Co-occupancy of two Pumilio molecules on a single hunchback NRE.

Pumilio controls a number of processes in eukaryotes, including the translational repression of hunchback (hb) mRNA in early Drosophila embryos. The Pumilio Puf domain binds to a pair of 32 nucleotide (nt) Nanos response elements (NRE1 and NRE2) within the 3' untranslated region of hb mRNA. Despite the elucidation of structures of human Pumilio Puf domain in complex with hb RNA elements, the nature of hb mRNA recognition remains unclear. In particular, the site that mediates regulation in vivo is significantly larger than the 8-10-nt RNA elements bound to single Puf molecules in crystal structures. Here we present biophysical and biochemical data that partially resolve the paradox. We show that each NRE is composed of two binding sites (Box A and Box B) and that two Puf domains can co-occupy a single NRE. The Puf domains have a higher affinity for the 3' Box B site than the 5' Box A site; binding to the intact NRE appears to be cooperative (at least in some experiments). We suggest that the 2 Pumilio:1 NRE complex is the functional regulatory unit in vivo.

[1]  Ann-Shyn Chiang,et al.  The staufen/pumilio Pathway Is Involved in Drosophila Long-Term Memory , 2003, Current Biology.

[2]  Robert E. Johnson,et al.  Human DNA Polymerase κ Encircles DNA: Implications for Mismatch Extension and Lesion Bypass , 2007 .

[3]  R. Wharton,et al.  Translational control of maternal Cyclin B mRNA by Nanos in the Drosophila germline , 2007, Development.

[4]  A. Aggarwal,et al.  Structure of Pumilio Reveals Similarity between RNA and Peptide Binding Motifs , 2001, Cell.

[5]  Marvin Wickens,et al.  A PUF family portrait: 3'UTR regulation as a way of life. , 2002, Trends in genetics : TIG.

[6]  R. Lehmann,et al.  The Pumilio protein binds RNA through a conserved domain that defines a new class of RNA-binding proteins. , 1997, RNA.

[7]  R. Wharton,et al.  Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in drosophila embryos , 1995, Cell.

[8]  P. Zamore,et al.  Crystal structure of a Pumilio homology domain. , 2001, Molecular cell.

[9]  A. Spradling,et al.  A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. , 1997, Development.

[10]  R. Lehmann,et al.  Involvement of the pumilio gene in the transport of an abdominal signal in the Drosophila embryo , 1987, Nature.

[11]  R. Wharton,et al.  Recruitment of Nanos to hunchback mRNA by Pumilio. , 1999, Genes & development.

[12]  Subhabrata Sanyal,et al.  The Translational Repressor Pumilio Regulates Presynaptic Morphology and Controls Postsynaptic Accumulation of Translation Factor eIF-4E , 2004, Neuron.

[13]  P. Schuck,et al.  Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. , 2000, Biophysical journal.

[14]  Deepak T Nair,et al.  Structures of human Pumilio with noncognate RNAs reveal molecular mechanisms for binding promiscuity. , 2008, Structure.

[15]  Marvin Wickens,et al.  A single spacer nucleotide determines the specificities of two mRNA regulatory proteins , 2005, Nature Structural &Molecular Biology.

[16]  A. Aggarwal,et al.  mRNA Regulation by Puf Domain Proteins , 2006, Science's STKE.

[17]  Peter Schuck,et al.  Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems. , 2002, Biophysical journal.

[18]  Masashi Yamada,et al.  Maternal Pumilio acts together with Nanos in germline development in Drosophila embryos , 1999, Nature Cell Biology.

[19]  The PUMILIO-RNA interaction: a single RNA-binding domain monomer recognizes a bipartite target sequence. , 1999, Biochemistry.

[20]  J. García de la Torre,et al.  Calculation of hydrodynamic properties of globular proteins from their atomic-level structure. , 2000, Biophysical journal.

[21]  R. Wharton,et al.  The Pumilio RNA-binding domain is also a translational regulator. , 1998, Molecular cell.

[22]  P. Brown,et al.  Extensive Association of Functionally and Cytotopically Related mRNAs with Puf Family RNA-Binding Proteins in Yeast , 2004, PLoS biology.

[23]  Matthew T Miller,et al.  Basis of altered RNA-binding specificity by PUF proteins revealed by crystal structures of yeast Puf4p , 2008, Nature Structural &Molecular Biology.

[24]  G. Struhl,et al.  RNA regulatory elements mediate control of Drosophila body pattern by the posterior morphogen nanos , 1991, Cell.

[25]  R. Mariuzza,et al.  Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition. , 2004, Analytical biochemistry.

[26]  Phillip D. Zamore,et al.  Modular Recognition of RNA by a Human Pumilio-Homology Domain , 2002, Cell.

[27]  R. Wharton,et al.  Drosophila Brain Tumor is a translational repressor. , 2001, Genes & development.

[28]  R Parker,et al.  The Puf3 protein is a transcript‐specific regulator of mRNA degradation in yeast , 2000, The EMBO journal.

[29]  Claudia Petritsch,et al.  nanos and pumilio Are Essential for Dendrite Morphogenesis in Drosophila Peripheral Neurons , 2004, Current Biology.