RNA recognition by a Staufen double‐stranded RNA‐binding domain

The double‐stranded RNA‐binding domain (dsRBD) is a common RNA‐binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem–loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA‐binding activity of dsRBD3 is required in vivo for Staufen‐dependent localization of bicoid and oskar mRNAs. Using high‐resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem–loop. The dsRBD recognizes the shape of A‐form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix α1 interacts with the single‐stranded loop that caps the RNA helix. Interactions between helix α1 and single‐stranded RNA may be important determinants of the specificity of dsRBD proteins.

[1]  G. Rubin,et al.  Genetic transformation of Drosophila with transposable element vectors. , 1982, Science.

[2]  D. St Johnston,et al.  A conserved double-stranded RNA-binding domain. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[3]  B. Bass,et al.  Binding properties of newly identified Xenopus proteins containing dsRNA-binding motifs , 1994, Current Biology.

[4]  M. Mathews,et al.  Two functionally distinct RNA-binding motifs in the regulatory domain of the protein kinase DAI , 1995, Molecular and cellular biology.

[5]  M. Mathews,et al.  Interactions between the double-stranded RNA binding motif and RNA: definition of the binding site for the interferon-induced protein kinase DAI (PKR) on adenovirus VA RNA. , 1995, RNA.

[6]  T. Pawson,et al.  Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. , 1994, Biochemistry.

[7]  S C Schultz,et al.  Molecular basis of double‐stranded RNA‐protein interactions: structure of a dsRNA‐binding domain complexed with dsRNA , 1998, The EMBO journal.

[8]  T. Cech,et al.  Minor-groove recognition of double-stranded RNA by the double-stranded RNA-binding domain from the RNA-activated protein kinase PKR. , 1996, Biochemistry.

[9]  Gabriele Varani,et al.  NMR investigation of RNA structure , 1996 .

[10]  Daniel St Johnston,et al.  staufen, a gene required to localize maternal RNAs in the Drosophila egg , 1991, Cell.

[11]  G. Varani,et al.  Refinement of the structure of protein–RNA complexes by residual dipolar coupling analysis , 1999, Journal of biomolecular NMR.

[12]  B. Golden,et al.  Ribosomal proteins S5 and L6: high-resolution crystal structures and roles in protein synthesis and antibiotic resistance. , 1998, Journal of molecular biology.

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

[14]  Julie Dawn Thompson,et al.  Detection of dsRNA-binding domains in RNA helicase A and Drosophila maleless: implications for monomeric RNA helicases , 1994, Nucleic Acids Res..

[15]  A. Bax,et al.  Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. , 1997, Science.

[16]  M. Palazzolo,et al.  Transposon-facilitated DNA sequencing. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Christopher W. Smith.  RNA : protein interactions : a practical approach , 1998 .

[18]  C. Nüsslein-Volhard,et al.  Multiple steps in the localization of bicoid RNA to the anterior pole of the Drosophila oocyte. , 1989, Development.

[19]  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.

[20]  K. Wüthrich,et al.  Heteronuclear filters in two-dimensional [1H, 1H]-NMR spectroscopy: combined use with isotope labelling for studies of macromolecular conformation and intermolecular interactions , 1990, Quarterly Reviews of Biophysics.

[21]  C. Doe,et al.  Staufen-dependent localization of prospero mRNA contributes to neuroblast daughter-cell fate , 1998, Nature.

[22]  T. Gibson,et al.  Structure of the dsRNA binding domain of E. coli RNase III. , 1995, The EMBO journal.

[23]  R. Lehmann,et al.  oskar organizes the germ plasm and directs localization of the posterior determinant nanos , 1991, Cell.

[24]  Daniel St Johnston,et al.  The intracellular localization of messenger RNAs , 1995, Cell.

[25]  M. Mathews,et al.  Functional characterization of the RNA-binding domain and motif of the double-stranded RNA-dependent protein kinase DAI (PKR). , 1995, Journal of molecular biology.

[26]  G. Varani,et al.  Determination of the NMR structure of the complex between U1A protein and its RNA polyadenylation inhibition element , 1998, Journal of biomolecular NMR.

[27]  R. Sauer,et al.  Origins of DNA-binding specificity: role of protein contacts with the DNA backbone. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[28]  G. Varani,et al.  Preparation of RNA:protein complexes for X-ray crystallography and NMR , 1998 .

[29]  M. Jantsch,et al.  Comparative Mutational Analysis of the Double-stranded RNA Binding Domains of Xenopus laevis RNA-binding Protein A* , 1996, The Journal of Biological Chemistry.

[30]  D. St Johnston,et al.  Miranda mediates asymmetric protein and RNA localization in the developing nervous system. , 1998, Genes & development.

[31]  Dominique Ferrandon,et al.  Staufen protein associates with the 3′UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner , 1994, Cell.

[32]  J. L. Smith,et al.  Multiple RNA regulatory elements mediate distinct steps in localization of oskar mRNA. , 1993, Development.

[33]  P. Legrain,et al.  Yeast RNase III as a key processing enzyme in small nucleolar RNAs metabolism. , 1998, Journal of molecular biology.

[34]  H. Noller,et al.  Site-Directed Hydroxyl Radical Probing of the rRNA Neighborhood of Ribosomal Protein S5 , 1996, Science.

[35]  G. Varani.  RNA - Protein Intermolecular Recognition , 1997 .

[36]  M. Mathews,et al.  Two RNA-binding motifs in the double-stranded RNA-activated protein kinase, DAI. , 1992, Genes & development.

[37]  P. Macdonald,et al.  oskar mRNA is localized to the posterior pole of the Drosophila oocyte , 1991, Cell.

[38]  G. Struhl,et al.  Cis- acting sequences responsible for anterior localization of bicoid mRNA in Drosophila embryos , 1988, Nature.

[39]  A. Brand,et al.  The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila , 1997, Current Biology.

[40]  Sherif Abou Elela,et al.  RNase III Cleaves Eukaryotic Preribosomal RNA at a U3 snoRNP-Dependent Site , 1996, Cell.

[41]  B. Williams,et al.  Structure of the double‐stranded RNA‐binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA‐mediated activation , 1998, The EMBO journal.

[42]  A. Murzin,et al.  NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N‐terminal domain of ribosomal protein S5. , 1995, The EMBO journal.