1 PABP-interacting proteins, PAIP1 and PAIP2, affect translation termination Alexandr Ivanov 1,2 , Ekaterina Shuvalova 1 , Tatiana Egorova 1 , Alexey Shuvalov 1 , Elizaveta Sokolova 1 , Nikita Bizyaev 1 , Ivan Shatsky 3 , Ilya Terenin 3,4,* and Elena Alkalaeva 1,* 1 Engelhardt Institute of Molecular Biology, the Russian Academy of Sciences, Moscow, Russia 2 Faculty of Bioengineering and Bioinformatics, M.V. Lomonosov Moscow State University, Moscow, Russia 3 Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia 4 Sechenov First Moscow State Medical University, Institute of Molecular Medicine, Moscow, Russian Federation * Corresponding authors: Elena Alkalaeva Email: alkalaeva@eimb.ru; Tel: +7 4991359977; Fax: +7 4991351405; Ilya Terenin E-mail: terenin@genebee.msu.ru Tel: +74959394857; Fax: +74959390338 Running title: PAIPs affect translation termination Keywords: PAIP1, PAIP2, PABP, eRF3, translation termination, stop codon readthrough, ribosome ABSTRACT Polyadenylate-binding protein (PABP) stimulates translation termination via interaction of its C- terminal domain with eukaryotic polypeptide chain release factor, eRF3. Additionally, two other proteins, poly(A)-binding protein-interacting protein 1 and 2 (PAIP1 and PAIP2), bind the same domain of PABP and regulate its translation-related activity. To study the biochemistry of eRF3 and PAIP1/2 competition for PABP binding, we quantified the effects of PAIPs on translation termination in the presence or absence of PABP. Our results demonstrated that both PAIP1 and PAIP2 prevented translation termination at premature termination codon (PTC), by controlling PABP activity. Moreover, PAIP1 and PAIP2 inhibited the activity of free PABP on translation termination in vitro. However, after binding the poly(A) tail, PABP became insensitive to suppression by PAIPs and efficiently activated translation termination in the presence of eRF3a. Additionally, we revealed that PAIP1 binds eRF3 in solution, which stabilizes the post-termination complex (postTC). These results indicated that PAIP1 and PAIP2 participate in translation termination and are important regulators of readthrough at PTC. INTRODUCTION PABP a multifunctional protein involved in RNA metabolism is a major mRNA-interacting protein in eukaryotic cells. The N-terminal half of the protein contains four RNA-recognition motifs (RRMs) (Fig. 1A), and two of these (RRM1/2) specifically bind 12 adenines on mRNA poly(A) tails, whereas the others (RRM3/4), non- specifically bind any RNA (1). Each RRM has two surfaces, with one capable of binding RNA and the other capable of interacting with the PABP- associated motif (PAM) 1 region of several proteins. The C-terminal domain of PABP (CTC), which binds the PAM2 motif of specific proteins, is joined with the N-terminal portion of PABP by an unstructured linker (Fig. 1A) (2). In animal cells, РАВР is controlled by the PABP-interacting proteins, PAIP1 and PAIP2. PAIP1 contains PAM1 and PAM2 motifs, as well as a domain homologous to the middle eIF4G domain (MIF4G) (Fig. 1A) (3). PAM1 binds the RRM1/2 motifs of PABP with high affinity, whereas PAM2 binds the CTC domain of PABP with low affinity (4). PAIP2 contains only PAM1 and PAM2 domains and binds PABP exclusively (Fig. 1A) (5, 6). Similar to PAIP1, the РАМ1 motif of PAIP2 efficiently binds the RRM2/3 of PABP, while the PAM2 motif binds the CTC domain of PABP with ~100-fold less affinity (5). РАВР is most known for protecting mRNA from degradation [reviewed in (7)], and РАВР regulates translation initiation. It binds the N- terminal region of eukaryotic initiation factor 4G (eIF4G), which mutually potentiates the activities of each protein (8). The interaction enhances both PABP binding to the poly(A) tail of mRNA and eIF4G interaction with the cap-binding initiation http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.006856 The latest version is at JBC Papers in Press. Published on April 16, 2019 as Manuscript RA118.006856 by guest on April 26, 2019 http://www.jbc.org/ Downloaded from