Structure and function of poly(A) binding proteins

Poly (A) tails are found at the 3' ends of almost all eukaryotic mRNAs. They are bound by two different poly (A) binding proteins, PABPC in the cytoplasm and PABPN1 in the nucleus. PABPC functions in the initiation of translation and in the regulation of mRNA decay. In both functions, an interaction with the m7G cap at the 5' end of the message plays an important role. PABPN1 is involved in the synthesis of poly (A) tails, increasing the processivity of poly (A) polymerase and contributing to defining the length of a newly synthesized poly (A) tail.     

Paip2A inhibits translation by competitively binding to the RNA recognition motifs of PABPC1 and promoting its dissociation from the poly(A) tail

Eukaryotic mRNAs possess a poly(A) tail at their 3′-end, to which poly(A)-binding protein C1 (PABPC1) binds and recruits other proteins that regulate translation. Enhanced poly(A)-dependent translation, which is also PABPC1 dependent, promotes cellular and viral proliferation. PABP-interacting protein 2A (Paip2A) effectively represses poly(A)-dependent translation by causing the dissociation of PABPC1 from the poly(A) tail; however, the underlying mechanism remains unknown. This study was conducted to investigate the functional mechanisms of Paip2A action by characterizing the PABPC1–poly(A) and PABPC1–Paip2A interactions. Isothermal titration calorimetry and NMR analyses indicated that both interactions predominantly occurred at the RNA recognition motif (RRM)2–RRM3 regions of PABPC1, which have comparable affinities for poly(A) and Paip2A (dissociation constant, K_d = 1 nM). However, the K_d values of isolated RRM2 were 200 and 4 μM in their interactions with poly(A) and Paip2A, respectively; K_d values of 5 and 1 μM were observed for the interactions of isolated RRM3 with poly(A) and Paip2A, respectively. NMR analyses also revealed that Paip2A can bind to the poly(A)-binding interfaces of the RRM2 and RRM3 regions of PABPC1. Based on these results, we propose the following functional mechanism for Paip2A: Paip2A initially binds to the RRM2 region of poly(A)-bound PABPC1, and RRM2-anchored Paip2A effectively displaces the RRM3 region from poly(A), resulting in dissociation of the whole PABPC1 molecule. Together, our findings provide insight into the translation repression effect of Paip2A and may aid in the development of novel anticancer and/or antiviral drugs.     

Modifiers of solid RNP granules control normal RNP dynamics and mRNA activity in early development

Ribonucleoproteins (RNPs) often coassemble into supramolecular bodies with regulated dynamics. The factors controlling RNP bodies and connections to RNA regulation are unclear. During Caenorhabditis elegans oogenesis, cytoplasmic RNPs can transition among diffuse, liquid, and solid states linked to mRNA regulation. Loss of CGH-1/Ddx6 RNA helicase generates solid granules that are sensitive to mRNA regulators. Here, we identified 66 modifiers of RNP solids induced by cgh-1 mutation. A majority of genes promote or suppress normal RNP body assembly, dynamics, or metabolism. Surprisingly, polyadenylation factors promote RNP coassembly in vivo, suggesting new functions of poly(A) tail regulation in RNP dynamics. Many genes carry polyglutatmine (polyQ) motifs or modulate polyQ aggregation, indicating possible connections with neurodegenerative disorders induced by CAG/polyQ expansion. Several RNP body regulators repress translation of mRNA subsets, suggesting that mRNAs are repressed by multiple mechanisms. Collectively, these findings suggest new pathways of RNP modification that control large-scale coassembly and mRNA activity during development.     

Poly(A)-binding proteins: Structure, domain organization, and activity regulation

RNA-binding proteins are of vital importance for mRNA functioning. Among these, poly(A)-binding proteins (PABPs) are of special interest due to their participation in virtually all mRNA-dependent events that is caused by their high affinity for A-rich mRNA sequences. Apart from mRNAs, PABPs interact with many proteins, thus promoting their involvement in cellular events. In the nucleus, PABPs play a role in polyadenylation, determine the length of the poly(A) tail, and may be involved in mRNA export. In the cytoplasm, they participate in regulation of translation initiation and either protect mRNAs from decay through binding to their poly(A) tails or stimulate this decay by promoting mRNA inter-actions with deadenylase complex proteins. This review presents modern notions of the role of PABPs in mRNA-dependent events; peculiarities of regulation of PABP amount in the cell and activities are also discussed.     

The “tale” of poly(A) binding protein: The MLLE domain and PAM2-containing proteins

The cytoplasmic poly(A) binding protein 1 (PABPC1) is an essential eukaryotic translational initiation factor first described over 40 years ago. Most studies of PABPC1 have focused on its N-terminal RRM domains, which bind the mRNA 3′ poly(A) tail and 5′ translation complex eIF4F via eIF4G; however, the protein also contains a C-terminal MLLE domain that binds a peptide motif, termed PAM2, found in many proteins involved in translation regulation and mRNA metabolism. Studies over the past decade have revealed additional functions of PAM2-containing proteins (PACs) in neurodegenerative diseases, circadian rhythms, innate defense, and ubiquitin-mediated protein degradation. Here, we summarize functional and structural studies of the MLLE/PAM2 interaction and discuss the diverse roles of PACs.     

hnRNPL and nucleolin bind LINE-1 RNA and function as host factors to modulate retrotransposition

Long INterspersed Element one (LINE-1, or L1), is a widely distributed, autonomous retrotransposon in mammalian genomes. During retrotransposition, L1 RNA functions first as a dicistronic mRNA and then as a template for cDNA synthesis. Previously, we defined internal ribosome entry sequences (IRESs) upstream of both ORFs (ORF1 and ORF2) in the dicistronic mRNA encoded by mouse L1. Here, RNA affinity chromatography was used to isolate cellular proteins that bind these regions of L1 RNA. Four proteins, the heterogeneous nuclear ribonucleoproteins (hnRNPs) R, Q and L, and nucleolin (NCL), appeared to interact specifically with the ORF2 IRES. These were depleted from HeLa cells to examine their effects on L1 IRES-mediated translation and L1 retrotransposition. NCL knockdown specifically reduced the ORF2 IRES activity, L1 and L1-assisted Alu retrotransposition without altering L1 RNA or protein abundance. These findings are consistent with NCL acting as an IRES trans-acting factor (ITAF) for ORF2 translation and hence a positive host factor for L1 retrotransposition. In contrast, hnRNPL knockdown dramatically increased L1 retrotransposition as well as L1 RNA and ORF1 protein, indicating that this cellular protein normally interferes with retrotransposition. Thus, hnRNPL joins a small, but growing list of cellular proteins that are potent negative regulators of L1 retrotransposition.     

RNA结合蛋白PABPC1的功能及生物学作用

多聚腺苷酸结合蛋白(poly (A) binding protein,PABP)家族通常被认为是mRNA poly (A)尾的一种保护屏障.其中细胞质多聚腺苷酸结合蛋白1 (cytoplasmic poly (A) binding protein-1,PABPC1)在高亲和力作用下能够与mRNA中富含腺苷酸的序列结合,在基因转录后调控中发挥着重要作用.同时PABPC1还参与mRNA的许多代谢通路,包括腺苷酸多聚化/脱腺苷酸化、m RNA转运、m RNA翻译、降解及mircoRNA相关调控.近年来关于PABPC1与生殖细胞的发育、心肌肥大和肿瘤的发生发展的报道屡见不鲜,可见PABPC1与细胞的生长发育有密切联系.本文将主要介绍PABPC1的结构、表达调控、功能及其生物学作用.     

Mitochondrial Dysfunction Reveals the Role of mRNA Poly(A) Tail Regulation in Oculopharyngeal Muscular Dystrophy Pathogenesis

Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.     

Quantitative Characterization of Tob Interactions Provides the Thermodynamic Basis for Translation Termination-coupled Deadenylase Regulation

Translation termination-coupled deadenylation is the first and often the rate-limiting step of eukaryotic mRNA decay in which two deadenylases, Ccr4-Caf1 and Pan2, play key roles. One of the deadenylases, Caf1, associates with Tob, which recruits Caf1 to the poly(A) tail through interactions with a cytoplasmic poly(A)-binding protein 1 (PABPC1). We previously proposed that the competition between Tob and eRF3 (a translation termination factor that interacts with PABPC1) is responsible for the regulation of deadenylase activity. However, the molecular mechanism of the regulation should be addressed by investigating the binding affinity and the cellular levels of these proteins. In this work, we characterized the human Tob interactions with Caf1 and a C-terminal domain of PABPC1 (PABC). Nuclear magnetic resonance (NMR) and Western blot analyses revealed that Tob consists of a structured N-terminal BTG-Tob domain and an unstructured C-terminal region with two conserved PAM2 (PABPC1-interacting motif 2) motifs. The BTG-TOB domain associates with Caf1, whereas the C-terminal PAM2 motif binds to PABC, with a K_d value of 20 μm. Furthermore, we demonstrated that the levels of eRF3 and Tob in HeLa cells are 4–5 μm and less than 0.2 μm, respectively. On the basis of these results, we propose a thermodynamic mechanism for the translation termination-coupled deadenylation mediated by the Tob-Caf1 complex.