The binding of TIA-1 to RNA C-rich sequences is driven by its C-terminal RRM domain.
T-cell intracellular antigen-1 (TIA-1) is a key DNA/RNA binding protein that regulates translation by sequestering target mRNAs in stress granules (SG) in response to stress conditions. TIA-1 possesses three RNA recognition motifs (RRM) along with a glutamine-rich domain, with the central domains (RRM2 and RRM3) acting as RNA binding platforms. While the RRM2 domain, which displays high affinity for U-rich RNA sequences, is primarily responsible for interaction with RNA, the contribution of RRM3 to bind RNA as well as the target RNA sequences that it binds preferentially are still unknown. Here we combined nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) techniques to elucidate the sequence specificity of TIA-1 RRM3. With a novel approach using saturation transfer difference NMR (STD-NMR) to quantify protein-nucleic acids interactions, we demonstrate that isolated RRM3 binds to both C- and U-rich stretches with micromolar affinity. In combination with RRM2 and in the context of full-length TIA-1, RRM3 significantly enhanced the binding to RNA, particularly to cytosine-rich RNA oligos, as assessed by biotinylated RNA pull-down analysis. Our findings provide new insight into the role of RRM3 in regulating TIA-1 binding to C-rich stretches, that are abundant at the 5' TOPs (5' terminal oligopyrimidine tracts) of mRNAs whose translation is repressed under stress situations.
Instituto de Bioquímica Vegetal y Fotosíntesis; Centro de Investigaciones Científicas Isla de la Cartuja; Universidad de Sevilla-CSIC; Sevilla, Spain.
Isabel
I
Cruz-Gallardo
Instituto de Bioquímica Vegetal y Fotosíntesis; Centro de Investigaciones Científicas Isla de la Cartuja; Universidad de Sevilla-CSIC; Sevilla, Spain.
Ángeles
Á
Aroca
Department of Biochemistry and Molecular Biology; Monash University; Clayton, Victoria, Australia.
Menachem J
MJ
Gunzburg
Department of Biochemistry and Molecular Biology; Monash University; Clayton, Victoria, Australia.
Andrew
A
Sivakumaran
Laboratory of Genetics; National Institute on Aging-Intramural Research Program; NIH; Baltimore, MD USA.
Je-Hyun
JH
Yoon
Instituto de Investigaciones Químicas; Centro de Investigaciones Científicas Isla de la Cartuja; Universidad de Sevilla-CSIC; Sevilla, Spain; School of Pharmacy; University of East Anglia; Norwich Research Park; Norwich, UK.
Jesús
J
Angulo
Swedish NMR Centre; University of Gothenburg; Gothenburg, Sweden.
Cecilia
C
Persson
Laboratory of Genetics; National Institute on Aging-Intramural Research Program; NIH; Baltimore, MD USA.
Myriam
M
Gorospe
Swedish NMR Centre; University of Gothenburg; Gothenburg, Sweden.
B Göran
BG
Karlsson
Department of Biochemistry and Molecular Biology; Monash University; Clayton, Victoria, Australia.
Jacqueline A
JA
Wilce
Instituto de Bioquímica Vegetal y Fotosíntesis; Centro de Investigaciones Científicas Isla de la Cartuja; Universidad de Sevilla-CSIC; Sevilla, Spain.
Irene
I
Díaz-Moreno
D001483:Base Sequence; D001665:Binding Sites; D020862:GC Rich Sequence; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D059372:Nucleotide Motifs; D039102:Poly(A)-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D056510:Position-Specific Scoring Matrices; D054730:Protein Interaction Domains and Motifs; D012313:RNA / Q000737:chemistry* / Q000235:genetics*
D039102:Poly(A)-Binding Proteins; D012313:RNA
DNA/RNA binding protein (D/RBP); RNA recognition motifs (RRM); TIA-1; saturation transfer difference-NMR (STD-NMR); scaffold independent analysis (SIA); surface plasmon resonance (SPR)