Target selection by natural and redesigned PUF proteins
Edited by Roy Parker, University of Colorado, Boulder, CO, and approved November 12, 2015 (received for review April 30, 2015)
Significance
Pumilio/fem-3 mRNA binding factor (PUF) proteins have become a leading scaffold in designing proteins to bind and control RNAs at will. We analyze the effects of that reengineering across the transcriptome in vivo for the first time to our knowledge. We show that yeast Puf2p, a noncanonical PUF protein, binds more than 1,000 mRNA targets. Puf2p binds multiple UAAU elements, unlike canonical PUF proteins. We design a modified Puf2p to bind UAAG rather than UAAU, which allows us to align the protein with the binding site. In vivo, the redesigned protein binds UAAG sites. Its altered specificity redistributes the protein away from 3′UTRs, such that the protein tracks with its sites, binds throughout the mRNA and represses a novel RNA network.
Abstract
Pumilio/fem-3 mRNA binding factor (PUF) proteins bind RNA with sequence specificity and modularity, and have become exemplary scaffolds in the reengineering of new RNA specificities. Here, we report the in vivo RNA binding sites of wild-type (WT) and reengineered forms of the PUF protein Saccharomyces cerevisiae Puf2p across the transcriptome. Puf2p defines an ancient protein family present throughout fungi, with divergent and distinctive PUF RNA binding domains, RNA-recognition motifs (RRMs), and prion regions. We identify sites in RNA bound to Puf2p in vivo by using two forms of UV cross-linking followed by immunopurification. The protein specifically binds more than 1,000 mRNAs, which contain multiple iterations of UAAU-binding elements. Regions outside the PUF domain, including the RRM, enhance discrimination among targets. Compensatory mutants reveal that one Puf2p molecule binds one UAAU sequence, and align the protein with the RNA site. Based on this architecture, we redesign Puf2p to bind UAAG and identify the targets of this reengineered PUF in vivo. The mutant protein finds its target site in 1,800 RNAs and yields a novel RNA network with a dramatic redistribution of binding elements. The mutant protein exhibits even greater RNA specificity than wild type. The redesigned protein decreases the abundance of RNAs in its redesigned network. These results suggest that reengineering using the PUF scaffold redirects and can even enhance specificity in vivo.
Data Availability
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE73274).
Acknowledgments
We thank M. Preston, C. Lapointe, A. Prasad, E. Sorokin, and B. Carrick for comments; L. Vanderploeg for assistance in figure preparation; and the University of Wisconsin Biotechnology Center DNA Sequencing Facility for assistance with performing RNA-seq. This work was supported by a gift from D.F.P., and NIH Grants R01 GM050942 (to M.W.) and T32 GM008349 (to D.F.P.). The synthesis of 4-thiouridine was supported by NIH Grant R01 CA073808 (to R.T.R.) and Canadian Institutes of Health Research (CIHR) Grant 289613 (to B.V.).
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References
1
M Wickens, DS Bernstein, J Kimble, R Parker, A PUF family portrait: 3'UTR regulation as a way of life. Trends Genet 18, 150–157 (2002).
2
A Galgano, et al., Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system. PLoS One 3, e3164 (2008).
3
AP Gerber, D Herschlag, PO Brown, Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast. PLoS Biol 2, E79 (2004).
4
AP Gerber, S Luschnig, MA Krasnow, PO Brown, D Herschlag, Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster. Proc Natl Acad Sci USA 103, 4487–4492 (2006).
5
B Zhang, et al., A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line. Nature 390, 477–484 (1997).
6
H Siemen, D Colas, HC Heller, O Brüstle, RA Pera, Pumilio-2 function in the mouse nervous system. PLoS One 6, e25932 (2011).
7
AC Goldstrohm, BA Hook, DJ Seay, M Wickens, PUF proteins bind Pop2p to regulate messenger RNAs. Nat Struct Mol Biol 13, 533–539 (2006).
8
Y Saint-Georges, et al., Yeast mitochondrial biogenesis: A role for the PUF RNA-binding protein Puf3p in mRNA localization. PLoS One 3, e2293 (2008).
9
P Kerner, SM Degnan, L Marchand, BM Degnan, M Vervoort, Evolution of RNA-binding proteins in animals: Insights from genome-wide analysis in the sponge Amphimedon queenslandica. Mol Biol Evol 28, 2289–2303 (2011).
10
CT Valley, et al., Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site. Proc Natl Acad Sci USA 109, 6054–6059 (2012).
11
X Wang, PD Zamore, TM Hall, Crystal structure of a Pumilio homology domain. Mol Cell 7, 855–865 (2001).
12
X Wang, J McLachlan, PD Zamore, TM Hall, Modular recognition of RNA by a human pumilio-homology domain. Cell 110, 501–512 (2002).
13
CG Cheong, TM Hall, Engineering RNA sequence specificity of Pumilio repeats. Proc Natl Acad Sci USA 103, 13635–13639 (2006).
14
PP Tam, et al., The Puf family of RNA-binding proteins in plants: Phylogeny, structural modeling, activity and subcellular localization. BMC Plant Biol 10, 44 (2010).
15
TMT Hall, Expanding the RNA-recognition code of PUF proteins. Nat Struct Mol Biol 21, 653–655 (2014).
16
Y Yosefzon, et al., Divergent RNA binding specificity of yeast Puf2p. RNA 17, 1479–1488 (2011).
17
S Alberti, R Halfmann, O King, A Kapila, S Lindquist, A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. Cell 137, 146–158 (2009).
18
DD Licatalosi, et al., HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456, 464–469 (2008).
19
M Hafner, et al., Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).
20
J Huerta-Cepas, S Capella-Gutiérrez, LP Pryszcz, M Marcet-Houben, T Gabaldón, PhylomeDB v4: Zooming into the plurality of evolutionary histories of a genome. Nucleic Acids Res 42, D897–D902 (2014).
21
JW Taylor, ML Berbee, Dating divergences in the Fungal Tree of Life: Review and new analyses. Mycologia 98, 838–849 (2006).
22
MA Freeberg, et al., Pervasive and dynamic protein binding sites of the mRNA transcriptome in Saccharomyces cerevisiae. Genome Biol 14, R13 (2013).
23
L Breiman, Random forests. Mach Learn 45, 5–32 (2001).
24
W Dang, et al., Inactivation of yeast Isw2 chromatin remodeling enzyme mimics longevity effect of calorie restriction via induction of genotoxic stress response. Cell Metab 19, 952–966 (2014).
25
ME Tanenbaum, LA Gilbert, LS Qi, JS Weissman, RD Vale, A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).
26
ZT Campbell, CT Valley, M Wickens, A protein-RNA specificity code enables targeted activation of an endogenous human transcript. Nat Struct Mol Biol 21, 732–738 (2014).
27
R Choudhury, YS Tsai, D Dominguez, Y Wang, Z Wang, Engineering RNA endonucleases with customized sequence specificities. Nat Commun 3, 1147 (2012).
28
Y Wang, C-G Cheong, TM Hall, Z Wang, Engineering splicing factors with designed specificities. Nat Methods 6, 825–830 (2009).
29
T Ozawa, Y Natori, M Sato, Y Umezawa, Imaging dynamics of endogenous mitochondrial RNA in single living cells. Nat Methods 4, 413–419 (2007).
30
AM Salazar, EJ Silverman, KP Menon, K Zinn, Regulation of synaptic Pumilio function by an aggregation-prone domain. J Neurosci 30, 515–522 (2010).
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Data Availability
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE73274).
Submission history
Published online: December 14, 2015
Published in issue: December 29, 2015
Keywords
Acknowledgments
We thank M. Preston, C. Lapointe, A. Prasad, E. Sorokin, and B. Carrick for comments; L. Vanderploeg for assistance in figure preparation; and the University of Wisconsin Biotechnology Center DNA Sequencing Facility for assistance with performing RNA-seq. This work was supported by a gift from D.F.P., and NIH Grants R01 GM050942 (to M.W.) and T32 GM008349 (to D.F.P.). The synthesis of 4-thiouridine was supported by NIH Grant R01 CA073808 (to R.T.R.) and Canadian Institutes of Health Research (CIHR) Grant 289613 (to B.V.).
Notes
This article is a PNAS Direct Submission.
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The authors declare no conflict of interest.
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Cite this article
Target selection by natural and redesigned PUF proteins, Proc. Natl. Acad. Sci. U.S.A.
112 (52) 15868-15873,
https://doi.org/10.1073/pnas.1508501112
(2015).
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