Structure and Mechanism of Dimer–Monomer Transition of a Plant Poly(A)-Binding Protein upon RNA Interaction: Insights into Its Poly(A) Tail Assembly

Abstract

Poly(A)-binding proteins (PABPs) play crucial roles in mRNA biogenesis, stability, transport and translational control in most eukaryotic cells. Although animal PABPs are well-studied proteins, the biological role, three-dimensional structure and RNA-binding mode of plant PABPs remain largely uncharacterized. Here, we report the structural features and RNA-binding mode of a Citrus sinensis PABP (CsPABPN1). CsPABPN1 has a domain architecture of nuclear PABPs (PABPNs) with a single RNA recognition motif (RRM) flanked by an acidic N-terminus and a GRPF-rich C-terminus. The RRM domain of CsPABPN1 displays virtually the same three-dimensional structure and poly(A)-binding mode of animal PABPNs. However, while the CsPABPN1 RRM domain specifically binds poly(A), the full-length protein also binds poly(U). CsPABPN1 localizes to the nucleus of plant cells and undergoes a dimer-monomer transition upon poly(A) interaction. We show that poly(A) binding by CsPABPN1 begins with the recognition of the RNA-binding sites RNP1 and RNP2, followed by interactions with residues of the β2 strands, which stabilize the dimer, thus leading to dimer dissociation. Like human PABPN1, CsPABPN1 also seems to form filaments in the presence of poly(A). Based on these data, we propose a structural model in which contiguous CsPABPN1 RRM monomers wrap around the RNA molecule creating a superhelical structure that could not only shield the poly(A) tail but also serve as a scaffold for the assembly of additional mRNA processing factors.

Role of the Citrus sinensis RNA deadenylase CsCAF1 in citrus canker resistance

“…However, given that PthA4 inhibited CsCAF1 deadenylase activity in vitro, we hypothesized that PthA4 could also inhibit CsCAF1-dependent deadenylation of CsLOB1 to stabilize this message. This idea is consistent with the fact that PthA4 interacts with several citrus proteins implicated in mRNA stabilization (Domingues et al, 2015;de Souza et al, 2012) and that the increased expression of CsCAF1 in citrus leaves infiltrated with the citrus canker bacteria correlates with a decrease in CsLOB1 expression ( Fig. 1A).…”

De novo design of RNA-binding proteins with a prion-like domain related to ALS/FTD proteinopathies

“…We assembled the base pairs necessary to encode the G / S-Y-G / S motif and minimize repeats of the nucleotide sequence to avoid formation of aberrant RNA structures. Additionally, we used the well-characterized PABP RRM from Citrus sinensis , which shares 43% identity with the human PABPN1 16 , to minimize the possibility of functional redundancy in mammalian cells. Furthermore, we added a NLS (MPKKKRKVGG) or NES (MLPPLERLTLDGG) to investigate compartment-specific cytotoxicity.…”

“…To address this, we generated artificial RNA-binding proteins harboring repeats of the G / S-Y-G / S-motif-enriched low-complexity sequence with and without enriched glutamine residues and signal sequences for nuclear import/export. To minimize superfluous functions in mammalian cells, a well-characterized plant-based RNA-recognition domain [poly(A)-binding protein (PABPs)] 16 was selected as a standard RNA-recognition motif (RRM). These proteins exhibited features similar to those of ALS-linked molecules, including insoluble aggregation, formation of cytoplasmic IBs and RNA-granule components, and instigation of cytotoxicity.…”

cDNA Library Screening Identifies Protein Interactors Potentially Involved in Non-Telomeric Roles of Arabidopsis Telomerase

“…Nucleic acid binding of RRM-containing proteins is often mediated by a pair of RRM domains ( Deo et al, 1999 ; Kwon and Chung, 2004 ). On the other hand, Xenopus laevis XlePABP2 and Citrus sinensis CsPABPN1, PABPNs that share similar structure with RRM protein, both bind poly(A) as monomers and undergo a dimer-monomer transition upon poly(A) binding ( Song et al, 2008 ; Domingues et al, 2015 ). We visualized RRM protein dimerization in tobacco BY-2 protoplasts and observed the same nuclear speckle pattern as with RRM-YFP localization.…”