Two Functionally Distinct Steps Mediate High Affinity Binding of U1A Protein to U1 Hairpin II RNA*

Binding of the U1A protein to its RNA target U1 hairpin II has been extensively studied as a model for a high affinity RNA/protein interaction. However, the mechanism and kinetics by which this complex is formed remain largely unknown. Here we use real-time biomolecular interaction analysis to dissect the roles various protein and RNA structural elements play in the formation of the U1A·U1 hairpin II complex. We show that neutralization of positive charges on the protein or increasing the salt concentration slows the association rate, suggesting that electrostatic interactions play an important role in bringing RNA and protein together. In contrast, removal of hydrogen bonding or stacking interactions within the RNA/protein interface, or reducing the size of the RNA loop, dramatically destabilizes the complex, as seen by a strong increase in the dissociation rate. Our data support a binding mechanism consisting of a rapid initial association based on electrostatic interactions and a subsequent locking step based on close-range interactions that occur during the induced fit of RNA and protein. Remarkably, these two steps can be clearly distinguished using U1A mutants containing single amino acid substitutions. Our observations explain the extraordinary affinity of U1A for its target and may suggest a general mechanism for high affinity RNA/protein interactions.

[1]  D G Myszka,et al.  Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors. , 1997, Methods in enzymology.

[2]  C. Oubridge,et al.  Identification of molecular contacts between the U1 A small nuclear ribonucleoprotein and U1 RNA. , 1991, The EMBO journal.

[3]  M. Sanderson.  High-speed digital microscopy. , 2000, Methods.

[4]  D G Myszka,et al.  Advances in surface plasmon resonance biosensor analysis. , 2000, Current opinion in biotechnology.

[5]  K. Hall,et al.  Crosslinking of an iodo-uridine-RNA hairpin to a single site on the human U1A N-terminal RNA binding domain. , 1995, RNA.

[6]  G. Varani,et al.  Solution structure of the N-terminal RNP domain of U1A protein: the role of C-terminal residues in structure stability and RNA binding. , 1996, Journal of molecular biology.

[7]  M. V. Regenmortel,et al.  Molecular recognition in the post-reductionist era. , 1999 .

[8]  G. Varani,et al.  Specificity of ribonucleoprotein interaction determined by RNA folding during complex formation , 1996, Nature.

[9]  J. Williamson.  Induced fit in RNA–protein recognition , 2000, Nature Structural Biology.

[10]  D. Scherly,et al.  Major determinants of the specificity of interaction between small nuclear ribonucleoproteins U1A and U2B" and their cognate RNAs , 1990, Nature.

[11]  D. Scherly,et al.  Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA. , 1989, The EMBO journal.

[12]  D. Draper.  Themes in RNA-protein recognition. , 1999, Journal of molecular biology.

[13]  P A Kollman,et al.  Structure and thermodynamics of RNA-protein binding: using molecular dynamics and free energy analyses to calculate the free energies of binding and conformational change. , 2000, Journal of molecular biology.

[14]  C. Burd,et al.  Conserved structures and diversity of functions of RNA-binding proteins. , 1994, Science.

[15]  L. Nilsson,et al.  Molecular dynamics simulations of the complex between human U1A protein and hairpin II of U1 small nuclear RNA and of free RNA in solution. , 1999, Biophysical journal.

[16]  H. Erickson,et al.  Kinetics of protein-protein association explained by Brownian dynamics computer simulation. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[17]  G. Varani,et al.  Structural basis of the RNA‐binding specificity of human U1A protein , 1997, The EMBO journal.

[18]  D. Myszka,et al.  CLAMP: a biosensor kinetic data analysis program. , 1998, Trends in biochemical sciences.

[19]  D G Myszka,et al.  Extending the range of rate constants available from BIACORE: interpreting mass transport-influenced binding data. , 1998, Biophysical journal.

[20]  David G. Myszka,et al.  HuD RNA Recognition Motifs Play Distinct Roles in the Formation of a Stable Complex with AU-Rich RNA , 2000, Molecular and Cellular Biology.

[21]  M. Luskin,et al.  The neuronal progenitor cells of the forebrain subventricular zone: intrinsic properties in vitro and following transplantation. , 1998, Methods.

[22]  K. Hall,et al.  RNA binding mediates the local cooperativity between the beta-sheet and the C-terminal tail of the human U1A RBD1 protein. , 1998, Journal of molecular biology.

[23]  J. Keene,et al.  U1-snRNP-A protein selects a ten nucleotide consensus sequence from a degenerate RNA pool presented in various structural contexts. , 1991, Nucleic acids research.

[24]  K. Hall,et al.  Interaction of N-terminal domain of U1A protein with an RNA stem/loop. , 1992, Nucleic acids research.

[25]  L. Kay,et al.  Changes in side-chain and backbone dynamics identify determinants of specificity in RNA recognition by human U1A protein. , 1999, Journal of molecular biology.

[26]  W. Boelens,et al.  The human U1 snRNP-Specific U1A protein inhibits polyadenylation of its own pre-mRNA , 1993, Cell.

[27]  K. Hall,et al.  Interaction of RNA hairpins with the human U1A N-terminal RNA binding domain. , 1994, Biochemistry.

[28]  G. Varani,et al.  NMR studies of U1 snRNA recognition by the N‐terminal RNP domain of the human U1A protein. , 1994, The EMBO journal.

[29]  Nobutoshi Ito,et al.  Crystal structure at 1.92 Å resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin , 1994, Nature.

[30]  L. Christensen.  Theoretical analysis of protein concentration determination using biosensor technology under conditions of partial mass transport limitation. , 1997, Analytical biochemistry.

[31]  P. Sillekens,et al.  cDNA cloning of the human U1 snRNA‐associated A protein: extensive homology between U1 and U2 snRNP‐specific proteins. , 1987, The EMBO journal.

[32]  Kazuki Kurimoto,et al.  Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein , 1999, Nature.

[33]  P. V. von Hippel,et al.  Diffusion-controlled macromolecular interactions. , 1985, Annual review of biophysics and biophysical chemistry.

[34]  F. Allain,et al.  Molecular basis of sequence‐specific recognition of pre‐ribosomal RNA by nucleolin , 2000, The EMBO journal.

[35]  J. Belasco,et al.  Analysis of RNA-binding proteins by in vitro genetic selection: identification of an amino acid residue important for locking U1A onto its RNA target. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[36]  P. Evans,et al.  The RNP domain: a sequence-specific RNA-binding domain involved in processing and transport of RNA. , 1995, Trends in biochemical sciences.

[37]  W. Boelens,et al.  A complex secondary structure in U1A pre‐mRNA that binds two molecules of U1A protein is required for regulation of polyadenylation. , 1993, The EMBO journal.

[38]  Rahul C. Deo,et al.  Recognition of Polyadenylate RNA by the Poly(A)-Binding Protein , 1999, Cell.

[39]  K. Hall,et al.  RNA hairpins with non-nucleotide spacers bind efficiently to the human U1A protein. , 1996, Journal of molecular biology.

[40]  J. Keene,et al.  Quantitative determination that one of two potential RNA-binding domains of the A protein component of the U1 small nuclear ribonucleoprotein complex binds with high affinity to stem-loop II of U1 RNA. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[41]  J. Brown,et al.  Molecular characterization of the spliceosomal proteins U1A and U2B″ from higher plants. , 1995, The EMBO journal.

[42]  H. Salz,et al.  The Drosophila sex determination gene snf encodes a nuclear protein with sequence and functional similarity to the mammalian U1A snRNP protein. , 1994, Genes & development.

[43]  Analysis of in vitro binding of U1-A protein mutants to U1 snRNA. , 1991, Nucleic acids research.

[44]  G. Varani,et al.  RNA recognition by RNP proteins during RNA processing. , 1998, Annual review of biophysics and biomolecular structure.