Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
RNA Biol ; 10(4): 579-89, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23603827

ABSTRACT

The RNA-binding protein TIAR is an mRNA-binding protein that acts as a translational repressor, particularly important under conditions of cellular stress. It binds to target mRNA and DNA via its RNA recognition motif (RRM) domains and is involved in both splicing regulation and translational repression via the formation of "stress granules." TIAR has also been shown to bind ssDNA and play a role in the regulation of transcription. Here we show, using surface plasmon resonance and nuclear magnetic resonance spectroscopy, specific roles of individual TIAR domains for high-affinity binding to RNA and DNA targets. We confirm that RRM2 of TIAR is the major RNA- and DNA-binding domain. However, the strong nanomolar affinity binding to U-rich RNA and T-rich DNA depends on the presence of the six amino acid residues found in the linker region C-terminal to RRM2. On its own, RRM1 shows preferred binding to DNA over RNA. We further characterize the interaction between RRM2 with the C-terminal extension and an AU-rich target RNA sequence using NMR spectroscopy to identify the amino acid residues involved in binding. We demonstrate that TIAR RRM2, together with its C-terminal extension, is the major contributor for the high-affinity (nM) interactions of TIAR with target RNA sequences.


Subject(s)
Amino Acid Motifs , DNA-Binding Proteins/metabolism , DNA/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Binding Sites , DNA/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Gene Expression Regulation , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Binding , RNA Splicing , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Sequence Alignment , Surface Plasmon Resonance
2.
Nucleic Acids Res ; 40(11): 5101-14, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22344691

ABSTRACT

Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.


Subject(s)
Cytosine/chemistry , Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Oligonucleotides/chemistry , Binding Sites , DNA/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Models, Molecular , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/chemistry , RNA, Messenger/metabolism
3.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 67(Pt 10): 1257-61, 2011 Oct 01.
Article in English | MEDLINE | ID: mdl-22102042

ABSTRACT

Polycytosine-binding proteins (PCBPs) are triple KH-domain proteins that play an important role in the regulation of translation of eukaryotic mRNA. They are also utilized by viral RNA and have been shown to interact with ssDNA. Underlying their function is the specific recognition of C-rich nucleotides by their KH domains. However, the structural basis of this recognition is only partially understood. Here, the preparation of a His-tagged KH domain is described, representing the first domain of PCBP1 that incorporates a C54S mutation as well as the addition of a C-terminal tryptophan. This construct has facilitated the preparation of highly diffracting crystals in complex with C-rich DNA (sequence ACCCCA). Crystals of the KH1-DNA complex were grown using the hanging-drop vapour-diffusion method in 0.1 M phosphate-citrate pH 4.2, 40%(v/v) PEG 300. X-ray diffraction data were collected to 1.77 Å resolution and the diffraction was consistent with space group P2(1), with unit-cell parameters a = 38.59, b = 111.88, c = 43.42 Å, α = γ = 90.0, ß = 93.37°. The structure of the KH1-DNA complex will further our insight into the basis of cytosine specificity by PCBPs.


Subject(s)
DNA/chemistry , Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Mutation , Crystallization , Crystallography, X-Ray , DNA/metabolism , DNA-Binding Proteins , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Humans , Models, Molecular , Protein Structure, Tertiary , RNA-Binding Proteins
4.
Nucleic Acids Res ; 39(3): 1117-30, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21233170

ABSTRACT

TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U-rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2'-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.


Subject(s)
Antigens, Surface/chemistry , DNA/chemistry , RNA-Binding Proteins/chemistry , RNA/chemistry , Adenine/analysis , Antigens, Surface/metabolism , DNA/metabolism , ELAV Proteins , ELAV-Like Protein 1 , Kinetics , Models, Molecular , Protein Binding , Protein Conformation , RNA/metabolism , RNA-Binding Proteins/metabolism , Scattering, Small Angle , Uracil/analysis , X-Ray Diffraction
SELECTION OF CITATIONS
SEARCH DETAIL
...