The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain

Cruz-Gallardo, Isabel; Aroca, Ángeles; Gunzburg, Menachem J.; Sivakumaran, Andrew; Yoon, Je‐Hyun; Angulo, Jesús; Persson, Cecilia; Gorospe, Myriam; Karlsson, Bengt; Wilce, Jacqueline A.; Dı́az-Moreno, Irene

doi:10.4161/rna.28801

Cited by 20 publications

(26 citation statements)

References 53 publications

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“…In particular, a pentameric C-rich consensus motif was established for TIA-1 RRM3 binding (26). We therefore set out to characterize the way in which these two adjacent RRMs interact with target RNAs comprised of both U-rich and C-rich regions.…”

Section: Resultsmentioning

confidence: 99%

TIA-1 RRM23 binding and recognition of target oligonucleotides

Waris

García‐Mauriño

Sivakumaran

et al. 2017

Nucleic Acids Research

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TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression.

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Section: Resultsmentioning

confidence: 99%

TIA-1 RRM23 binding and recognition of target oligonucleotides

Waris

García‐Mauriño

Sivakumaran

et al. 2017

Nucleic Acids Research

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“…[45] Unlabeled and 15 Nl abeled RRM2 were expressed in Escherichia coli BL21(D3) cells as His-tagged proteins and they were purified by nickel affinity chromatography (Ni Sepharose 6F ast Flow,G EH ealthcare) according to earlier protocols. [2,30] The His-tag was cleaved from the proteins using TEV protease (AcTEV Protease, Life Te chnologies) and removed by as econd purification step of nickel affinityc hromatography. [30] Protein samples were concentrated to 0.3 mm in 20 mm potassium phosphate buffer with 50 mm KCl (pH 7.5).…”

Section: Experimental Section Sample Preparationmentioning

confidence: 99%

“…[2,30] The His-tag was cleaved from the proteins using TEV protease (AcTEV Protease, Life Te chnologies) and removed by as econd purification step of nickel affinityc hromatography. [30] Protein samples were concentrated to 0.3 mm in 20 mm potassium phosphate buffer with 50 mm KCl (pH 7.5). RNA and DNA oligonucleotides were chemically synthesized (IDT,I ntegrated DNA Te chnologies;1m mol synthesis) and solubilized in 20 mm potassium phosphate buffer with 50 mm KCl (pH 7.0) to final concentrations in the range of 2-7.4 mm.B oth protein and nucleic acid samples were resuspended in the same buffer to avoid changes of the pK a values derived from differences in the ionic strength.…”

Section: Experimental Section Sample Preparationmentioning

confidence: 99%

“…RRM2 drives the interaction with RNA showing the highest affinities by pyrimidinerich sequences in comparison with RRM3, [27][28][29] but the latter enhances the overall TIA-1 binding affinity by RNA, especially with C-rich motifs. [30] The RRM1 domain does not contribute significantly to RNA binding, [28,29] but it recognizes T-rich DNA sequences along with RRM2 in the TIAR protein. [31,32] The PRD, in turn, promotes the aggregation of TIA-1 and mediates the SG assembly.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Non‐Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH‐Modulation of Protein–Nucleic Acid Interfaces

Cruz-Gallardo

Conte

Velázquez‐Campoy

et al. 2015

Chemistry A European J

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A useful (2) J(N-H) coupling-based NMR spectroscopic approach is proposed to unveil, at the molecular level, the contribution of the imidazole groups of histidines from RNA/DNA-binding proteins on the modulation of binding to nucleic acids by pH. Such protonation/deprotonation events have been monitored on the single His96 located at the second RNA/DNA recognition motif (RRM2) of T-cell intracellular antigen-1 (TIA-1) protein. The pKa values of the His96 ionizable groups were substantially higher in the complexes with short U-rich RNA and T-rich DNA oligonucleotides than those of the isolated TIA-1 RRM2. Herein, the methodology applied to determine changes in pKa of histidine side chains upon DNA/RNA binding, gives valuable information to understand the pH effect on multidomain DNA/RNA-binding proteins that shuttle among different cellular compartments.

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“…[25][26][27] In this process, the recognition motif RRM2 in the TIA protein, facilitated by RRM3, attaches to the uridinerich regions proximal to the intron's 5 0 splice site, and the Qrich domain binds to the N-terminal region of the subunit U1-C in U1 snRNP through a process facilitated by RRM1. 11,15,[27][28][29] There are also some studies that point to the participation of TIA proteins in the recruitment of U6 snRNP. 30,31 The cellular relevance of TIA proteins in the regulation of constitutive and alternative splicing was confirmed by mapping the binding of these regulators to RNA using an experimental approach in HeLa cells consisting of in vivo irradiation of the cells with UV light and subsequent immunoprecipitation of the RNA-protein complexes.…”

Section: Regulators And/or Modulators Of Gene Expressionmentioning

confidence: 99%