In Saccharomyces cerevisiae, the Nrd1-dependent termination and processing pathways play an important role in surveillance and processing of non-coding ribonucleic acids (RNAs). The termination and subsequent processing is dependent on the Nrd1 complex consisting of two RNA-binding proteins Nrd1 and Nab3 and Sen1 helicase. It is established that Nrd1 and Nab3 cooperatively recognize specific termination elements within nascent RNA, GUA[A/G] and UCUU[G], respectively. Interestingly, some transcripts do not require GUA[A/G] motif for transcription termination in vivo and binding in vitro, suggesting the existence of alternative Nrd1-binding motifs. Here we studied the structure and RNA-binding properties of Nrd1 using nuclear magnetic resonance (NMR), fluorescence anisotropy and phenotypic analyses in vivo. We determined the solution structure of a two-domain RNA-binding fragment of Nrd1, formed by an RNA-recognition motif and helix-loop bundle. NMR and fluorescence data show that not only GUA[A/G] but also several other G-rich and AU-rich motifs are able to bind Nrd1 with affinity in a low micromolar range. The broad substrate specificity is achieved by adaptable interaction surfaces of the RNA-recognition motif and helix-loop bundle domains that sandwich the RNA substrates. Our findings have implication for the role of Nrd1 in termination and processing of many non-coding RNAs arising from bidirectional pervasive transcription.

CEITEC Central European Institute of Technology Masaryk University Brno 62500 Czech Republic National Centre for Biomolecular Research Faculty of Science Masaryk University Brno 62500 Czech Republic

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Richard P., Manley J.L. Transcription termination by nuclear RNA polymerases. Genes Dev. 2009;23:1247–1269. PubMed PMC

Bentley D. The mRNA assembly line: transcription and processing machines in the same factory. Curr. Opin. Cell Biol. 2002;14:336–342. PubMed

Steinmetz E.J., Conrad N.K., Brow D.A., Corden J.L. RNA-binding protein Nrd1 directs poly(A)-independent 3′-end formation of RNA polymerase II transcripts. Nature. 2001;413:327–331. PubMed

Arigo J.T., Eyler D.E., Carroll K.L., Corden J.L. Termination of cryptic unstable transcripts is directed by yeast RNA-binding proteins Nrd1 and Nab3. Mol. Cell. 2006;23:841–851. PubMed

Thiebaut M., Kisseleva-Romanova E., Rougemaille M., Boulay J., Libri D. Transcription termination and nuclear degradation of cryptic unstable transcripts: a role for the nrd1-nab3 pathway in genome surveillance. Mol. Cell. 2006;23:853–864. PubMed

Vasiljeva L., Buratowski S. Nrd1 interacts with the nuclear exosome for 3′ processing of RNA polymerase II transcripts. Mol. Cell. 2006;21:239–248. PubMed

Vanacova S., Stefl R. The exosome and RNA quality control in the nucleus. EMBO Rep. 2007;8:651–657. PubMed PMC

Steinmetz E.J., Brow D.A. Repression of gene expression by an exogenous sequence element acting in concert with a heterogeneous nuclear ribonucleoprotein-like protein, Nrd1, and the putative helicase Sen1. Mol. Cell. Biol. 1996;16:6993–7003. PubMed PMC

Steinmetz E.J., Brow D.A. Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association. Proc. Natl. Acad. Sci. U.S.A. 1998;95:6699–6704. PubMed PMC

Carroll K.L., Pradhan D.A., Granek J.A., Clarke N.D., Corden J.L. Identification of cis elements directing termination of yeast nonpolyadenylated snoRNA transcripts. Mol. Cell. Biol. 2004;24:6241–6252. PubMed PMC

Carroll K.L., Ghirlando R., Ames J.M., Corden J.L. Interaction of yeast RNA-binding proteins Nrd1 and Nab3 with RNA polymerase II terminator elements. RNA. 2007;13:361–373. PubMed PMC

Hobor F., Pergoli R., Kubicek K., Hrossova D., Bacikova V., Zimmermann M., Pasulka J., Hofr C., Vanacova S., Stefl R. Recognition of transcription termination signal by the nuclear polyadenylated RNA-binding (NAB) 3 protein. J. Biol. Chem. 2011;286:3645–3657. PubMed PMC

Porrua O., Hobor F., Boulay J., Kubicek K., D'Aubenton-Carafa Y., Gudipati R.K., Stefl R., Libri D. In vivo SELEX reveals novel sequence and structural determinants of Nrd1-Nab3-Sen1-dependent transcription termination. EMBO J. 2012;31:3935–3948. PubMed PMC

Lunde B.M., Horner M., Meinhart A. Structural insights into cis element recognition of non-polyadenylated RNAs by the Nab3-RRM. Nucleic Acids Res. 2011;39:337–346. PubMed PMC

Vasiljeva L., Kim M., Terzi N., Soares L.M., Buratowski S. Transcription termination and RNA degradation contribute to silencing of RNA polymerase II transcription within heterochromatin. Mol. Cell. 2008;29:313–323. PubMed

Jasnovidova O., Stefl R. The CTD code of RNA polymerase II: a structural view. Wiley Interdiscip. Rev. RNA. 2013;4:1–16. PubMed

Kubicek K., Cerna H., Holub P., Pasulka J., Hrossova D., Loehr F., Hofr C., Vanacova S., Stefl R. Serine phosphorylation and proline isomerization in RNAP II CTD control recruitment of Nrd1. Genes Dev. 2012;26:1891–1896. PubMed PMC

Vanacova S., Wolf J., Martin G., Blank D., Dettwiler S., Friedlein A., Langen H., Keith G., Keller W. A new yeast poly(A) polymerase complex involved in RNA quality control. PLoS Biol. 2005;3:e189. PubMed PMC

LaCava J., Houseley J., Saveanu C., Petfalski E., Thompson E., Jacquier A., Tollervey D. RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell. 2005;121:713–724. PubMed

Wyers F., Rougemaille M., Badis G., Rousselle J.C., Dufour M.E., Boulay J., Regnault B., Devaux F., Namane A., Seraphin B., et al. Cryptic pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell. 121:725–737. PubMed

Wlotzka W., Kudla G., Granneman S., Tollervey D. The nuclear RNA polymerase II surveillance system targets polymerase III transcripts. EMBO J. 2011;30:1790–1803. PubMed PMC

Jamonnak N., Creamer T.J., Darby M.M., Schaughency P., Wheelan S.J., Corden J.L. Yeast Nrd1, Nab3, and Sen1 transcriptome-wide binding maps suggest multiple roles in post-transcriptional RNA processing. RNA. 2011;17:2011–2025. PubMed PMC

Creamer T.J., Darby M.M., Jamonnak N., Schaughency P., Hao H., Wheelan S.J., Corden J.L. Transcriptome-wide binding sites for components of the Saccharomyces cerevisiae non-poly(A) termination pathway: Nrd1, Nab3, and Sen1. PLoS Genet. 2011;7 PubMed PMC

Lepore N., L. L.D. A functional interface at the rDNA connects rRNA synthesis, pre-rRNA processing and nucleolar surveillance in budding yeast. PLoS ONE. 2011;6 PubMed PMC

Honorine R., Mosrin-Huaman C., Hervouet-Coste N., Libri D., Rahmouni A.R. Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment, as revealed by the targeting of Rho-induced aberrant transcripts. Nucleic Acids Res. 2011;39:2809–2820. PubMed PMC

Jenks M.H., O'Rourke T.W., Reines D. Properties of an intergenic terminator and start site switch that regulate IMD2 transcription in yeast. Mol. Cell. Biol. 2008;28:3883–3893. PubMed PMC

Ciais D., Bohnsack M.T., Tollervey D. The mRNA encoding the yeast ARE-binding protein Cth2 is generated by a novel 3′ processing. Nucleic Acids Res. 2008;36:3075–3084. PubMed PMC

Noel J.F., Larose S., Abou Elela S., Wellinger R.J. Budding yeast telomerase RNA transcription termination is dictated by the Nrd1/Nab3 non-coding RNA termination pathway. Nucleic Acids Res. 2012;40:5625–5636. PubMed PMC

Gardner K.H., Kay L.E. The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins. Annu. Rev. Biophys. Biomol. Struct. 1998;27:357–406. PubMed

Goto N.K., Gardner K.H., Mueller G.A., Willis R.C., Kay L.E. A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. J. Biomol. NMR. 1999;13:369–374. PubMed

Tugarinov V., Kanelis V., Kay L.E. Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat. Protoc. 2006;1:749–754. PubMed

Sattler M., Schleucher J., Griesinger C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 1999;34:93–158.

Tugarinov V., Kay L.E., Ibraghimov I., Orekhov V.Y. High-resolution four-dimensional 1H-13C NOE spectroscopy using methyl-TROSY, sparse data acquisition, and multidimensional decomposition. J. Am. Chem. Soc. 2005;127:2767–2775. PubMed

Montelione G.T., Lyons B.A., Emerson S.D., Tashiro M. An efficient triple resonance experiment using C-13 isotropic mixing for determining sequence-specific resonance assignments of isotopically-enriched proteins. J. Am. Chem. Soc. 1992;114:10974–10975.

Kay L.E., Xu G.Y., Singer A.U., Muhandiram D.R., Forman-Kay J.D. A gradient-enhanced HCCH TOCSY experiment for recording side-chain H-1 and C-13 correlations in H2O samples of proteins. J. Magn. Reson. B. 1993;101:333–337.

Morris M.J., Wingate K.L., Silwal J., Leeper T.C., Basu S. The porphyrin TmPyP4 unfolds the extremely stable G-quadruplex in MT3-MMP mRNA and alleviates its repressive effect to enhance translation in eukaryotic cells. Nucleic Acids Res. 2012;40:4137–4145. PubMed PMC

Korzhnev D.M., Billeter M., Arseniev A.S., Orekhov V.Y. NMR studies of Brownian tumbling and internal motions in proteins. Prog. Nucl. Magn. Reson. Spectrosc. 2001;38:197–266.

Guntert P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 2004;278:353–378. PubMed

Herrmann T., Guntert P., Wuthrich K. Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol. 2002;319:209–227. PubMed

Case D.A., Darden T.A., Cheatham T.E., III, Simmerling C.L., Wang J., Duke R.E., Luo R., Walker Ross C., Zhang W., Merz K.M., et al. AMBER 12. San Francisco, CA: University of California; 2012.

Hornak V., Abel R., Okur A., Strockbine B., Roitberg A., Simmerling C. Comparison of multiple Amber force fields and development of improved protein backbone parameters. Proteins. 2006;65:712–725. PubMed PMC

Cornell W.D., Cieplak P., Bayly C.I., Gould I.R., Merz K.M., Ferguson D.M., Spellmeyer D.C., Fox T., Caldwell J.W., Kollman P.A. A 2nd generation force-field for the simulation of proteins, nucleic-acids, and organic-molecules. J. Am. Chem. Soc. 1995;117:5179–5197.

Stefl R., Oberstrass F.C., Hood J.L., Jourdan M., Zimmermann M., Skrisovska L., Maris C., Peng L., Hofr C., Emeson R.B., et al. The solution structure of the ADAR2 dsRBM-RNA complex reveals a sequence-specific readout of the minor groove. Cell. 2010;143:225–237. PubMed PMC

Laskowski R.A., Rullmannn J.A., MacArthur M.W., Kaptein R., Thornton J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR. 1996;8:477–486. PubMed

Vriend G. What If—a molecular modeling and drug design program. J. Mol. Graph. 1990;8:52–56. PubMed

Koradi R., Billeter M., Wuthrich K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 1996;14:51, 29–55. PubMed

Letunic I., Doerks T., Bork P. SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Res. 2012;40:D302–D305. PubMed PMC

Akerud T., Thulin E., Van Etten R.L., Akke M. Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding. J. Mol. Biol. 2002;322:137–152. PubMed

Guntert P., Mumenthaler C., Wuthrich K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 1997;273:283–298. PubMed

Maris C., Dominguez C., Allain F.H. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J. 2005;272:2118–2131. PubMed

Stefl R., Skrisovska L., Allain F.H. RNA sequence- and shape-dependent recognition by proteins in the ribonucleoprotein particle. EMBO Rep. 2005;6:33–38. PubMed PMC

Swanson M.S., Nakagawa T.Y., LeVan K., Dreyfuss G. Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins. Mol. Cell. Biol. 1987;7:1731–1739. PubMed PMC

Adam S.A., Nakagawa T., Swanson M.S., Woodruff T.K., Dreyfuss G. mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol. Cell. Biol. 1986;6:2932–2943. PubMed PMC

Dreyfuss G., Swanson M.S., Pinol-Roma S. Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation. Trends Biochem. Sci. 1988;13:86–91. PubMed

Handa N., Nureki O., Kurimoto K., Kim I., Sakamoto H., Shimura Y., Muto Y., Yokoyama S. Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein. Nature. 1999;398:579–585. PubMed

Clery A., Blatter M., Allain F.H. RNA recognition motifs: boring? Not quite. Curr. Opin. Struct. Biol. 2008;18:290–298. PubMed

Daubner G.M., Clery A., Allain F.H. RRM-RNA recognition: NMR or crystallography…and new findings. Curr. Opin. Struct. Biol. 2013;23:100–108. PubMed

Barraud P., Allain F.H. Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology. J. Biomol. NMR. 2013;55:119–138. PubMed

Teplova M., Yuan Y.R., Phan A.T., Malinina L., Ilin S., Teplov A., Patel D.J. Structural basis for recognition and sequestration of UUU(OH) 3′ termini of nascent RNA polymerase III transcripts by La, a rheumatic disease autoantigen. Mol. Cell. 2006;21:75–85. PubMed PMC

Huang Y., Bayfield M.A., Intine R.V., Marala R.J. Separate RNA-binding surfaces on the multifunctional La protein mediate distinguishable activities in tRNA. Nat. Struct. Mol. Biol. 2006;13:611–618. PubMed

Oberstrass F.C., Auweter S.D., Erat M., Hargous Y., Henning A., Wenter P., Reymond L., Amir-Ahmady B., Pitsch S., Black D.L., et al. Structure of PTB bound to RNA: specific binding and implications for splicing regulation. Science. 2005;309:2054–2057. PubMed

Sickmier E.A., Frato K.E., Shen H., Paranawithana S.R., Green M.R., Klelkopf C.L. Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65. Mol. Cell. 2006;23:49–59. PubMed PMC

Deka P., Rajan P.K., Perez-Canadillas J.M., Varani G. Protein and RNA dynamics play key roles in determining the specific recognition of GU-rich polyadenylation regulatory elements by human Cstf-64 protein. J. Mol. Biol. 2005;347:719–733. PubMed

Perez-Canadillas J.M., Varani G. Recognition of GU-rich polyadenylation regulatory elements by human CstF-64 protein. EMBO J. 2003;22:2821–2830. PubMed PMC

Schulz D., Schwalb B., Kiesel A., Baejen C., Torkler P., Gagneur J., Soeding J., Cramer P. Transcriptome surveillance by selective termination of noncoding RNA synthesis. Cell. 2013;155:1075–1087. PubMed

Dominguez C., Fisette J.F., Chabot B., Allain F.H. Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs. Nat. Struct. Mol. Biol. 2010;17:853–861. PubMed

Samatanga B., Dominguez C., Jelesarov I., Allain F.H. The high kinetic stability of a G-quadruplex limits hnRNP F qRRM3 binding to G-tract RNA. Nucleic Acids Res. 2013;41:2505–2516. PubMed PMC

Neil H., Malabat C., D'Aubenton-Carafa Y., Xu Z., Steinmetz L.M., Jacquier A. Widespread bidirectional promoters are the major source of cryptic transcripts in yeast. Nature. 2009;457:1038–1042. PubMed

Zobrazit více v PubMed

PDB
2M88