Engineering a conserved RNA regulatory protein repurposes its biological function in vivo

Bhat, Vandita D.; McCann, Kathleen L.; Wang, Yeming; Fonseca, Dallas R.; Shukla, Tarjani; Alexander, Jacqueline C.; Qiu, Chen; Wickens, Marv; Lo, Te-Wen; Tanaka Hall, Traci M.; Campbell, Zachary T.

doi:10.7554/eLife.43788

Title: Engineering a conserved RNA regulatory protein repurposes its biological function in vivo

Journal Article · Thu Jan 17 00:00:00 EST 2019 · eLife

DOI:https://doi.org/10.7554/eLife.43788· OSTI ID:1506498

Bhat, Vandita D. ^[1];

^[2]; Wang, Yeming ^[2]; Fonseca, Dallas R. ^[3]; Shukla, Tarjani ^[1]; Alexander, Jacqueline C. ^[3]; Qiu, Chen ^[2]; Wickens, Marv ^[4]; Lo, Te-Wen ^[3];

^[2];

^[1]

Department of Biological Sciences, University of Texas Dallas, Richardson, United States
Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, United States
Department of Biology, Ithaca College, Ithaca, United States
Department of Biochemistry, University of Wisconsin-Madison, Madison, United States

PUF (PUmilio/FBF) RNA-binding proteins recognize distinct elements. In C. elegans, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we first determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of puf-8. This work highlights the critical role of motif length in the specification of biological function.

Cite

Export

Save

Research Organization:: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: National Institutes of Health (NIH)

OSTI ID:: 1506498

Journal Information:: eLife, Vol. 8, Issue 01, 2019; ISSN 2050-084X

Publisher:: eLife Sciences Publications, Ltd.

Country of Publication:: United States

Language:: ENGLISH

References (64)

Engineered proteins with Pumilio/ fem-3 mRNA binding factor scaffold to manipulate RNA metabolism Wang, Yang; Wang, Zefeng; Tanaka Hall, Traci M. FEBS Journal, Vol. 280, Issue 16 https://doi.org/10.1111/febs.12367	journal	June 2013
Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins Castello, Alfredo; Fischer, Bernd; Eichelbaum, Katrin Cell, Vol. 149, Issue 6 https://doi.org/10.1016/j.cell.2012.04.031	journal	June 2012
The staufen/pumilio Pathway Is Involved in Drosophila Long-Term Memory Dubnau, Josh; Chiang, Ann-Shyn; Grady, Lori Current Biology, Vol. 13, Issue 4 https://doi.org/10.1016/S0960-9822(03)00064-2	journal	February 2003
Expanding RNA binding specificity and affinity of engineered PUF domains Zhao, Yang-Yang; Mao, Miao-Wei; Zhang, Wen-Jing Nucleic Acids Research, Vol. 46, Issue 9 https://doi.org/10.1093/nar/gky134	journal	February 2018
Basis of altered RNA-binding specificity by PUF proteins revealed by crystal structures of yeast Puf4p Miller, Matthew T.; Higgin, Joshua J.; Tanaka Hall, Traci M. Nature Structural & Molecular Biology, Vol. 15, Issue 4 https://doi.org/10.1038/nsmb.1390	journal	March 2008
A 5' cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs Zhu, D.; Stumpf, C. R.; Krahn, J. M. Proceedings of the National Academy of Sciences, Vol. 106, Issue 48 https://doi.org/10.1073/pnas.0812079106	journal	November 2009
Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions Lo, Te-Wen; Pickle, Catherine S.; Lin, Steven Genetics, Vol. 195, Issue 2 https://doi.org/10.1534/genetics.113.155382	journal	August 2013
A compendium of RNA-binding motifs for decoding gene regulation Ray, Debashish; Kazan, Hilal; Cook, Kate B. Nature, Vol. 499, Issue 7457 https://doi.org/10.1038/nature12311	journal	July 2013
Alternate Modes of Cognate RNA Recognition by Human PUMILIO Proteins Lu, Gang; Hall, Traci M. Tanaka Structure, Vol. 19, Issue 3 https://doi.org/10.1016/j.str.2010.12.019	journal	March 2011
Concentration and localization of co-expressed ELAV/Hu proteins control specificity of mRNA processing Zaharieva, Emanuela; Haussmann, Irmgard U.; Bräuer, Ulrike Molecular and Cellular Biology https://doi.org/10.1128/MCB.00473-15	journal	June 2015
A protein-RNA specificity code enables targeted activation of an endogenous human transcript Campbell, Zachary T.; Valley, Cary T.; Wickens, Marvin Nature Structural & Molecular Biology, Vol. 21, Issue 8 https://doi.org/10.1038/nsmb.2847	journal	June 2014
Divergence of Pumilio/ fem-3 mRNA Binding Factor (PUF) Protein Specificity through Variations in an RNA-binding Pocket Qiu, Chen; Kershner, Aaron; Wang, Yeming Journal of Biological Chemistry, Vol. 287, Issue 9 https://doi.org/10.1074/jbc.M111.326264	journal	December 2011
Binding specificity and mRNA targets of a C. elegans PUF protein, FBF-1 Bernstein, D. RNA, Vol. 11, Issue 4 https://doi.org/10.1261/rna.7255805	journal	April 2005
Extensive Association of Functionally and Cytotopically Related mRNAs with Puf Family RNA-Binding Proteins in Yeast Gerber, André P.; Herschlag, Daniel; Brown, Patrick O. PLoS Biology, Vol. 2, Issue 3 https://doi.org/10.1371/journal.pbio.0020079	journal	March 2004
The Caenorhabditis elegans pumilio homolog, puf-9, is required for the 3′UTR-mediated repression of the let-7 microRNA target gene, hbl-1 Nolde, Mona J.; Saka, Nazli; Reinert, Kristy L. Developmental Biology, Vol. 305, Issue 2 https://doi.org/10.1016/j.ydbio.2007.02.040	journal	May 2007
A universal code for RNA recognition by PUF proteins Filipovska, Aleksandra; Razif, Muhammad F. M.; Nygård, Karoline K. A. Nature Chemical Biology, Vol. 7, Issue 7 https://doi.org/10.1038/nchembio.577	journal	May 2011
Improved methods for building protein models in electron density maps and the location of errors in these models Jones, T. A.; Zou, J. Y.; Cowan, S. W. Acta Crystallographica Section A Foundations of Crystallography, Vol. 47, Issue 2, p. 110-119 https://doi.org/10.1107/S0108767390010224	journal	March 1991
Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein Wang, Y.; Opperman, L.; Wickens, M. Proceedings of the National Academy of Sciences, Vol. 106, Issue 48 https://doi.org/10.1073/pnas.0812076106	journal	November 2009
PUF-8 negatively regulates RAS/MAPK signalling to promote differentiation of C. elegans germ cells Vaid, S.; Ariz, M.; Chaturbedi, A. Development, Vol. 140, Issue 8 https://doi.org/10.1242/dev.088013	journal	March 2013
A conserved PUF–Ago–eEF1A complex attenuates translation elongation Friend, Kyle; Campbell, Zachary T.; Cooke, Amy Nature Structural & Molecular Biology, Vol. 19, Issue 2 https://doi.org/10.1038/nsmb.2214	journal	January 2012
RNA-protein interactions in the yeast three-hybrid system: Affinity, sensitivity, and enhanced library screening Hook, B. RNA, Vol. 11, Issue 2 https://doi.org/10.1261/rna.7202705	journal	February 2005
The RNA-binding proteomes from yeast to man harbour conserved enigmRBPs Beckmann, Benedikt M.; Horos, Rastislav; Fischer, Bernd Nature Communications, Vol. 6, Issue 1 https://doi.org/10.1038/ncomms10127	journal	December 2015
Analyzing mRNA–protein complexes using a yeast three-hybrid system Bernstein, D. Methods, Vol. 26, Issue 2 https://doi.org/10.1016/S1046-2023(02)00015-4	journal	February 2002
The CCP4 suite programs for protein crystallography Acta Crystallographica Section D Biological Crystallography, Vol. 50, Issue 5, p. 760-763 https://doi.org/10.1107/S0907444994003112	journal	September 1994
Crystal Structure of a Pumilio Homology Domain Wang, Xiaoqiang; Zamore, Phillip D.; Hall, Traci M. Tanaka Molecular Cell, Vol. 7, Issue 4 https://doi.org/10.1016/S1097-2765(01)00229-5	journal	April 2001
Inducible Control of mRNA Transport Using Reprogrammable RNA-Binding Proteins Abil, Zhanar; Gumy, Laura F.; Zhao, Huimin ACS Synthetic Biology, Vol. 6, Issue 6 https://doi.org/10.1021/acssynbio.7b00025	journal	March 2017
PUF-8, a Pumilio Homolog, Inhibits the Proliferative Fate in the Caenorhabditis elegans Germline Racher, Hilary; Hansen, Dave G3: Genes\|Genomes\|Genetics, Vol. 2, Issue 10 https://doi.org/10.1534/g3.112.003350	journal	October 2012
Protein-RNA networks revealed through covalent RNA marks Lapointe, Christopher P.; Wilinski, Daniel; Saunders, Harriet A. J. Nature Methods, Vol. 12, Issue 12 https://doi.org/10.1038/nmeth.3651	journal	November 2015
A Caenorhabditis elegans PUF protein family with distinct RNA binding specificity Stumpf, C. R.; Kimble, J.; Wickens, M. RNA, Vol. 14, Issue 8 https://doi.org/10.1261/rna.1095908	journal	June 2008
Enzymatic assembly of DNA molecules up to several hundred kilobases Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan Nature Methods, Vol. 6, Issue 5, p. 343-345 https://doi.org/10.1038/nmeth.1318	journal	April 2009
Recurrent rewiring and emergence of RNA regulatory networks Wilinski, Daniel; Buter, Natascha; Klocko, Andrew D. Proceedings of the National Academy of Sciences, Vol. 114, Issue 14 https://doi.org/10.1073/pnas.1617777114	journal	March 2017
A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans Crittenden, Sarah L.; Bernstein, David S.; Bachorik, Jennifer L. Nature, Vol. 417, Issue 6889 https://doi.org/10.1038/nature754	journal	May 2002
Live-cell imaging of viral RNA genomes using a Pumilio-based reporter Tilsner, Jens; Linnik, Olga; Christensen, Nynne M. The Plant Journal, Vol. 57, Issue 4 https://doi.org/10.1111/j.1365-313X.2008.03720.x	journal	February 2009
A PUF family portrait: 3′UTR regulation as a way of life Wickens, Marvin; Bernstein, David S.; Kimble, Judith Trends in Genetics, Vol. 18, Issue 3 https://doi.org/10.1016/S0168-9525(01)02616-6	journal	March 2002
A single spacer nucleotide determines the specificities of two mRNA regulatory proteins Opperman, Laura; Hook, Brad; DeFino, Mia Nature Structural & Molecular Biology, Vol. 12, Issue 11 https://doi.org/10.1038/nsmb1010	journal	October 2005
Redundant control of the Caenorhabditis elegans sperm/oocyte switch by PUF-8 and FBF-1, two distinct PUF RNA-binding proteins Bachorik, J. L.; Kimble, J. Proceedings of the National Academy of Sciences, Vol. 102, Issue 31 https://doi.org/10.1073/pnas.0504593102	journal	July 2005
Identification of a Conserved Interface between PUF and CPEB Proteins Campbell, Zachary T.; Menichelli, Elena; Friend, Kyle Journal of Biological Chemistry, Vol. 287, Issue 22 https://doi.org/10.1074/jbc.M112.352815	journal	April 2012
The Pfam protein families database: towards a more sustainable future Finn, Robert D.; Coggill, Penelope; Eberhardt, Ruth Y. Nucleic Acids Research, Vol. 44, Issue D1 https://doi.org/10.1093/nar/gkv1344	journal	December 2015
A Protein⋅Protein Interaction Platform Involved in Recruitment of GLD-3 to the FBF⋅fem-3 mRNA Complex Wu, Joann; Campbell, Zachary T.; Menichelli, Elena Journal of Molecular Biology, Vol. 425, Issue 4 https://doi.org/10.1016/j.jmb.2012.11.013	journal	February 2013
Biochemical Characterization of the Caenorhabditis elegans FBF⋅CPB-1 Translational Regulation Complex Identifies Conserved Protein Interaction Hotspots Menichelli, Elena; Wu, Joann; Campbell, Zachary T. Journal of Molecular Biology, Vol. 425, Issue 4 https://doi.org/10.1016/j.jmb.2012.11.012	journal	February 2013
Engineering RNA sequence specificity of Pumilio repeats Cheong, C. -G.; Hall, T. M. T. Proceedings of the National Academy of Sciences, Vol. 103, Issue 37 https://doi.org/10.1073/pnas.0606294103	journal	September 2006
Targeted translational regulation using the PUF protein family scaffold Cooke, A.; Prigge, A.; Opperman, L. Proceedings of the National Academy of Sciences, Vol. 108, Issue 38 https://doi.org/10.1073/pnas.1105151108	journal	September 2011
Structure of Pumilio Reveals Similarity between RNA and Peptide Binding Motifs Edwards, Thomas A.; Pyle, Scott E.; Wharton, Robin P. Cell, Vol. 105, Issue 2 https://doi.org/10.1016/S0092-8674(01)00318-X	journal	April 2001
hnRNP Proteins and the Biogenesis of mRNA Dreyfuss, Gideon; Matunis, Michael J.; Pinol-Roma, Serafin Annual Review of Biochemistry, Vol. 62, Issue 1 https://doi.org/10.1146/annurev.bi.62.070193.001445	journal	June 1993
Imaging dynamics of endogenous mitochondrial RNA in single living cells Ozawa, Takeaki; Natori, Yutaka; Sato, Moritoshi Nature Methods, Vol. 4, Issue 5 https://doi.org/10.1038/nmeth1030	journal	April 2007
Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio Weidmann, Chase A.; Qiu, Chen; Arvola, René M. eLife, Vol. 5 https://doi.org/10.7554/eLife.17096	journal	August 2016
The establishment of Caenorhabditis elegans germline pattern is controlled by overlapping proximal and distal somatic gonad signals Pepper, Anita S. -R; Lo, Te-Wen; Killian, Darrell J. Developmental Biology, Vol. 259, Issue 2 https://doi.org/10.1016/S0012-1606(03)00203-3	journal	July 2003
RNA regulatory networks diversified through curvature of the PUF protein scaffold Wilinski, Daniel; Qiu, Chen; Lapointe, Christopher P. Nature Communications, Vol. 6, Issue 1 https://doi.org/10.1038/ncomms9213	journal	September 2015
Heterogeneous nuclear ribonucleoproteins (hnRNPs) in cellular processes: Focus on hnRNP E1's multifunctional regulatory roles Chaudhury, A.; Chander, P.; Howe, P. H. RNA, Vol. 16, Issue 8 https://doi.org/10.1261/rna.2254110	journal	June 2010
Target selection by natural and redesigned PUF proteins Porter, Douglas F.; Koh, Yvonne Y.; VanVeller, Brett Proceedings of the National Academy of Sciences, Vol. 112, Issue 52 https://doi.org/10.1073/pnas.1508501112	journal	December 2015
FBF-1 and FBF-2 Regulate the Size of the Mitotic Region in the C. elegans Germline Lamont, Liana B.; Crittenden, Sarah L.; Bernstein, David Developmental Cell, Vol. 7, Issue 5 https://doi.org/10.1016/j.devcel.2004.09.013	journal	November 2004
Dedifferentiation of Primary Spermatocytes into Germ Cell Tumors in C. elegans Lacking the Pumilio-like Protein PUF-8 Subramaniam, Kuppuswamy; Seydoux, Geraldine Current Biology, Vol. 13, Issue 2 https://doi.org/10.1016/S0960-9822(03)00005-8	journal	January 2003
Cooperativity in RNA-Protein Interactions: Global Analysis of RNA Binding Specificity Campbell, Zachary T.; Bhimsaria, Devesh; Valley, Cary T. Cell Reports, Vol. 1, Issue 5 https://doi.org/10.1016/j.celrep.2012.04.003	journal	May 2012
Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice Barragán-Iglesias, Paulino; Lou, Tzu-Fang; Bhat, Vandita D. Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-017-02449-5	journal	January 2018
Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site Valley, C. T.; Porter, D. F.; Qiu, C. Proceedings of the National Academy of Sciences, Vol. 109, Issue 16 https://doi.org/10.1073/pnas.1200521109	journal	March 2012
Architecture and dynamics of overlapped RNA regulatory networks Lapointe, Christopher P.; Preston, Melanie A.; Wilinski, Daniel RNA, Vol. 23, Issue 11 https://doi.org/10.1261/rna.062687.117	journal	August 2017
A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line Zhang, Beilin; Gallegos, Maria; Puoti, Alessandro Nature, Vol. 390, Issue 6659 https://doi.org/10.1038/37297	journal	December 1997
Engineering splicing factors with designed specificities Wang, Yang; Cheong, Cheom-Gil; Tanaka Hall, Traci M. Nature Methods, Vol. 6, Issue 11 https://doi.org/10.1038/nmeth.1379	journal	October 2009
[20] Processing of X-ray diffraction data collected in oscillation mode Otwinowski, Zbyszek; Minor, Wladek Macromolecular Crystallography Part A, 307-326 https://doi.org/10.1016/S0076-6879(97)76066-X	book	January 1997
Engineering RNA endonucleases with customized sequence specificities Choudhury, Rajarshi; Tsai, Yihsuan S.; Dominguez, Daniel Nature Communications, Vol. 3, Issue 1 https://doi.org/10.1038/ncomms2154	journal	January 2012
Stacking interactions in PUF-RNA complexes Koh, Y. Y.; Wang, Y.; Qiu, C. RNA, Vol. 17, Issue 4 https://doi.org/10.1261/rna.2540311	journal	March 2011
Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains Dong, Shuyun; Wang, Yang; Cassidy-Amstutz, Caleb Journal of Biological Chemistry, Vol. 286, Issue 30 https://doi.org/10.1074/jbc.M111.244889	journal	June 2011
Preferential expression of scores of functionally and evolutionarily diverse DNA and RNA-binding proteins during Oxytricha trifallax macronuclear development Neeb, Zachary T.; Hogan, Daniel J.; Katzman, Sol PLOS ONE, Vol. 12, Issue 2 https://doi.org/10.1371/journal.pone.0170870	journal	February 2017
Modular Recognition of RNA by a Human Pumilio-Homology Domain Wang, Xiaoqiang; McLachlan, Juanita; Zamore, Phillip D. Cell, Vol. 110, Issue 4 https://doi.org/10.1016/S0092-8674(02)00873-5	journal	August 2002

Similar Records

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

Journal Article · Thu Aug 19 00:00:00 EDT 2010 · Proc. Natl. Acad. Sci. USA · OSTI ID:1506498

Wang, Yeming; Opperman, Laura; Wickens, Marvin; +1 more

Structural basis for specific recognition of multiple mRNA targets by a PUF regulatory protein

Journal Article · Wed Nov 02 00:00:00 EDT 2011 · Proceedings of the National Academy of Sciences of the United States of America · OSTI ID:1506498

Wang, Yeming; Opperman, Laura; Wickens, Marvin; +1 more

Basis of altered RNA-binding specificity by PUF proteins revealed by crystal structures of yeast Puf4p

Journal Article · Fri Jun 06 00:00:00 EDT 2008 · Nat. Struct. Mol. Biol. · OSTI ID:1506498

Miller, Matthew T; Higgin, Joshua J; Hall, Traci M Tanaka

Title: Engineering a conserved RNA regulatory protein repurposes its biological function in vivo

Citation Formats

References (64)

Similar Records

Related Subjects