Abstract
RNA-binding proteins are key players in the regulation of gene expression. In this Progress article, we discuss state-of-the-art technologies that can be used to study individual RNA-binding proteins or large complexes such as the ribosome. We also describe how these approaches can be used to study interactions with different types of RNAs, including nascent transcripts, mRNAs, microRNAs and ribosomal RNAs, in order to investigate transcription, RNA processing and translation. Finally, we highlight current challenges in data analysis and the future steps that are needed to obtain a quantitative and high-resolution picture of protein–RNA interactions on a genome-wide scale.
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Change history
31 January 2012
In two instances in the same sentence of the above article, the use of 'mRNA' and 'microRNA (miRNA)' had been reversed. The sentence has now been corrected so that it reads: “Although the direct pairing of an miRNA with its target mRNA cannot yet be deduced from these data, the detection of Argonaute binding sites in both miRNAs and mRNAs enabled the discovery of endogenous mRNA target sites.” The editors apologize for this error.
References
Moore, M. J. From birth to death: the complex lives of eukaryotic mRNAs. Science 309, 1514–1518 (2005).
Keene, J. D. RNA regulons: coordination of post-transcriptional events. Nature Rev. Genet. 8, 533–543 (2007).
Trifillis, P., Day, N. & Kiledjian, M. Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins. RNA 5, 1071–1082 (1999).
Brooks, S. A. & Rigby, W. F. Characterization of the mRNA ligands bound by the RNA binding protein hnRNP A2 utilizing a novel in vivo technique. Nucleic Acids Res. 28, e49 (2000).
Tenenbaum, S. A., Carson, C. C., Lager, P. J. & Keene, J. D. Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc. Natl Acad. Sci. 97, 14085–14090 (2000).
Mili, S. & Steitz, J. A. Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA 10, 1692–1694 (2004).
Ule, J. et al. CLIP identifies NOVA-regulated RNA networks in the brain. Science 302, 1212–1215 (2003).
Ule, J., Jensen, K., Mele, A. & Darnell, R. B. CLIP: A method for identifying protein–RNA interaction sites in living cells. Methods 37, 376–386 (2005).
Darnell, R. B. HITS-CLIP: panoramic views of protein-RNA regulation in living cells. Wiley Interdiscip. Rev. RNA 1, 266–286 (2010).
Wang, Z. et al. iCLIP predicts the dual splicing effects of TIA-RNA interactions. PLoS Biol. 8, e1000530 (2010).
Granneman, S., Kudla, G., Petfalski, E. & Tollervey, D. Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs. Proc. Natl Acad. Sci. USA 106, 9613–9618 (2009).
Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).
Guil, S. & Caceres, J. F. The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a. Nature Struct. Mol. Biol. 14, 591–596 (2007).
König, J. et al. The fungal RNA-binding protein Rrm4 mediates long-distance transport of ubi1 and rho3 mRNAs. EMBO J. 28, 1855–1866 (2009).
Licatalosi, D. D. et al. HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456, 464–469 (2008).
Yeo, G. W. et al. An RNA code for the FOX2 splicing regulator revealed by mapping RNA–protein interactions in stem cells. Nature Struct. Mol. Biol. 16, 130–137 (2009).
Chi, S. W., Zang, J. B., Mele, A. & Darnell, R. B. Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps. Nature 460, 479–486 (2009).
Zisoulis, D. G. et al. Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nature Struct. Mol. Biol. 17, 173–179 (2010).
Leung, A. K. et al. Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs. Nature Struct. Mol. Biol. 18, 237–244 (2011).
Kudla, G., Granneman, S., Hahn, D., Beggs, J. D. & Tollervey, D. Cross-linking, ligation, and sequencing of hybrids reveals RNA–RNA interactions in yeast. Proc. Natl Acad. Sci. USA 108, 10010–10015 (2011).
König, J. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nature Struct. Mol. Biol. 17, 909–915 (2010).
Kishore, S. et al. A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins. Nature Methods 8, 559–564 (2011).
Zhang, C. & Darnell, R. B. Mapping in vivo protein–RNA interactions at single-nucleotide resolution from HITS-CLIP data. Nature Biotechnol. 29, 607–614 (2011).
Urlaub, H., Hartmuth, K. & Lührmann, R. A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles. Methods 26, 170–181 (2002).
Kivioja, T. et al. Counting absolute numbers of molecules using unique molecular identifiers. Nature Methods 20 Nov 2011 (doi:10.1038/nmeth.1778).
Hafner, M. et al. RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA 17, 1697–1712 (2011).
Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. & Weissman, J. S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009).
Guo, H., Ingolia, N. T., Weissman, J. S. & Bartel, D. P. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010).
Fuda, N. J., Ardehali, M. B. & Lis, J. T. Defining mechanisms that regulate RNA polymerase II transcription in vivo. Nature 461, 186–192 (2009).
Churchman, L. S. & Weissman, J. S. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368–373 (2011).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25, 1105–1111 (2009).
Hoffmann, S. et al. Fast mapping of short sequences with mismatches, insertions and deletions using index structures. PLoS Comput. Biol. 5, e1000502 (2009).
Wu, T. D. & Nacu, S. Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26, 873–881 (2010).
Khorshid, M., Rodak, C. & Zavolan, M. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins. Nucleic Acids Res. 39, D245–D252 (2011).
Corcoran, D. L. et al. PARalyzer: Definition of RNA binding sites from PAR-CLIP short-read sequence data. Genome Biol. 12, R79 (2011).
Yang, J. H. et al. starBase: a database for exploring microRNA-mRNA interaction maps from Argonaute CLIP–seq and Degradome-seq data. Nucleic Acids Res. 39, D202–D209 (2011).
Bailey, T. L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).
Siddharthan, R., Siggia, E. D. & van Nimwegen, E. PhyloGibbs: a Gibbs sampling motif finder that incorporates phylogeny. PLoS Comput. Biol. 1, e67 (2005).
Ule, J. et al. An RNA map predicting NOVA-dependent splicing regulation. Nature 444, 580–586 (2006).
Witten, J. T. & Ule, J. Understanding splicing regulation through RNA splicing maps. Trends Genet. 27, 89–97 (2011).
Lebedeva, S. et al. Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR. Mol. Cell 43, 340–352 (2011).
Mukherjee, N. et al. Integrative regulatory mapping indicates that the RNA-binding protein HuR couples pre-mRNA processing and mRNA stability. Mol. Cell 43, 327–339 (2011).
Schadt, E. E., Turner, S. & Kasarskis, A. A window into third-generation sequencing. Hum. Mol. Genet. 19, R227–R240 (2010).
Acknowledgements
This work was supported by the Medical Research Council, the European Molecular Biology Laboratory (grant number U105185858), the European Research Council (206726-CLIP) and by a Human Frontiers Science Program Long-Term fellowship and an EMBL EIPOD fellowship to J.K. and K.Z., respectively.
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Glossary
- Argonaute proteins
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Core components of the RNA-mediated silencing pathways. They provide the platform for target mRNA recognition by small non-coding RNAs and harbour the catalytic activity for mRNA cleavage.
- Differential display
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A PCR-based approach that was used to study differences in RNA populations. It has now been superseded by microarray and RNA sequencing approaches.
- Global run-on sequencing
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(GRO-seq). A technique that combines nuclear run-on assays with high-throughput sequencing to obtain genome-wide information about active transcription.
- Heterogeneous nuclear ribonucleoprotein
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(HNRNP). The core protein components of heterogeneous nuclear ribonucleoprotein particles that associate with all nascent transcripts. They are involved in diverse aspects of post-transcriptional regulation.
- k-mers
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Nucleic acid sequences with a number of nucleotides of length k.
- NOVA
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A regulator of a biologically coherent set of RNAs important for synaptic function. It is involved in the neurological disorder paraneoplastic opsoclonus myoclonus ataxia.
- Ribonomics
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The genome-scale study of protein–RNA interactions and their functional consequences.
- Ribonucleoprotein particles
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(RNPs). Complexes consisting of protein and RNA components.
- Small nuclear RNAs
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(snRNAs). A class of non-coding RNAs that are found in the nucleus of eukaryotic cells and that constitute core components of all subunits of the spliceosome.
- Small nucleolar RNAs
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(snoRNAs). A class of small non-coding RNAs that are involved in guiding chemical modifications of other RNAs, such as ribosomal or transfer RNAs.
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König, J., Zarnack, K., Luscombe, N. et al. Protein–RNA interactions: new genomic technologies and perspectives. Nat Rev Genet 13, 77–83 (2012). https://doi.org/10.1038/nrg3141
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DOI: https://doi.org/10.1038/nrg3141
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