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• W1588341390 abstract "RNA recognitionmotifs (RRMs) are characterized by highly conserved regions located centrally on a β-sheet, which forms the RNA binding surface. Variable flanking regions, such as the loop connecting β-strands 2 and 3, are thought to be important in determining the RNA-binding specificities of individual RRMs. The N-terminal RRM of the spliceosomal U1A protein mediates binding to an RNA hairpin (U1hpII) in the U1 small nuclear RNA. In this complex, the β2-β3 loop protrudes through the 10-nucleotide RNA loop. Shortening of the RNA loop strongly perturbs binding, suggesting that an optimal “fit” of the β2-β3 loop into the RNA loop is an important factor in complexation. To understand this interaction further, we mutated or deleted loop residues Lys50and Met51, which protrude centrally into the RNA loop but do not make any direct contacts to the bases. Using BIACORE, we analyzed the ability of these U1A mutants to bind to wild type RNAs, or RNAs with shortened loops. Alanine replacement mutations only modestly affected binding to wild type U1hpII. Interestingly, simultaneous replacement of Lys50 and Met51 with alanine appeared to alleviate the loss of binding caused by shortening of the RNA loop. Deletion of Lys50 or Met51 caused a dramatic loss in stability of the U1A·U1hpII complex. However, deletion of both residues simultaneously was much less deleterious. Simulated annealing molecular dynamics analyses suggest this is due to the ability of this mutant to rearrange flanking amino acids to substitute for the two deleted residues. The double deletion mutant also exhibited substantially reduced negative effects of RNA loop shortening, suggesting the rearranged loop is better able to accommodate a short RNA loop. Our results indicate that one of the roles of the β2-β3 loop is to provide a steric fit into the RNA loop, thereby stabilizing the RNA·protein complex. RNA recognitionmotifs (RRMs) are characterized by highly conserved regions located centrally on a β-sheet, which forms the RNA binding surface. Variable flanking regions, such as the loop connecting β-strands 2 and 3, are thought to be important in determining the RNA-binding specificities of individual RRMs. The N-terminal RRM of the spliceosomal U1A protein mediates binding to an RNA hairpin (U1hpII) in the U1 small nuclear RNA. In this complex, the β2-β3 loop protrudes through the 10-nucleotide RNA loop. Shortening of the RNA loop strongly perturbs binding, suggesting that an optimal “fit” of the β2-β3 loop into the RNA loop is an important factor in complexation. To understand this interaction further, we mutated or deleted loop residues Lys50and Met51, which protrude centrally into the RNA loop but do not make any direct contacts to the bases. Using BIACORE, we analyzed the ability of these U1A mutants to bind to wild type RNAs, or RNAs with shortened loops. Alanine replacement mutations only modestly affected binding to wild type U1hpII. Interestingly, simultaneous replacement of Lys50 and Met51 with alanine appeared to alleviate the loss of binding caused by shortening of the RNA loop. Deletion of Lys50 or Met51 caused a dramatic loss in stability of the U1A·U1hpII complex. However, deletion of both residues simultaneously was much less deleterious. Simulated annealing molecular dynamics analyses suggest this is due to the ability of this mutant to rearrange flanking amino acids to substitute for the two deleted residues. The double deletion mutant also exhibited substantially reduced negative effects of RNA loop shortening, suggesting the rearranged loop is better able to accommodate a short RNA loop. Our results indicate that one of the roles of the β2-β3 loop is to provide a steric fit into the RNA loop, thereby stabilizing the RNA·protein complex. RNA recognition motif ribonucleoprotein RNA binding domain The RNA recognition motif (RRM),1 also known as theribonucleoprotein (RNP) consensus domain or RNA binding domain (RBD), is the most common and best characterized RNA binding domain. It is present in one or more copies in hundreds of RNA binding proteins (1Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1723) Google Scholar, 2Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar, 3Perez-Canadillas J.M. Varani G. Curr. Opin. Struct. Biol. 2001; 11: 53-58Crossref PubMed Scopus (113) Google Scholar). Proteins carrying RRM domains play critical roles in a wide variety of cellular processes, including RNA processing and packaging, mRNA export, translation, and RNA degradation. RRM domains are about 90 amino acids long and fold into a globular structure consisting of a four-stranded antiparallel β-sheet (the RNA binding surface) backed by two α-helices (see Fig. 1, A and B). RRM domains are characterized by the presence of two highly conserved stretches of 8 and 6 amino acids, known as the RNP1 and RNP2 consensus sequences (see Fig. 1A). These consensus sequences lie strategically in the center of the β-sheet surface and contain conserved aromatic residues critical for RNA binding (2Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar, 4Nagai K. Oubridge C. Jessen T.H., Li, J. Evans P.R. Nature. 1990; 348: 515-520Crossref PubMed Scopus (550) Google Scholar, 5Oubridge C. Ito N. Evans P.R. Teo C.H. Nagai K. Nature. 1994; 372: 432-438Crossref PubMed Scopus (781) Google Scholar, 6Jessen T.H. Oubridge C. Teo C.H. Pritchard C. Nagai K. EMBO J. 1991; 10: 3447-3456Crossref PubMed Scopus (166) Google Scholar, 7Kranz J.K. Hall K.B. J. Mol. Biol. 1998; 275: 465-481Crossref PubMed Scopus (44) Google Scholar, 8Kranz J.K., Lu, J. Hall K.B. Protein Sci. 1996; 5: 1567-1583Crossref PubMed Scopus (35) Google Scholar, 9Katsamba P.S. Myszka D.G. Laird-Offringa I.A. J. Biol. Chem. 2001; 276: 21476-21481Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10Blakaj D.M. McConnell K.J. Beveridge D.L. Baranger A.M. J. Am. Chem. Soc. 2001; 123: 2548-2551Crossref PubMed Scopus (54) Google Scholar, 11Stump W.T. Hall K.B. RNA (N. Y.). 1995; 1: 55-63PubMed Google Scholar, 12Nolan S.J. Shiels J.C. Tuite J.B. Cecere K.L. Baranger A.M. J. Am. Chem. Soc. 1999; 121: 8951-8952Crossref Scopus (47) Google Scholar, 13De Guzman R.N. Turner R.B. Summers M.F. Biopolymers. 1998; 48: 181-195Crossref PubMed Scopus (56) Google Scholar, 14Allain F.H. Bouvet P. Dieckmann T. Feigon J. EMBO J. 2000; 19: 6870-6881Crossref PubMed Scopus (172) Google Scholar). Because the highly conserved nature of the RNP sequences precludes a major role in controlling the specificity of the interaction (1Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1723) Google Scholar, 2Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar, 11Stump W.T. Hall K.B. RNA (N. Y.). 1995; 1: 55-63PubMed Google Scholar,15Tang Y. Nilsson L. Biophys. J. 1999; 77: 1284-1305Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Allain F.H. Howe P.W. Neuhaus D. Varani G. EMBO J. 1997; 16: 5764-5772Crossref PubMed Scopus (146) Google Scholar), the less conserved loop regions surrounding the β-sheet surface are thought to be the predominant determinants of the target specificity of individual RRM proteins. The mechanism by which these flanking regions confer specificity is of considerable interest. Here we describe studies of one such region, the β2-β3 loop, which plays an important role in RNA binding by the U1A protein.U1A, the A protein of the U1 small nuclear ribonucleoprotein, is the most extensively studied RRM protein, and is often used as a paradigm for RRM domain/RNA interaction. Its N-terminal RRM is necessary and sufficient for high affinity binding to U1 hairpin II (U1hpII), a stem-loop structure in the U1 small nuclear RNA (see Fig.1D) (17Scherly D. Boelens W. van Venrooij W.J. Dathan N.A. Hamm J. Mattaj I.W. EMBO J. 1989; 8: 4163-4170Crossref PubMed Scopus (255) Google Scholar, 18Lutz-Freyermuth C. Query C.C. Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6393-6397Crossref PubMed Scopus (135) Google Scholar). The β2-β3 loop of U1A (amino acids 46–52, see Fig. 1, A–C) inserts into the RNA loop, causing the RNA loop residues to splay out (5Oubridge C. Ito N. Evans P.R. Teo C.H. Nagai K. Nature. 1994; 372: 432-438Crossref PubMed Scopus (781) Google Scholar). This allows the presentation of the bases to highly conserved amino acids within the RNP sequences and the establishment of the critical interactions that mediate high affinity binding (5Oubridge C. Ito N. Evans P.R. Teo C.H. Nagai K. Nature. 1994; 372: 432-438Crossref PubMed Scopus (781) Google Scholar). The interaction between the RNA and protein loops also appears to be important for providing stability to the RNA·protein complex, as suggested by the increased dissociation rate of complexes containing a Leu49 → Met U1A mutant or RNAs with shortened loops (9Katsamba P.S. Myszka D.G. Laird-Offringa I.A. J. Biol. Chem. 2001; 276: 21476-21481Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 19Laird-Offringa I.A. Belasco J.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11859-11863Crossref PubMed Scopus (55) Google Scholar). Here we describe experiments aimed at testing whether proper complex stability might require a snug “fit” of the RNA and protein loops. We hypothesized that a reduction in the bulk of the β2-β3loop might allow it to interact better with RNAs with shorter loops. Using a surface plasmon resonance-based biosensor (BIACORE) (20Morton T.A. Myszka D.G. Methods Enzymol. 1998; 295: 268-294Crossref PubMed Scopus (267) Google Scholar, 21Myszka D.G. J. Mol. Recognit. 1999; 12: 279-284Crossref PubMed Scopus (649) Google Scholar, 22Myszka D.G. Methods Enzymol. 2000; 323: 325-340Crossref PubMed Scopus (188) Google Scholar) we investigated the effects of β2-β3 loop mutants on the interaction of U1A with U1hpII RNAs carrying loops of different sizes. We show that removal of two amino acids from the β2-β3 loop favors the interaction with smaller RNA hairpins, supporting a steric role of this part of U1A. The RNA recognition motif (RRM),1 also known as theribonucleoprotein (RNP) consensus domain or RNA binding domain (RBD), is the most common and best characterized RNA binding domain. It is present in one or more copies in hundreds of RNA binding proteins (1Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1723) Google Scholar, 2Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar, 3Perez-Canadillas J.M. Varani G. Curr. Opin. Struct. Biol. 2001; 11: 53-58Crossref PubMed Scopus (113) Google Scholar). Proteins carrying RRM domains play critical roles in a wide variety of cellular processes, including RNA processing and packaging, mRNA export, translation, and RNA degradation. RRM domains are about 90 amino acids long and fold into a globular structure consisting of a four-stranded antiparallel β-sheet (the RNA binding surface) backed by two α-helices (see Fig. 1, A and B). RRM domains are characterized by the presence of two highly conserved stretches of 8 and 6 amino acids, known as the RNP1 and RNP2 consensus sequences (see Fig. 1A). These consensus sequences lie strategically in the center of the β-sheet surface and contain conserved aromatic residues critical for RNA binding (2Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar, 4Nagai K. Oubridge C. Jessen T.H., Li, J. Evans P.R. Nature. 1990; 348: 515-520Crossref PubMed Scopus (550) Google Scholar, 5Oubridge C. Ito N. Evans P.R. Teo C.H. Nagai K. Nature. 1994; 372: 432-438Crossref PubMed Scopus (781) Google Scholar, 6Jessen T.H. Oubridge C. Teo C.H. Pritchard C. Nagai K. EMBO J. 1991; 10: 3447-3456Crossref PubMed Scopus (166) Google Scholar, 7Kranz J.K. Hall K.B. J. Mol. Biol. 1998; 275: 465-481Crossref PubMed Scopus (44) Google Scholar, 8Kranz J.K., Lu, J. Hall K.B. Protein Sci. 1996; 5: 1567-1583Crossref PubMed Scopus (35) Google Scholar, 9Katsamba P.S. Myszka D.G. Laird-Offringa I.A. J. Biol. Chem. 2001; 276: 21476-21481Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 10Blakaj D.M. McConnell K.J. Beveridge D.L. Baranger A.M. J. Am. Chem. Soc. 2001; 123: 2548-2551Crossref PubMed Scopus (54) Google Scholar, 11Stump W.T. Hall K.B. RNA (N. Y.). 1995; 1: 55-63PubMed Google Scholar, 12Nolan S.J. Shiels J.C. Tuite J.B. Cecere K.L. Baranger A.M. J. Am. Chem. Soc. 1999; 121: 8951-8952Crossref Scopus (47) Google Scholar, 13De Guzman R.N. Turner R.B. Summers M.F. Biopolymers. 1998; 48: 181-195Crossref PubMed Scopus (56) Google Scholar, 14Allain F.H. Bouvet P. Dieckmann T. Feigon J. EMBO J. 2000; 19: 6870-6881Crossref PubMed Scopus (172) Google Scholar). Because the highly conserved nature of the RNP sequences precludes a major role in controlling the specificity of the interaction (1Burd C.G. Dreyfuss G. Science. 1994; 265: 615-621Crossref PubMed Scopus (1723) Google Scholar, 2Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (250) Google Scholar, 11Stump W.T. Hall K.B. RNA (N. Y.). 1995; 1: 55-63PubMed Google Scholar,15Tang Y. Nilsson L. Biophys. J. 1999; 77: 1284-1305Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Allain F.H. Howe P.W. Neuhaus D. Varani G. EMBO J. 1997; 16: 5764-5772Crossref PubMed Scopus (146) Google Scholar), the less conserved loop regions surrounding the β-sheet surface are thought to be the predominant determinants of the target specificity of individual RRM proteins. The mechanism by which these flanking regions confer specificity is of considerable interest. Here we describe studies of one such region, the β2-β3 loop, which plays an important role in RNA binding by the U1A protein. U1A, the A protein of the U1 small nuclear ribonucleoprotein, is the most extensively studied RRM protein, and is often used as a paradigm for RRM domain/RNA interaction. Its N-terminal RRM is necessary and sufficient for high affinity binding to U1 hairpin II (U1hpII), a stem-loop structure in the U1 small nuclear RNA (see Fig.1D) (17Scherly D. Boelens W. van Venrooij W.J. Dathan N.A. Hamm J. Mattaj I.W. EMBO J. 1989; 8: 4163-4170Crossref PubMed Scopus (255) Google Scholar, 18Lutz-Freyermuth C. Query C.C. Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6393-6397Crossref PubMed Scopus (135) Google Scholar). The β2-β3 loop of U1A (amino acids 46–52, see Fig. 1, A–C) inserts into the RNA loop, causing the RNA loop residues to splay out (5Oubridge C. Ito N. Evans P.R. Teo C.H. Nagai K. Nature. 1994; 372: 432-438Crossref PubMed Scopus (781) Google Scholar). This allows the presentation of the bases to highly conserved amino acids within the RNP sequences and the establishment of the critical interactions that mediate high affinity binding (5Oubridge C. Ito N. Evans P.R. Teo C.H. Nagai K. Nature. 1994; 372: 432-438Crossref PubMed Scopus (781) Google Scholar). The interaction between the RNA and protein loops also appears to be important for providing stability to the RNA·protein complex, as suggested by the increased dissociation rate of complexes containing a Leu49 → Met U1A mutant or RNAs with shortened loops (9Katsamba P.S. Myszka D.G. Laird-Offringa I.A. J. Biol. Chem. 2001; 276: 21476-21481Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 19Laird-Offringa I.A. Belasco J.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11859-11863Crossref PubMed Scopus (55) Google Scholar). Here we describe experiments aimed at testing whether proper complex stability might require a snug “fit” of the RNA and protein loops. We hypothesized that a reduction in the bulk of the β2-β3loop might allow it to interact better with RNAs with shorter loops. Using a surface plasmon resonance-based biosensor (BIACORE) (20Morton T.A. Myszka D.G. Methods Enzymol. 1998; 295: 268-294Crossref PubMed Scopus (267) Google Scholar, 21Myszka D.G. J. Mol. Recognit. 1999; 12: 279-284Crossref PubMed Scopus (649) Google Scholar, 22Myszka D.G. Methods Enzymol. 2000; 323: 325-340Crossref PubMed Scopus (188) Google Scholar) we investigated the effects of β2-β3 loop mutants on the interaction of U1A with U1hpII RNAs carrying loops of different sizes. We show that removal of two amino acids from the β2-β3 loop favors the interaction with smaller RNA hairpins, supporting a steric role of this part of U1A. We thank members of the Laird-Offringa laboratory for helpful criticism." @default.
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• W1588341390 title "Complex role of the β2-β3 Loop in the Interaction of U1A with U1 Hairpin II RNA" @default.
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