Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation.

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.

Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

Inhibition of nonsense-mediated mRNA decay by the cytoplasmic poly(A)-binding protein

The expression of a gene from transcription of the DNA into pre-messenger RNA (pre-mRNA) over translation of messenger RNA (mRNA) into protein is constantly monitored for errors. This quality control is necessary to guarantee successful gene expression. One quality control mechanism important to this thesis is called nonsense-mediated mRNA decay (NMD). NMD is a cellular process that eliminates mRNA transcripts harboring premature translation termination codons (PTCs). Furthermore, NMD is known to regulate certain transcripts with long 3′ UTRs. However, some mRNA transcripts are known to evade NMD. The mechanism of NMD activation has been subjected to many studies whereas NMD evasion or suppression still remains rather elusive. It has previously been shown that the cytoplasmic poly(A)-binding protein (PABPC1) is able to suppress NMD of certain transcripts. In this study I show that PABPC1 is able to suppress NMD of a long 3′ UTR-carrying reporter when tethered immediately downstream of the termination codon. I further am able to show the importance of the interaction between PABPC1 and eIF4G for NMD suppression, whereas the interaction between PABPC1 and eRF3a seems dispensable. These results indicate an involvement of efficient translation termination and potentially ribosome recycling in NMD suppression. I am able to show that if PABPC1 is too far removed from the terminating ribosome NMD is activated. After showing the importance of PABPC1 recruitment directly downstream of a terminating ribosome in NMD suppression, I am further able to demonstrate several different methods by which PABPC1 can be recruited. Fold-back of the poly(A)-tail mediated by two interacting proteins on opposite ends of a 3′ UTR manages to bring PABPC1 bound to the poly(A)-tail into close proximity of the terminating ribosome and therefore suppress NMD. Furthermore, small PAM2 peptides that are known to interact with the MLLE domain of PABPC1 are able to strongly suppress NMD initiated by either a long 3′ UTR or an EJC. I am also able to show the NMD antagonizing power of recruited PABPC1 for the known endogenous NMD target β-globin PTC39, which is responsible for the disease β-thalassemia. This shows the potential medical implications and application of suppressing NMD by recruiting PABPC1 into close proximity of a terminating ribosome.

Loss of nuclear poly(A)-binding protein 1 causes defects in myogenesis and mRNA biogenesis

The nuclear poly(A)-binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays a critical role in polyadenylation. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing and weakness in the proximal limb muscles. Although significant data from in vitro biochemical assays define the function of PABPN1 in control of poly(A) tail length, little is known about the role of PABPN1 in mammalian cells. To assess the function of PABPN1 in mammalian cells and specifically in cells affected in OPMD, we examined the effects of PABPN1 depletion using siRNA in primary mouse myoblasts from extraocular, pharyngeal and limb muscles. PABPN1 knockdown significantly decreased cell proliferation and myoblast differentiation during myogenesis in vitro. At the molecular level, PABPN1 depletion in myoblasts led to a shortening of mRNA poly(A) tails, demonstrating the cellular function of PABPN1 in polyadenylation control in a mammalian cell. In addition, PABPN1 depletion caused nuclear accumulation of poly(A) RNA, revealing that PABPN1 is required for proper poly(A) RNA export from the nucleus. Together, these experiments demonstrate that PABPN1 plays an essential role in myoblast proliferation and differentiation, suggesting that it is required for muscle regeneration and maintenance in vivo.

Genetic interactions of yeast eukaryotic translation initiation factor 5a (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIFSA domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIFSA may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.

Loss of nuclear poly(A)-binding protein 1 (PABPN1) causes defects in myogenesis and mRNA biogenesis

The nuclear poly(A)-binding protein 1 (PABPN1) is a ubiquitously expressed protein that plays a critical role in polyadenylation. Short expansions of the polyalanine tract in the N-terminus of PABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onset disease characterized by eyelid drooping, difficulty in swallowing and weakness in the proximal limb muscles. Although significant data from in vitro biochemical assays define the function of PABPN1 in control of poly(A) tail length, little is known about the role of PABPN1 in mammalian cells. To assess the function of PABPN1 in mammalian cells and specifically in cells affected in OPMD, we examined the effects of PABPN1 depletion using siRNA in primary mouse myoblasts from extraocular, pharyngeal and limb muscles. PABPN1 knockdown significantly decreased cell proliferation and myoblast differentiation during myogenesis in vitro. At the molecular level, PABPN1 depletion in myoblasts led to a shortening of mRNA poly(A) tails, demonstrating the cellular function of PABPN1 in polyadenylation control in a mammalian cell. In addition, PABPN1 depletion caused nuclear accumulation of poly(A) RNA, revealing that PABPN1 is required for proper poly(A) RNA export from the nucleus. Together, these experiments demonstrate that PABPN1 plays an essential role in myoblast proliferation and differentiation, suggesting that it is required for muscle regeneration and maintenance in vivo.

Structure and function of the C-terminal PABC domain of human poly(A)-binding protein

We have determined the solution structure of the C-terminal quarter of human poly(A)-binding protein (hPABP). The protein fragment contains a protein domain, PABC [for poly(A)-binding protein C-terminal domain], which is also found associated with the HECT family of ubiquitin ligases. By using peptides derived from PABP interacting protein (Paip) 1, Paip2, and eRF3, we show that PABC functions as a peptide binding domain. We use chemical shift perturbation analysis to identify the peptide binding site in PABC and the major elements involved in peptide recognition. From comparative sequence analysis of PABC-binding peptides, we formulate a preliminary PABC consensus sequence and identify human ataxin-2, the protein responsible for type 2 spinocerebellar ataxia (SCA2), as a potential PABC ligand.

X-ray structure of the human hyperplastic discs protein: An ortholog of the C-terminal domain of poly(A)-binding protein

The poly(A)-binding protein (PABP) recognizes the 3′ mRNA poly(A) tail and plays an essential role in eukaryotic translation initiation and mRNA stabilization/degradation. PABP is a modular protein, with four N-terminal RNA-binding domains and an extensive C terminus. The C-terminal region of PABP is essential for normal growth in yeast and has been implicated in mediating PABP homo-oligomerization and protein–protein interactions. A small, proteolytically stable, highly conserved domain has been identified within this C-terminal segment. Remarkably, this domain is also present in the hyperplastic discs protein (HYD) family of ubiquitin ligases. To better understand the function of this conserved region, an x-ray structure of the PABP-like segment of the human HYD protein has been determined at 1.04-Å resolution. The conserved domain adopts a novel fold resembling a right-handed supercoil of four α-helices. Sequence profile searches and comparative protein structure modeling identified a small ORF from the Arabidopsis thaliana genome that encodes a structurally similar but distantly related PABP/HYD domain. Phylogenetic analysis of the experimentally determined (HYD) and homology modeled (PABP) protein surfaces revealed a conserved feature that may be responsible for binding to a PABP interacting protein, Paip1, and other shared interaction partners.

Human testis expresses a specific poly(A)-binding protein

In testis mRNA stability and translation initiation are extensively under the control of poly(A)-binding proteins (PABP). Here we have cloned a new human testis-specific PABP (PABP3) of 631 amino acids (70.1 kDa) with 92.5% identical residues to the ubiquitous PABP1. A northern blot of multiple human tissues hybridised with PABP3- and PABP1-specific oligonucleotide probes revealed two PABP3 mRNAs (2.1 and 2.5 kb) detected only in testis, whereas PABP1 mRNA (3.2 kb) was present in all tested tissues. In human adult testis, PABP3 mRNA expression was restricted to round spermatids, whereas PABP1 was expressed in these cells as well as in pachytene spermatocytes. PABP3-specific antibodies identified a protein of 70 kDa in human testis extracts. This protein binds poly(A) with a slightly lower affinity as compared to PABP1. The human PABP3 gene is intronless with a transcription start site 61 nt upstream from the initiation codon. A sequence of 256 bp upstream from the transcription start site drives the promoter activity of PABP3 and its tissue-specific expression. The expression of PABP3 might be a way to bypass PABP1 translational repression and to produce the amount of PABP needed for active mRNA translation in spermatids.

Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: identification by automated liquid chromatography-tandem mass spectrometry.

The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for cap-independent initiation of translation. Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined. In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES. The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry. The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells. PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein. Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis. The closely related PCBP1 showed no stable interaction with the RNA. Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2.

Simultaneous purification of biotin-binding proteins-I and -II from chicken egg yolk and their characterization

Chicken egg yolk biotin-binding protein-I (BBP-I) has been purified to homogeneity along with the tetrameric BBP-II by a common protocol. The purification includes delipidation of egg yolk by butanol extraction, DEAE-Sephacel chromatography, treatment with guanidinium chloride and biotin-aminohexyl-Sepharose affinity chromatography. The identity of purified BBP-I was ascertained by its physicochemical properties as well as by its immunological cross-reactivity and precursor-product relationship with BBP-II.

LPS-binding protein and IL-6 Mark paradoxical tuberculosis immune reconstitution inflammatory syndrome in HIV patients

Background: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) remains a poorly understood complication in HIV-TB co-infected patients initiating antiretroviral therapy (ART). The role of the innate immune system in TB-IRIS is becoming increasingly apparent, however the potential involvement in TB-IRIS of a leaky gut and proteins that interfere with TLR stimulation by binding PAMPs has not been investigated before. Here we aimed to investigate the innate nature of the cytokine response in TB-IRIS and to identify novel potential biomarkers. Methods: From a large prospective cohort of HIV-TB co-infected patients receiving TB treatment, we compared 40 patients who developed TB-IRIS during the first month of ART with 40 patients matched for age, sex and baseline CD4 count who did not. We analyzed plasma levels of lipopolysaccharide (LPS)-binding protein (LBP), LPS, sCD14, endotoxin-core antibody, intestinal fatty acid-binding protein (I-FABP) and 18 pro-and anti-inflammatory cytokines before and during ART. Results: We observed lower baseline levels of IL-6 (p = 0.041), GCSF (p = 0.036) and LBP (p = 0.016) in TB-IRIS patients. At IRIS event, we detected higher levels of LBP, IL-1RA, IL-4, IL-6, IL-7, IL-8, G-CSF (p ≤ 0.032) and lower I-FABP levels (p = 0.013) compared to HIV-TB co-infected controls. Only IL-6 showed an independent effect in multivariate models containing significant cytokines from pre-ART (p = 0.039) and during TB-IRIS (p = 0.034). Conclusion: We report pre-ART IL-6 and LBP levels as well as IL-6, LBP and I-FABP levels during IRIS-event as potential biomarkers in TB-IRIS. Our results show no evidence of the possible contribution of a leaky gut to TB-IRIS and indicate that IL-6 holds a distinct role in the disturbed innate cytokine profile before and during TB-IRIS. Future clinical studies should investigate the importance and clinical relevance of these markers for the diagnosis and treatment of TB-IRIS. Copyright: © 2013 Goovaerts et al.

mRNA binding protein mrnp 41 localizes to both nucleus and cytoplasm

We have identified and molecularly characterized a human protein with a Mr of 40,880 Da. After UV irradiation of HeLa cells, this protein was cross-linked to poly(A)-containing mRNA and was therefore designated mrnp 41 (for mRNA binding protein of 41 kDa). Cell fractionation and immunoblotting showed mrnp 41 in both the cytoplasm and the nucleus and particularly in the nuclear envelope. Immunofluorescence microscopy localized mrnp 41 to distinct foci in the nucleoplasm, to the nuclear rim, and to meshwork-like structures throughout the cytoplasm. The cytoplasmic meshwork staining was disrupted by prior treatment of cells with the actin filament- or microtubule-disrupting drugs cytochalasin or nocodazole, respectively, suggesting association of mrnp 41 with the cytoskeleton. Double immunofluorescence with antibodies against mrnp 41 and the cytoplasmic poly(A) binding protein showed colocalization to the cytoplasmic meshwork. Immunogold electronmicroscopy confirmed mrnp 41’s cytoplasmic and nucleoplasmic localization and revealed a striking labeling of nuclear pore complexes. Together these data suggest that mrnp 41 may function in nuclear export of mRNPs and/or in cytoplasmic transport on, or attachment to, the cytoskeleton. Consistent with a role of mrnp 41 in nuclear export are previous reports that mutations in homologs of mrnp 41 in Schizosaccharomyces pombe, designated Rae1p, or in Saccharomyces cerevisiae, designated Gle2p, result in mRNA accumulation in the nucleus although it is presently not known whether these homologs are mRNA binding proteins as well.

Selenium binding protein 1 in ovarian cancer

Selenium binding protein I (SELENBP1) was identified to be the most significantly down-regulated protein in ovarian cancer cells by a membrane proteome profiling analysis. SELENBP1 expression levels in 4 normal ovaries, 8 benign ovarian tumors, 12 borderline ovarian tumors and 141 invasive ovarian cancers were analyzed with immunohistochemical assay. SELENBP1 expression was reduced in 87% cases of invasive ovarian cancer (122/141) and was significantly reduced in borderline tumors and invasive cancers (p < 0.001). Cox multivariate analysis within the 141 invasive cancer tissues showed that SELENBP1 expression score was a potential prognostic indicator for unfavorable prognosis of ovarian cancer (hazard ratio [HR], 2.18; 95% CI = L22-190; p = 0.009). Selenium can disrupt the androgen pathway, which has been implicated in modulating SELENBP1 expression. We investigated the effects of selenium and androgen on normal human ovarian surrace epithelial (HOSE) cells and cancer cells. Interestingly, SELENBP1 mRNA and protein levels were reduced by androgen and elevated by selenium treatment in the normal HOSE cells, whereas reversed responses were observed in the ovarian cancer cell lines. These results suggest that changes of SELENBP1 expression in malignant ovarian cancer are an indicator of aberration of selenium/androgen pathways and may reveal prognostic information of ovarian cancer. (c) 2005 Wiley-Liss, Inc.

Bimolecular interaction of Insulin-Like Growth Factor (IGF) binding protein-2 with αvβ3 negatively modulates IGF-I-Mediated migration and tumor growth

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the αvβ3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and αvβ3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7β3, which overexpressed the αvβ3 integrin. Collectively, these results indicate that αvβ3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.