Structural insights into cis element recognition of non-polyadenylated RNAs by the Nab3-RRM

Transcription termination of non-polyadenylated RNAs in Saccharomyces cerevisiae occurs through the action of the Nrd1–Nab3–Sen1 complex. Part of the decision to terminate via this pathway occurs via direct recognition of sequences within the nascent transcript by RNA recognition motifs (RRMs) within Nrd1 and Nab3. Here we present the 1.6 Å structure of Nab3-RRM bound to its UCUU recognition sequence. The crystal structure reveals clear density for a UCU trinucleotide and a fourth putative U binding site. Nab3-RRM establishes a clear preference for the central cytidine of the UCUU motif, which forms pseudo-base pairing interactions primarily through hydrogen bonds to main chain atoms and one serine hydroxyl group. Specificity for the flanking uridines is less defined; however, binding experiments confirm that these residues are also important for high affinity binding. Comparison of the Nab3-RRM to other structures of RRMs bound to polypyrimidine RNAs showed that this mode of recognition is similar to what is observed for the polypyrimidine-tract binding RRMs, and that the serine residue involved in pseudo-base pairing is only found in RRMs that bind to polypyrimidine RNAs that contain a cytosine base, suggesting a possible mechanism for discriminating between cytosine and uracil bases in RRMs that bind to polypyrimidine-containing RNA.

[1]  K. Henrick,et al.  Inference of macromolecular assemblies from crystalline state. , 2007, Journal of molecular biology.

[2]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

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

[4]  Jack Snoeyink,et al.  Nucleic Acids Research Advance Access published April 22, 2007 MolProbity: all-atom contacts and structure validation for proteins and nucleic acids , 2007 .

[5]  Michael R Green,et al.  Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65. , 2006, Molecular cell.

[6]  Patricia Richard,et al.  Transcription termination by nuclear RNA polymerases. , 2009, Genes & development.

[7]  Wolfgang Kabsch,et al.  Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants , 1993 .

[8]  D. Brow,et al.  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 , 1996, Molecular and cellular biology.

[9]  Gabriele Varani,et al.  RNA is rarely at a loss for companions; as soon as RNA , 2008 .

[10]  J M Thornton,et al.  LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. , 1995, Protein engineering.

[11]  J. Granek,et al.  Identification of cis Elements Directing Termination of Yeast Nonpolyadenylated snoRNA Transcripts , 2004, Molecular and Cellular Biology.

[12]  Jay Painter,et al.  Electronic Reprint Biological Crystallography Optimal Description of a Protein Structure in Terms of Multiple Groups Undergoing Tls Motion Biological Crystallography Optimal Description of a Protein Structure in Terms of Multiple Groups Undergoing Tls Motion , 2005 .

[13]  C. Dominguez,et al.  The RNA recognition motif, a plastic RNA‐binding platform to regulate post‐transcriptional gene expression , 2005, The FEBS journal.

[14]  D. Brow,et al.  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. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. Black,et al.  Structure of PTB Bound to RNA: Specific Binding and Implications for Splicing Regulation , 2005, Science.

[16]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[17]  O. Rando,et al.  Distinct pathways for snoRNA and mRNA termination. , 2006, Molecular cell.

[18]  Jay Painter,et al.  TLSMD web server for the generation of multi-group TLS models , 2006 .

[19]  A. Fatica,et al.  Functional Analysis of Yeast snoRNA and snRNA 3′-End Formation Mediated by Uncoupling of Cleavage and Polyadenylation , 2002, Molecular and Cellular Biology.

[20]  S. Buratowski,et al.  The Nrd1–Nab3–Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain , 2008, Nature Structural &Molecular Biology.

[21]  L. Tong,et al.  Protein factors in pre-mRNA 3′-end processing , 2008, Cellular and Molecular Life Sciences.

[22]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[23]  E. Birney,et al.  Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. , 1993, Nucleic acids research.

[24]  M. Swanson,et al.  A yeast heterogeneous nuclear ribonucleoprotein complex associated with RNA polymerase II. , 2000, Genetics.

[25]  D. Libri,et al.  Phosphorylation of the RNA polymerase II C-terminal domain dictates transcription termination choice , 2008, Nature Structural &Molecular Biology.

[26]  D. Brow,et al.  RNA-binding protein Nrd1 directs poly(A)-independent 3′-end formation of RNA polymerase II transcripts , 2001, Nature.

[27]  Geoffrey J. Barton,et al.  Jalview Version 2—a multiple sequence alignment editor and analysis workbench , 2009, Bioinform..

[28]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[29]  R. Stefl,et al.  1H, 13C, and 15N chemical shift assignments for the RNA recognition motif of Nab3 , 2010, Biomolecular NMR assignments.

[30]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[31]  R. Ghirlando,et al.  Interaction of yeast RNA-binding proteins Nrd1 and Nab3 with RNA polymerase II terminator elements. , 2007, RNA.

[32]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

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

[34]  J. Corden,et al.  Termination of cryptic unstable transcripts is directed by yeast RNA-binding proteins Nrd1 and Nab3. , 2006, Molecular cell.

[35]  M. Culbertson,et al.  Multiple protein/protein and protein/RNA interactions suggest roles for yeast DNA/RNA helicase Sen1p in transcription, transcription-coupled DNA repair and RNA processing. , 2004, Nucleic acids research.

[36]  Y. Hargous,et al.  Molecular basis of RNA recognition and TAP binding by the SR proteins SRp20 and 9G8 , 2006, The EMBO journal.

[37]  Jing Zhao,et al.  Formation of mRNA 3′ Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis , 1999, Microbiology and Molecular Biology Reviews.

[38]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[39]  P. Kuzmič,et al.  Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. , 1996, Analytical biochemistry.

[40]  D. Libri,et al.  Transcription termination and nuclear degradation of cryptic unstable transcripts: a role for the nrd1-nab3 pathway in genome surveillance. , 2006, Molecular cell.