes in the efficiency of individual pre-rRNA processing steps.
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References
Henras AK, Plisson-Chastang C, O’Donohue M-F et al (2014) An overview of pre-ribosomal RNA processing in eukaryotes. Wiley Interdiscip Rev RNA 6:225–242
Tafforeau L, Zorbas C, Langhendries J-L et al (2013) The complexity of human ribosome biogenesis revealed by systematic nucleolar screening of pre-rRNA processing factors. Mol Cell 51:539–551
Mansour FH, Pestov DG (2013) Separation of long RNA by agarose-formaldehyde gel electrophoresis. Anal Biochem 441:18–20
Chomczynski P (1992) One-hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal Biochem 201:134–139
Wang M, Anikin L, Pestov DG (2014) Two orthogonal cleavages separate subunit RNAs in mouse ribosome biogenesis. Nucleic Acids Res 42:11180–11191
Chomczynski P (1992) Solubilization in formamide protects RNA from degradation. Nucleic Acids Res 20:3791–3792
Acknowledgement
This work was supported by NIH grant GM074091 to DGP.
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© 2016 Springer Science+Business Media New York
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Wang, M., Pestov, D.G. (2016). Quantitative Northern Blot Analysis of Mammalian rRNA Processing. 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_12
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DOI: https://doi.org/10.1007/978-1-4939-3792-9_12
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3790-5
Online ISBN: 978-1-4939-3792-9
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