scholarly journals Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Janine Weber ◽  
Han Bao ◽  
Christoph Hartlmüller ◽  
Zhiqin Wang ◽  
Almut Windhager ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Point Mutations ◽  
Structural Basis ◽  
Binding Modes ◽  
Nucleic Acid Binding

The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

scholarly journals Crystal structure and functional characterization of the human RBM25 PWI domain and its flanking basic region

2013 ◽  
Vol 450 (1) ◽  
pp. 85-94 ◽  
Author(s):  
Deshun Gong ◽  
Fan Yang ◽  
Fudong Li ◽  
Dandan Qian ◽  
Minhao Wu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Alternative Splicing ◽  
Nucleic Acid ◽  
Rna Binding ◽  
Functional Characterization ◽  
Binding Motif ◽  
Nucleic Acid Binding ◽  
Binding Surface ◽  
Basic Region

Human RBM25 (RNA-binding motif protein 25) is a novel splicing factor that contains a PWI domain, a newly identified RNA/DNA-binding domain, and regulates Bcl-x pre-mRNA alternative splicing. The flanking basic region has been suggested to serve as a co-operative partner of the PWI domain in the binding of nucleic acids, but the structure of this basic region is unknown. In the present paper, we report the crystal structure of the RBM25 PWI domain and its flanking basic region. The PWI domain is revealed to comprise a conserved four-helix bundle, and the flanking basic region forms two α-helices and associates with helix H4 of the PWI domain. These interactions promote directly the formation of an enlarged nucleic-acid-binding platform. Structure-guided mutagenesis reveals a positively charged nucleic-acid-binding surface in the RBM25 PWI domain that is entirely different from that in the SRm160 PWI domain. Furthermore, we show that the promotion of the pro-apoptotic Bcl-xS isoform expression by RBM25 is facilitated by the PWI domain in vivo. Thus the present study suggests that the PWI domain plays an important role in the regulation of Bcl-x pre-mRNA alternative splicing.

scholarly journals Structural basis of nucleic acid binding byNicotiana tabacumglycine-rich RNA-binding protein: implications for its RNA chaperone function

2014 ◽  
Vol 42 (13) ◽  
pp. 8705-8718 ◽  
Author(s):  
Fariha Khan ◽  
Mark A. Daniëls ◽  
Gert E. Folkers ◽  
Rolf Boelens ◽  
S. M. Saqlan Naqvi ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Structural Basis ◽  
Nucleic Acid Binding ◽  
Rna Chaperone ◽  
Chaperone Function

scholarly journals Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 872 ◽  
Author(s):  
Clemens Grimm ◽  
Jann-Patrick Pelz ◽  
Cornelius Schneider ◽  
Katrin Schäffler ◽  
Utz Fischer
Keyword(s):  
Crystal Structure ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Turnover Rates ◽  
Mrna Transcription ◽  
Terminal Part ◽  
Protein Output

Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains.

scholarly journals Challenges and perspectives for structural biology of lncRNAs—the example of the Xist lncRNA A-repeats

2019 ◽  
Vol 11 (10) ◽  
pp. 845-859 ◽  
Author(s):  
Alisha N Jones ◽  
Michael Sattler
Keyword(s):  
High Resolution ◽  
Structural Biology ◽  
Recent Progress ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Features ◽  
Molecular Features

Abstract Following the discovery of numerous long non-coding RNA (lncRNA) transcripts in the human genome, their important roles in biology and human disease are emerging. Recent progress in experimental methods has enabled the identification of structural features of lncRNAs. However, determining high-resolution structures is challenging as lncRNAs are expected to be dynamic and adopt multiple conformations, which may be modulated by interaction with protein binding partners. The X-inactive specific transcript (Xist) is necessary for X inactivation during dosage compensation in female placental mammals and one of the best-studied lncRNAs. Recent progress has provided new insights into the domain organization, molecular features, and RNA binding proteins that interact with distinct regions of Xist. The A-repeats located at the 5′ end of the transcript are of particular interest as they are essential for mediating silencing of the inactive X chromosome. Here, we discuss recent progress with elucidating structural features of the Xist lncRNA, focusing on the A-repeats. We discuss the experimental and computational approaches employed that have led to distinct structural models, likely reflecting the intrinsic dynamics of this RNA. The presence of multiple dynamic conformations may also play an important role in the formation of the associated RNPs, thus influencing the molecular mechanism underlying the biological function of the Xist A-repeats. We propose that integrative approaches that combine biochemical experiments and high-resolution structural biology in vitro with chemical probing and functional studies in vivo are required to unravel the molecular mechanisms of lncRNAs.

scholarly journals Structural analysis reveals features of the spindle checkpoint kinase Bub1–kinetochore subunit Knl1 interaction

2012 ◽  
Vol 196 (4) ◽  
pp. 451-467 ◽  
Author(s):  
Veronica Krenn ◽  
Annemarie Wehenkel ◽  
Xiaozheng Li ◽  
Stefano Santaguida ◽  
Andrea Musacchio
Keyword(s):  
Crystal Structure ◽  
Structural Analysis ◽  
Convex Surface ◽  
Point Mutations ◽  
Tetratricopeptide Repeats ◽  
Checkpoint Protein ◽  
Checkpoint Kinases ◽  
Shed Light

The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR–KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1.

scholarly journals Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins

1987 ◽  
Vol 7 (8) ◽  
pp. 2947-2955
Author(s):  
A Y Jong ◽  
M W Clark ◽  
M Gilbert ◽  
A Oehm ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Nucleic Acid ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nucleic Acid Binding ◽  
Dna And Rna ◽  
C Terminus

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

Benzo(a)pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo

Science ◽  
1976 ◽  
Vol 193 (4253) ◽  
pp. 592-595 ◽  
Author(s):  
I. Weinstein ◽  
A. Jeffrey ◽  
K. Jennette ◽  
S. Blobstein ◽  
R. Harvey ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Binding ◽  
Diol Epoxides

scholarly journals The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization.

1993 ◽  
Vol 4 (11) ◽  
pp. 1189-1204 ◽  
Author(s):  
M A Heine ◽  
M L Rankin ◽  
P J DiMario
Keyword(s):  
Nucleic Acid ◽  
Binding Activity ◽  
Full Length ◽  
Nucleic Acid Binding ◽  
Binding Assays ◽  
Nucleic Acid Probes ◽  
E Coli ◽  
Indirect Immunofluorescence Staining

Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain. Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes. Whether produced in E. coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity. Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei. The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli. Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.

Nucleic-acid-binding properties of hnRNP-U/SAF-A, a nuclear-matrix protein which binds DNA and RNA in vivo and in vitro

1994 ◽  
Vol 221 (2) ◽  
pp. 749-757 ◽  
Author(s):  
Frank O. FACKELMAYER ◽  
Kirsten DAHM ◽  
Andrea RENZ ◽  
Uwe RAMSPERGER ◽  
Arndt RICHTER
Keyword(s):  
Nucleic Acid ◽  
Nuclear Matrix ◽  
Matrix Protein ◽  
Nucleic Acid Binding ◽  
Nuclear Matrix Protein ◽  
Binding Properties ◽  
Dna And Rna ◽  
Hnrnp U

scholarly journals Structural basis for multifunctional roles of human Ints3 C-terminal domain

2020 ◽  
pp. jbc.RA120.016393
Author(s):  
Jian Li ◽  
Xinli Ma ◽  
Surajit Banerjee ◽  
Sankar Baruah ◽  
Nicholas J Schnicker ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Genome Stability ◽  
Double Strand Breaks ◽  
Structural Basis ◽  
Nucleic Acid Binding ◽  
Ataxia Telangiectasia Mutated ◽  
Interacting Protein ◽  
Strand Breaks ◽  
Terminal Domain

Proper repair of damaged DNA is critical for the maintenance of genome stability. A complex composed of Integrator subunit 3 (Ints3), single-stranded DNA-binding protein 1 (SSB1) and SSB-interacting protein 1 (SSBIP1) is required for efficient homologous recombination-dependent repair of double-strand breaks (DSBs) and ataxia-telangiectasia mutated (ATM)-dependent signaling pathways. It is known that in this complex the Ints3 N-terminal domain scaffolds SSB1 and SSBIP1. However, the molecular basis for the function of the Ints3 C-terminal domain remains unclear. Here, we present the crystal structure of the Ints3 C-terminal domain, uncovering a HEAT-repeat superhelical fold. Using structure and mutation analysis, we show that the C-terminal domain exists as a stable dimer. A basic groove and a cluster of conserved residues on two opposite sides of the dimer bind single-stranded RNA/DNA (ssRNA/ssDNA) and Integrator complex subunit 6 (Ints6), respectively. Dimerization is required for nucleic acid binding, but not for Ints6 binding. Additionally, in vitro experiments using HEK 293T cells demonstrate that Ints6 interaction is critical for maintaining SSB1 protein level. Taken together, our findings establish the structural basis of a multifunctional Ints3 C-terminal module, allowing us to propose a novel mode of nucleic acid recognition by helical repeat protein and paving the way for future mechanistic studies.

Sign in / Sign up

Export Citation Format

Share Document