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1.
Genes Dev ; 28(13): 1498-514, 2014 Jul 01.
Article in English | MEDLINE | ID: mdl-24990967

ABSTRACT

Cytoplasmic changes in polyA tail length is a key mechanism of translational control and is implicated in germline development, synaptic plasticity, cellular proliferation, senescence, and cancer progression. The presence of a U-rich cytoplasmic polyadenylation element (CPE) in the 3' untranslated regions (UTRs) of the responding mRNAs gives them the selectivity to be regulated by the CPE-binding (CPEB) family of proteins, which recognizes RNA via the tandem RNA recognition motifs (RRMs). Here we report the solution structures of the tandem RRMs of two human paralogs (CPEB1 and CPEB4) in their free and RNA-bound states. The structures reveal an unprecedented arrangement of RRMs in the free state that undergo an original closure motion upon RNA binding that ensures high fidelity. Structural and functional characterization of the ZZ domain (zinc-binding domain) of CPEB1 suggests a role in both protein-protein and protein-RNA interactions. Together with functional studies, the structures reveal how RNA binding by CPEB proteins leads to an optimal positioning of the N-terminal and ZZ domains at the 3' UTR, which favors the nucleation of the functional ribonucleoprotein complexes for translation regulation.


Subject(s)
Models, Molecular , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , mRNA Cleavage and Polyadenylation Factors/chemistry , mRNA Cleavage and Polyadenylation Factors/metabolism , 3' Untranslated Regions , Amino Acid Motifs , Crystallography, X-Ray , Cytoplasm/metabolism , Gene Expression Regulation , Humans , Protein Structure, Tertiary , RNA/chemistry , RNA, Messenger/metabolism , Ribonucleoproteins/metabolism
2.
Chembiochem ; 14(4): 457-66, 2013 Mar 04.
Article in English | MEDLINE | ID: mdl-23362130

ABSTRACT

Current solution NMR techniques enable structural investigations of proteins in molecular particles with sizes up to several hundred kDa. However, the large molecular weight of proteins in such systems results in increased numbers of NMR signals, and the resulting spectral overlap typically imposes limitations. For multidomain proteins, segmental isotope labeling of individual domains facilitates the spectral interpretation by reducing the number of signals, but for large domains with small signal dispersion, signal overlap can persist. To overcome limitations arising from spectral overlap, we present a strategy that combines cell-free expression and ligation of the expressed proteins to produce multidomain proteins with selective amino acid-type labeling in individual domains. The bottleneck of intrinsically low cell-free expression yields of precursor molecules was overcome by introducing new fusion constructs that allowed milligram production of ligation-competent domains labeled in one or multiple amino acid types. Ligation-competent unlabeled partner domains were produced in vivo, and subsequent domain ligation was achieved by using an on-column strategy. This approach is illustrated with two multidomain RNA-binding proteins, that is, the two C-terminal RNA-recognition motifs of the human polypyrimidine tract-binding protein, and two highly homologous helix-turn-helix domains of the human glutamyl-prolyl-tRNA synthetase.


Subject(s)
Amino Acids/analysis , Amino Acyl-tRNA Synthetases/chemistry , Isotope Labeling/methods , Nuclear Magnetic Resonance, Biomolecular/methods , Polypyrimidine Tract-Binding Protein/chemistry , Humans , Protein Structure, Tertiary
3.
Cell ; 143(2): 225-37, 2010 Oct 15.
Article in English | MEDLINE | ID: mdl-20946981

ABSTRACT

Sequence-dependent recognition of dsDNA-binding proteins is well understood, yet sequence-specific recognition of dsRNA by proteins remains largely unknown, despite their importance in RNA maturation pathways. Adenosine deaminases that act on RNA (ADARs) recode genomic information by the site-selective deamination of adenosine. Here, we report the solution structure of the ADAR2 double-stranded RNA-binding motifs (dsRBMs) bound to a stem-loop pre-mRNA encoding the R/G editing site of GluR-2. The structure provides a molecular basis for how dsRBMs recognize the shape, and also more surprisingly, the sequence of the dsRNA. The unexpected direct readout of the RNA primary sequence by dsRBMs is achieved via the minor groove of the dsRNA and this recognition is critical for both editing and binding affinity at the R/G site of GluR-2. More generally, our findings suggest a solution to the sequence-specific paradox faced by many dsRBM-containing proteins that are involved in post-transcriptional regulation of gene expression.


Subject(s)
Adenosine Deaminase/chemistry , RNA, Double-Stranded/chemistry , Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Amino Acid Sequence , Animals , Cell Line , Humans , Mice , Models, Molecular , Molecular Sequence Data , Mutation , Nuclear Magnetic Resonance, Biomolecular , RNA Precursors/metabolism , RNA, Double-Stranded/metabolism , RNA-Binding Proteins , Rats , Receptors, AMPA/genetics , Sequence Alignment
4.
J Biomol NMR ; 46(1): 51-65, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19690964

ABSTRACT

In the last 15 years substantial advances have been made to place isotope labels in native and glycosylated proteins for NMR studies and structure determination. Key developments include segmental isotope labeling using Native Chemical Ligation, Expressed Protein Ligation and Protein Trans-Splicing. These advances are pushing the size limit of NMR spectroscopy further making larger proteins accessible for this technique. It is just emerging that segmental isotope labeling can be used to define inter-domain interactions in NMR structure determination. Labeling of post-translational modified proteins like glycoproteins remains difficult but some promising developments were recently achieved. Key achievements are segmental and site-specific labeling schemes that improve resonance assignment and structure determination of the glycan moiety. We adjusted the focus of this perspective article to concentrate on the NMR applications based on recent developments rather than on labeling methods themselves to illustrate the considerable potential for biomolecular NMR.


Subject(s)
Glycoproteins/chemistry , Isotope Labeling/methods , Nuclear Magnetic Resonance, Biomolecular/methods , Protein Conformation , Proteins/chemistry , Glycoproteins/metabolism , Glycosylation , Protein Processing, Post-Translational , Proteins/metabolism
5.
J Mol Biol ; 375(1): 151-64, 2008 Jan 04.
Article in English | MEDLINE | ID: mdl-17936301

ABSTRACT

The study of multidomain or large proteins in solution by NMR spectroscopy has been made possible in recent years by the development of new spectroscopic methods. However, resonance overlap found in large proteins remains a limiting factor, making resonance assignments and structure determination of large proteins very difficult. In this study, we present an expressed protein ligation protocol that can be used for the segmental isotopic labeling of virtually any multidomain or high molecular mass protein, independent of both the folding state and the solubility of the protein fragments, as well as independent of whether the fragments are interacting. The protocol was applied successfully to two different multidomain proteins containing RNA recognition motifs (RRMs), heterogeneous nuclear ribonucleoprotein L and Npl3p. High yields of segmentally labeled proteins could be obtained, allowing characterization of the interdomain interactions with NMR spectroscopy. We found that the RRMs of heterogeneous nuclear ribonucleoprotein L interact, whereas those of Npl3p are independent. Subsequently, the structures of the two RRMs of Npl3p were determined on the basis of samples in which each RRM was expressed individually. The two Npl3p RRMs adopt the expected beta alpha beta beta alpha beta fold.


Subject(s)
Amino Acid Motifs , Fungal Proteins/chemistry , Heterogeneous-Nuclear Ribonucleoprotein L/chemistry , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/chemistry , RNA-Binding Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Amino Acid Sequence , Amino Acid Substitution , Carbon Isotopes/metabolism , Cysteine/metabolism , Electrophoresis, Polyacrylamide Gel , Heterogeneous-Nuclear Ribonucleoprotein L/genetics , Heterogeneous-Nuclear Ribonucleoprotein L/isolation & purification , Heterogeneous-Nuclear Ribonucleoprotein L/metabolism , Humans , Hydrogen-Ion Concentration , Isotope Labeling/methods , Models, Chemical , Models, Molecular , Molecular Sequence Data , Molecular Weight , Nitrogen Isotopes/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA-Binding Proteins/genetics , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/metabolism , Static Electricity , Temperature
6.
EMBO Rep ; 8(4): 372-9, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17318228

ABSTRACT

The RBMY (RNA-binding motif gene on Y chromosome) protein encoded by the human Y chromosome is important for normal sperm development. Although its precise molecular RNA targets are unknown at present, it is suggested that human RBMY (hRBMY) participates in splicing in the testis. Using systematic evolution of ligands by exponential enrichment, we found that RNA stem-loops capped by a C(A)/(U)CAA pentaloop are high-affinity binding targets for hRBMY. Subsequent nuclear magnetic resonance structural determination of the hRBMY RNA recognition motif (RRM) in complex with a high-affinity target showed two distinct modes of RNA recognition. First, the RRM beta-sheet surface binds to the RNA loop in a sequence-specific fashion. Second, the beta2-beta3 loop of the hRBMY inserts into the major groove of the RNA stem. The first binding mode might be conserved in the paralogous protein heterogeneous nuclear RNP G, whereas the second mode of binding is found only in hRBMY. This structural difference could be at the origin of the function of RBMY in spermatogenesis.


Subject(s)
Nuclear Proteins/chemistry , RNA-Binding Proteins/chemistry , RNA/chemistry , Testis/metabolism , Amino Acid Motifs , Amino Acid Sequence , Directed Molecular Evolution , Electrophoretic Mobility Shift Assay , Humans , Male , Molecular Sequence Data , Mutagenesis , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Structure, Secondary , RNA/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , SELEX Aptamer Technique , Sequence Analysis, Protein
7.
EMBO J ; 25(21): 5126-37, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-17036044

ABSTRACT

The sequence-specific RNA-binding proteins SRp20 and 9G8 are the smallest members of the serine- and arginine-rich (SR) protein family, well known for their role in splicing. They also play a role in mRNA export, in particular of histone mRNAs. We present the solution structures of the free 9G8 and SRp20 RNA recognition motifs (RRMs) and of SRp20 RRM in complex with the RNA sequence 5'CAUC3'. The SRp20-RNA structure reveals that although all 4 nt are contacted by the RRM, only the 5' cytosine is primarily recognized in a specific way. This might explain the numerous consensus sequences found by SELEX (systematic evolution of ligands by exponential enrichment) for the RRM of 9G8 and SRp20. Furthermore, we identify a short arginine-rich peptide adjacent to the SRp20 and 9G8 RRMs, which does not contact RNA but is necessary and sufficient for interaction with the export factor Tip-associated protein (TAP). Together, these results provide a molecular description for mRNA and TAP recognition by SRp20 and 9G8.


Subject(s)
Cell Nucleus/metabolism , Models, Molecular , Nucleocytoplasmic Transport Proteins/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Active Transport, Cell Nucleus/physiology , Amino Acid Motifs , Humans , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins , Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/genetics , Peptides/chemistry , Peptides/genetics , Peptides/metabolism , Protein Binding/genetics , Protein Structure, Tertiary , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Serine-Arginine Splicing Factors
8.
Structure ; 14(2): 345-55, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16472753

ABSTRACT

Adenosine deaminases that act on RNA (ADARs) site-selectively modify adenosines to inosines within RNA transcripts, thereby recoding genomic information. How ADARs select specific adenosine moieties for deamination is poorly understood. Here, we report NMR structures of the two double-stranded RNA binding motifs (dsRBMs) of rat ADAR2 and an NMR chemical shift perturbation study of the interaction of the two dsRBMs with a 71 nucleotide RNA encoding the R/G site of the GluR-B. We have identified the protein and the RNA surfaces involved in complex formation, allowing us to present an NMR-based model of the complex. We have found that dsRBM1 recognizes a conserved pentaloop, whereas dsRBM2 recognizes two bulged bases adjacent to the editing site, demonstrating RNA structure-dependent recognition by the ADAR2 dsRBMs. In vitro mutagenesis studies with both the protein and the RNA further support our structural findings.


Subject(s)
Adenosine Deaminase/chemistry , Models, Molecular , RNA, Double-Stranded/chemistry , RNA-Binding Proteins/chemistry , Adenosine Deaminase/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Dimerization , Humans , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA Editing , RNA, Double-Stranded/metabolism , RNA-Binding Proteins/metabolism , Rats , Sequence Alignment
10.
EMBO Rep ; 6(1): 33-8, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15643449

ABSTRACT

At all stages of its life (from transcription to translation), an RNA transcript interacts with many different RNA-binding proteins. The composition of this supramolecular assembly, known as a ribonucleoprotein particle, is diverse and highly dynamic. RNA-binding proteins control the generation, maturation and lifespan of the RNA transcript and thus regulate and influence the cellular function of the encoded gene. Here, we review our current understanding of protein-RNA recognition mediated by the two most abundant RNA-binding domains (the RNA-recognition motif and the double-stranded RNA-binding motif) plus the zinc-finger motif, the most abundant nucleic-acid-binding domain. In addition, we discuss how not only the sequence but also the shape of the RNA are recognized by these three classes of RNA-binding protein.


Subject(s)
Nucleic Acid Conformation , RNA/chemistry , RNA/metabolism , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Animals , Base Sequence , Humans , RNA/genetics , Substrate Specificity , Zinc Fingers
11.
Genome ; 47(2): 316-24, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15060584

ABSTRACT

Telomere-binding proteins participate in forming a functional nucleoprotein structure at chromosome ends. Using a genomic approach, two Arabidopsis thaliana genes coding for candidate Myb-like telomere binding proteins were cloned and expressed in E. coli. Both proteins, termed AtTBP2 (accession Nos. T46051 (protein database) and GI:638639 (nucleotide database); 295 amino acids, 32 kDa, pI 9.53) and AtTBP3 (BAB08466, GI:9757879; 299 amino acids, 33 kDa, pI 9.88), contain a single Myb-like DNA-binding domain at the N-terminus, and a histone H1/H5-like DNA-binding domain in the middle of the protein sequence. Both proteins are expressed in various A. thaliana tissues. Using the two-hybrid system interaction between the proteins AtTBP2 and AtTBP3 and self interactions of each of the proteins were detected. Gel-retardation assays revealed that each of the two proteins is able to bind the G-rich strand and double-stranded DNA of plant telomeric sequence with an affinity proportional to a number of telomeric repeats. Substrates bearing a non-telomeric DNA sequence positioned between two telomeric repeats were bound with an efficiency depending on the length of interrupting sequence. The ability to bind variant telomere sequences decreased with sequence divergence from the A. thaliana telomeric DNA. None of the proteins alone or their mixture affects telomerase activity in vitro. Correspondingly, no interaction was observed between any of two proteins and the Arabidopsis telomerase reverse transcriptase catalytic subunit TERT (accession No. AF172097) using two-hybrid assay.


Subject(s)
Arabidopsis Proteins/metabolism , Plant Proteins/metabolism , Proto-Oncogene Proteins c-myb/metabolism , Telomere-Binding Proteins/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Base Sequence , Binding, Competitive , Cloning, Molecular , Electrophoretic Mobility Shift Assay , Gene Expression , Molecular Sequence Data , Plant Proteins/genetics , Protein Binding , Protein Interaction Mapping , Protein Structure, Tertiary , Proto-Oncogene Proteins c-myb/genetics , Telomerase/metabolism , Telomere/genetics , Telomere/metabolism , Telomere-Binding Proteins/genetics , Two-Hybrid System Techniques
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