Nuclear poly(A)-binding Proteins Research Articles

Nuclear and cytoplasmic poly(A) binding proteins (PABPs) favor distinct transcripts and isoforms.

The poly(A)-tail appended to the 3′-end of most eukaryotic transcripts plays a key role in their stability, nuclear transport, and translation. These roles are largely mediated by Poly(A) Binding Proteins (PABPs) that coat poly(A)-tails and interact with various proteins involved in the biogenesis and function of RNA. While it is well-established that the nuclear PABP (PABPN) binds newly synthesized poly(A)-tails and is replaced by the cytoplasmic PABP (PABPC) on transcripts exported to the cytoplasm, the distribution of transcripts for different genes or isoforms of the same gene on these PABPs has not been investigated on a genome-wide scale. Here, we analyzed the identity, splicing status, poly(A)-tail size, and translation status of RNAs co-immunoprecipitated with endogenous PABPN or PABPC in human cells. At steady state, many protein-coding and non-coding RNAs exhibit strong bias for association with PABPN or PABPC. While PABPN-enriched transcripts more often were incompletely spliced and harbored longer poly(A)-tails and PABPC-enriched RNAs had longer half-lives and higher translation efficiency, there are curious outliers. Overall, our study reveals the landscape of RNAs bound by PABPN and PABPC, providing new details that support and advance the current understanding of the roles these proteins play in poly(A)-tail synthesis, maintenance, and function.

Altered rRNA processing disrupts nuclear RNA homeostasis via competition for the poly(A)-binding protein Nab2.

RNA-binding proteins (RBPs) are key mediators of RNA metabolism. Whereas some RBPs exhibit narrow transcript specificity, others function broadly across both coding and non-coding RNAs. Here, in Saccharomyces cerevisiae, we demonstrate that changes in RBP availability caused by disruptions to distinct cellular processes promote a common global breakdown in RNA metabolism and nuclear RNA homeostasis. Our data shows that stabilization of aberrant ribosomal RNA (rRNA) precursors in an enp1-1 mutant causes phenotypes similar to RNA exosome mutants due to nucleolar sequestration of the poly(A)-binding protein (PABP) Nab2. Decreased nuclear PABP availability is accompanied by genome-wide changes in RNA metabolism, including increased pervasive transcripts levels and snoRNA processing defects. These phenotypes are mitigated by overexpression of PABPs, inhibition of rDNA transcription, or alterations in TRAMP activity. Our results highlight the need for cells to maintain poly(A)-RNA levels in balance with PABPs and other RBPs with mutable substrate specificity across nucleoplasmic and nucleolar RNA processes.

Nuclear, Cytosolic, and Surface-Localized Poly(A)-Binding Proteins of Plasmodium yoelii.

Malaria is a devastating illness that causes approximately 500,000 deaths annually. The malaria-causing parasite (Plasmodium genus) uses the process of translational repression to regulate its growth, development, and transmission. As poly(A)-binding proteins (PABP) have been identified as critical components of RNA metabolism and translational repression in model eukaryotes and in Plasmodium, we have identified and investigated two PABPs in Plasmodium yoelii, PyPABP1 and PyPABP2. In contrast to most single-celled eukaryotes, Plasmodium closely resembles metazoans and encodes both a nuclear PABP and a cytosolic PABP; here, we provide multiple lines of evidence in support of this observation. The conserved domain architectures of PyPABP1 and PyPABP2 resemble those of yeast and metazoans, while multiple independent binding assays demonstrated their ability to bind very strongly and specifically to poly(A) sequences. Interestingly, we also observed that purified PyPABP1 forms homopolymeric chains despite exhaustive RNase treatment in vitro. Finally, we show by indirect immunofluorescence assays (IFAs) that PyPABP1 and PyPABP2 are cytoplasm- and nucleus-associated PABPs during the blood stages of the life cycle. Surprisingly, however, PyPABP1 was instead observed to also be localized on the surface of transmitted salivary gland sporozoites and to be deposited in trails when parasites glide on a substrate. This is the third RNA-binding protein verified to be found on the sporozoite surface, and the data may point to an unappreciated RNA-centered interface between the host and parasite. IMPORTANCE Malaria remains one of the great global health problems. The parasite that causes malaria (Plasmodium genus) relies upon exquisite control of its transmission between vertebrate hosts and mosquitoes. One crucial way that it does so is by proactively producing mRNAs needed to establish the new infection but by silencing and storing them until they are needed. One key protein in this process of translational repression in model eukaryotes is poly(A)-binding protein (PABP). Here we have shown that Plasmodium yoelii utilizes both a nuclear PABP and a cytosolic PABP, both of which bind specifically to polyadenylated RNA sequences. Moreover, we find that the cytosolic PABP forms chains in vitro, consistent with its appreciated role in coating the poly(A) tails of mRNA. Finally, we have also verified that, surprisingly, the cytosolic PABP is found on the surface of Plasmodium sporozoites. Taking the data together, we propose that Plasmodium utilizes a more metazoan-like strategy for RNA metabolism using specialized PABPs.

Poly(A)-binding proteins are required for translational regulation in vertebrate oocytes and early embryos.

Poly(A)-binding proteins (PABPs) function in the timely regulation of gene expression during oocyte maturation, fertilisation and early embryo development in vertebrates. To this end, PABPs bind to poly(A) tails or specific sequences of maternally stored mRNAs to protect them from degradation and to promote their translational activities. To date, two structurally different PABP groups have been identified: (1) cytoplasmic PABPs, including poly(A)-binding protein, cytoplasmic 1 (PABPC1), embryonic poly(A)-binding protein (EPAB), induced PABP and poly(A)-binding protein, cytoplasmic 3; and (2) nuclear PABPs, namely embryonic poly(A)-binding protein 2 and nuclear poly(A)-binding protein 1. Many studies have been undertaken to characterise the spatial and temporal expression patterns and subcellular localisations of PABPC1 and EPAB in vertebrate oocytes and early embryos. In the present review, we comprehensively evaluate and discuss the expression patterns and particular functions of the EPAB and PABPC1 genes, especially in mouse and human oocytes and early embryos.

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

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.

Poly(A) Tail-Mediated Gene Regulation by Opposing Roles of Nab2 and Pab2 Nuclear Poly(A)-Binding Proteins in Pre-mRNA Decay

The 3' end of most eukaryotic transcripts is decorated by poly(A)-binding proteins (PABPs), which influence the fate of mRNAs throughout gene expression. However, despite the fact that multiple PABPs coexist in the nuclei of most eukaryotes, how functional interplay between these nuclear PABPs controls gene expression remains unclear. By characterizing the ortholog of the Nab2/ZC3H14 zinc finger PABP in Schizosaccharomyces pombe, we show here that the two major fission yeast nuclear PABPs, Pab2 and Nab2, have opposing roles in posttranscriptional gene regulation. Notably, we find that Nab2 functions in gene-specific regulation in a manner opposite to that of Pab2. By studying the ribosomal-protein-coding gene rpl30-2, which is negatively regulated by Pab2 via a nuclear pre-mRNA decay pathway that depends on the nuclear exosome subunit Rrp6, we show that Nab2 promotes rpl30-2 expression by acting at the level of the unspliced pre-mRNA. Our data support a model in which Nab2 impedes Pab2/Rrp6-mediated decay by competing with Pab2 for polyadenylated transcripts in the nucleus. The opposing roles of Pab2 and Nab2 reveal that interplay between nuclear PABPs can influence gene regulation.

A Proline‐Tyrosine Nuclear Localization Signal (PY‐NLS) Is Required for the Nuclear Import of Fission Yeast PAB2, but Not of Human PABPN1

Nuclear poly(A)-binding proteins (PABPs) are evolutionarily conserved proteins that play key roles in eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, the major nuclear PABP, Pab2, functions in the maturation of small nucleolar RNAs as well as in nuclear RNA decay. Despite knowledge about its nuclear functions, nothing is known about how Pab2 is imported into the nucleus. Here, we show that Pab2 contains a proline-tyrosine nuclear localization signal (PY-NLS) that is necessary and sufficient for its nuclear localization and function. Consistent with the role of karyopherin β2 (Kapβ2)-type receptors in the import of PY-NLS cargoes, we show that the fission yeast ortholog of human Kapβ2, Kap104, binds to recombinant Pab2 and is required for Pab2 nuclear localization. The absence of arginine methylation in a basic region N-terminal to the PY-core motif of Pab2 did not affect its nuclear localization. However, in the context of a sub-optimal PY-NLS, we found that Pab2 was more efficiently targeted to the nucleus in the absence of arginine methylation, suggesting that this modification can affect the import kinetics of a PY-NLS cargo. Although a sequence resembling a PY-NLS motif can be found in the human Pab2 ortholog, PABPN1, our results indicate that neither a functional PY-NLS nor Kapβ2 activity are required to promote entry of PABPN1 into the nucleus of human cells. Our findings describe the mechanism by which Pab2 is imported into the nucleus, providing the first example of a PY-NLS import system in fission yeast. In addition, this study suggests the existence of alternative or redundant nuclear import pathways for human PABPN1.

Depletion of Nuclear Poly(A) Binding Protein PABPN1 Produces a Compensatory Response by Cytoplasmic PABP4 and PABP5 in Cultured Human Cells

BackgroundIn vertebrates, poly(A) binding protein (PABP) is known to exist in five different isoforms. PABPs are primarily cytosolic with the exception of the nuclear PABP (PABPN1), which is located in the nucleus. Within the nucleus, PABPN1 is believed to bind to the poly(A) tail of nascent mRNA and along with cleavage and polyadenylation specificity factor (CPSF) define the length of the newly synthesized poly(A) tail.Methodology/Principal FindingsThe cellular role of PABP1 has been extensively studied over the years; however, the function of other PABPs remains poorly defined. In order to understand the role of PABPN1 in cellular mRNA metabolism and it’s interrelation with other PABPs, we depleted PABPN1 using RNAi in HeLa and HEK293 cells. Our results show that PABPN1 depletion did not have any effect on the poly(A) tail length, nuclear export of mRNA, mRNA translation, and transcription. Rather, PABPN1 depletion resulted in a compensatory response as observed by increased level of PABP5 and nuclear accumulation of PABP4. In addition, PABP4 was associated with the poly(A) tract of pre-mRNA and CPSF in PABPN1 depleted cells. Nevertheless, PABPN1 depletion significantly affected cell survival as evidenced by an increase in apoptosis markers: phosphorylated p53 and PUMA and as judged by the expression of ER stress marker GRP78.ConclusionOur results suggest that although function of PABPN1 may be compensated by nuclear translocation of PABP4 and perhaps by increase in the cytoplasmic abundance of PABP5, these were not sufficient to prevent apoptosis of cells. Thus PABPN1 may have a novel anti apoptotic role in mammalian cells.

The Nuclear Poly(A)-Binding Protein Interacts with the Exosome to Promote Synthesis of Noncoding Small Nucleolar RNAs

Poly(A)-binding proteins (PABPs) are important to eukaryotic gene expression. In the nucleus, the PABP PABPN1 is thought to function in polyadenylation of pre-mRNAs. Deletion of fission yeast pab2, the homolog of mammalian PABPN1, results in transcripts with markedly longer poly(A) tails, but the nature of the hyperadenylated transcripts and the mechanism that leads to RNA hyperadenylation remain unclear. Here we report that Pab2 functions in the synthesis of noncoding RNAs, contrary to the notion that PABPs function exclusively on protein-coding mRNAs. Accordingly, the absence of Pab2 leads to the accumulation of polyadenylated small nucleolar RNAs (snoRNAs). Our findings suggest that Pab2 promotes poly(A) tail trimming from pre-snoRNAs by recruiting the nuclear exosome. This work unveils a function for the nuclear PABP in snoRNA synthesis and provides insights into exosome recruitment to polyadenylated RNAs.

Poly(A)-binding proteins: multifunctional scaffolds for the post-transcriptional control of gene expression

The poly(A)-binding proteins (PABPs) are highly conserved polypeptides that bind to poly(A) tails of mRNAs. Although they lack catalytic activity, PABPs have several roles, including actions in regulating the length of the poly(A) tail, in regulating the export of mRNAs from the nucleus, in translation initiation and termination, in recycling of ribosomes, and in mRNA stability.