Characterization of the multimeric structure of poly(A)-binding protein on a poly(A) tail

Abstract

Eukaryotic mature mRNAs possess a poly adenylate tail (poly(A)), to which multiple molecules of poly(A)-binding protein C1 (PABPC1) bind. PABPC1 regulates translation and mRNA metabolism by binding to regulatory proteins. To understand functional mechanism of the regulatory proteins, it is necessary to reveal how multiple molecules of PABPC1 exist on poly(A). Here, we characterize the structure of the multiple molecules of PABPC1 on poly(A), by using transmission electron microscopy (TEM), chemical cross-linking, and NMR spectroscopy. The TEM images and chemical cross-linking results indicate that multiple PABPC1 molecules form a wormlike structure in the PABPC1-poly(A) complex, in which the PABPC1 molecules are linearly arrayed. NMR and cross-linking analyses indicate that PABPC1 forms a multimer by binding to the neighbouring PABPC1 molecules via interactions between the RNA recognition motif (RRM) 2 in one molecule and the middle portion of the linker region of another molecule. A PABPC1 mutant lacking the interaction site in the linker, which possesses an impaired ability to form the multimer, reduced the in vitro translation activity, suggesting the importance of PABPC1 multimer formation in the translation process. We therefore propose a model of the PABPC1 multimer that provides clues to comprehensively understand the regulation mechanism of mRNA translation.

Molecular Basis for poly(A) RNP Architecture and Recognition by the Pan2-Pan3 Deadenylase

“…Also accessible are the other known protein-protein interaction surfaces in the Pan2cat-Pan3-90A RNP assembly, namely the Pan3-binding site for the microRNA regulatory cofactor TNRC6/GW182 ( Christie et al., 2013 ) and the Pab1-binding site for the translation initiation factor eIF4G ( Safaee et al., 2012 ). In general, the zigzagging arrangement of the poly(A) RNP that we observe in the cryo-EM reconstruction is consistent with the RNA-binding footprint of the poly(A)-binding protein ( Webster et al., 2018 ), with the hinges between RRM domains that had been inferred by single-molecule studies ( Lee et al., 2014 ) and with the overall worm-like appearance in negative-stain studies ( Sawazaki et al., 2018 ).

Figure 5 The Pan2 WD40 Domain Senses the Length of the poly(A) RNP (A) Views of the interaction between the Pan2 WD40 domain and the second RNP oligomerization interface (i.e., RRM4-linker helix of the second Pab1 protomer in light green and the RRM1-RRM2 module of the third protomer in green).

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Influenza A virus NS1 protein binds as a dimer to the RNA-free PABP1 but not to the PABP1•Poly(A) RNA Complex

“…short such that only one PABP1 molecule can directly bind to it, and the second PABP1 molecule binds by weaker protein-protein interaction. 14,34,35 This interpretation is consistent with the fact that PABP1 monomer binds with a high affinity to poly(A) RNA, whereas PABP1 dimers are formed only at much higher concentrations. 36,37 After establishing the EMSA to monitor the binding of PABP1 to poly(A)18, we analyzed the effect of NS1 on the PABP1•poly(A)18 complex.…”

Leukemic presentation of ALK-positive anaplastic large cell lymphoma with a novel partner, poly(A) binding protein cytoplasmic 1 (PABPC1), responding to single-agent crizotinib

“…The poly(A)-bound PABPC1 forms multimers via mutual intermolecular interactions, which reportedly involve the linker region. 10,11 The PABPC1-ALK fusion encodes a 1001-amino-acid chimeric protein with a predicted molecular mass of 112 kDa. In the PABPC1-ALK fusion reported herein, the 5' partner of the fusion protein retained the N-terminus and the portion of the linker region of PABPC1 that has been shown to play a role in the PABPC1 multimers on poly(A) ( Figure 2C).…”