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Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells

Nature Biotechnology volume 29, pages 436–442 (2011)Cite this article

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Abstract

Cellular RNA levels are determined by the interplay of RNA production, processing and degradation. However, because most studies of RNA regulation do not distinguish the separate contributions of these processes, little is known about how they are temporally integrated. Here we combine metabolic labeling of RNA at high temporal resolution with advanced RNA quantification and computational modeling to estimate RNA transcription and degradation rates during the response of mouse dendritic cells to lipopolysaccharide. We find that changes in transcription rates determine the majority of temporal changes in RNA levels, but that changes in degradation rates are important for shaping sharp 'peaked' responses. We used sequencing of the newly transcribed RNA population to estimate temporally constant RNA processing and degradation rates genome wide. Degradation rates vary significantly between genes and contribute to the observed differences in the dynamic response. Certain transcripts, including those encoding cytokines and transcription factors, mature faster. Our study provides a quantitative approach to study the integrative process of RNA regulation.

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Figure 1: Changes in transcription rates during the response of dendritic cells to LPS.
Figure 2: Changes in transcription rate account for most expression changes; changes in degradation rate contribute to 'peaked' responses.
Figure 3: Genome-wide analysis of RNA transcription and degradation rates using RNA- and 4sU-Seq.
Figure 4: Genome-wide analysis of RNA processing rates.

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Acknowledgements

We thank S. Schwartz for assistance in analyzing splicing signals, J. Bochicchio for project management and the Broad Sequencing Platform for all sequencing work. I.A. was supported by the Human Frontiers Science Program. Work was supported by the Howard Hughes Medical Institute, a National Institutes of Health PIONEER DP1-00003958-01 award, a Burroughs Wellcome Fund Career Award at the Scientific Interface and the Merkin Foundation for Stem Cell Research at the Broad Institute (A.R.) by a US-Israel Bi-national Science Foundation award (N.F. and A.R.) and the EU FP7 “MODEL-IN” consortium grant (N.F.).

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Author notes

  1. Nir Friedman, Ido Amit and Aviv Regev: These authors contributed equally to this work.

Authors and Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Michal Rabani, Joshua Z Levin, Lin Fan, Xian Adiconis, Raktima Raychowdhury, Manuel Garber, Andreas Gnirke, Chad Nusbaum, Nir Hacohen, Ido Amit & Aviv Regev

  2. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Michal Rabani

  3. School of Computer Science, Hebrew University, Jerusalem, Israel

    Nir Friedman

  4. Institute of Life Sciences, Hebrew University, Jerusalem, Israel

    Nir Friedman

  5. Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Aviv Regev

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Contributions

M.R., I.A. and A.R. conceived and designed the study. M.R. and I.A. conducted the experiments. M.R., N.F. and A.R. designed the computational methods. M.R. developed and implemented the computational methods. R.R. made the cell cultures. J.Z.L., X.A., L.F., A.G. and C.N. constructed and sequenced the cDNA libraries. N.H. contributed experimental methods and reagents. M.G. contributed computational methods for RNA-Seq analysis.

Corresponding authors

Correspondence to Nir Friedman, Ido Amit or Aviv Regev.

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Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Notes and Supplementary Figs. 1–24 (PDF 6164 kb)

Supplementary Table 1

The 254 genes in the 'signature' set. (XLS 28 kb)

Supplementary Table 2

nCounter measurements data. (XLS 375 kb)

Supplementary Table 3

Standard RNA-Seq sequencing libraries statistics. (XLS 14 kb)

Supplementary Table 4

4sU-Seq sequencing libraries statistics. (XLS 15 kb)

Supplementary Table 5

Functional enrichments in the 8 expression clusters. (XLS 51 kb)

Supplementary Table 6

Functional enrichments in 10 deciles with distinct (fixed) degradation rates and in the group of genes that reject the 'constant degradation' model. (XLS 173 kb)

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Rabani, M., Levin, J., Fan, L. et al. Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells. Nat Biotechnol 29, 436–442 (2011). https://doi.org/10.1038/nbt.1861

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