Multimerization of Staufen1 in live cells.
Transport of mRNA is an efficient mechanism to target proteins to specific regions of a cell. Although it is well documented that mRNAs are transported in ribonucleoprotein (RNP) complexes, several of the mechanisms involved in complex formation and localization are poorly understood. Staufen (Stau) 1, a double-stranded RNA-binding protein, is a well accepted marker of mRNA transport complexes. In this manuscript, we provide evidence that Stau1 self-associates in live cells using immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays. The double-stranded RNA-binding domains dsRBD3 and dsRBD4 contributed about half of the signal, suggesting that Stau1 RNA-binding activity is involved in Stau1 self-association. Protein-protein interaction also occurred, via dsRBD5 and dsRBD2, as shown by in vitro pull-down, yeast two-hybrid, and BRET assays in live cells. Interestingly, Stau1 self-association contributes to the formation of oligomeric complexes as evidenced by the coexpression of split Renilla luciferase halves covalently linked to Stau1 in a protein complementation assay (PCA) combined with a BRET assay with Stau1-YFP. Moreover, we showed that these higher-order Stau1-containing complexes carry RNAs when the RNA stain SYTO 14 was used as the energy acceptor in the PCA/BRET assay. The oligomeric composition of Stau1-containing complexes and the presence of specific mRNAs have been confirmed by biochemical approaches involving two successive immunoprecipitations of Stau1-tagged molecules followed by qRT-PCR amplification. Altogether, these results indicate that Stau1 self-associates in mRNPs via its multiple functional domains that can select mRNAs to be transported and establish protein-protein interaction.
Martel
Catherine
C
Département de Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
Dugré-Brisson
Samuel
S
Boulay
Karine
K
Breton
Billy
B
Lapointe
Gabriel
G
Armando
Sylvain
S
Trépanier
Véronique
V
Duchaîne
Thomas
T
Bouvier
Michel
M
Desgroseillers
Luc
L
D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008163:Luminescent Measurements; D055503:Protein Multimerization; D034443:RNA Transport; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism
D003598:Cytoskeletal Proteins; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C492185:STAU1 protein, human; C026452:messenger ribonucleoprotein