Crystal structure of the two N-terminal RRM domains of Pub1 and the poly(U)-binding properties of Pub1.
Yeast poly(U)-binding protein (Pub1) is a major nuclear and cytoplasmic protein that contains three RNA recognition motif (RRM) domains (termed Pub1RRM1, Pub1RRM2 and Pub1RRM3). Pub1 has been implicated as a regulator of cellular mRNA decay. Nearly 10% of all yeast mRNA decay occurs in a Pub1-dependent manner. Pub1 binds to and stabilizes AU-rich element (ARE) and ARE-like sequence-containing transcripts by protecting them from degradation through the deadenylation-dependent pathway, and also binds to and stabilizes stabilizer element (STE)-containing transcripts by preventing their degradation via the nonsense-mediated decay (NMD) pathway. RNA-binding analyses showed that Pub1 binds to poly(U) in vitro. Here we show the crystal structures of Pub1RRM2 and the first two tandem RRM domains (Pub1RRM12). Crystallography showed that the structure of Pub1RRM12 is a domain-swapped dimer. Size exclusion chromatography assay and analytical ultracentrifugation (AUC) showed that Pub1RRM12 is a monomer in solution. Kinetic analysis showed that all three individual RRM domains can bind to poly(U) with similar affinities and Pub1RRM12 binds to a long poly(U) segment with higher affinity. Mutagenesis analysis revealed that residues on the beta-sheets of Pub1RRM1 and Pub1RRM2 are critical for poly(U) binding.
Li
Heng
H
Hefei National Laboratory for Physical Sciences at Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China.
Shi
Hui
H
Wang
Hong
H
Zhu
Zhiqiang
Z
Li
Xu
X
Gao
Yongxiang
Y
Cui
Yingji
Y
Niu
Liwen
L
Teng
Maikun
M
D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray / Q000379:methods*; D039102:Poly(A)-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014461:Ultracentrifugation
C083327:PUB1 protein, S cerevisiae; D039102:Poly(A)-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins