Expression and prognostic roles of PABPC1 in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide. The present study was aimed to identify potential hub genes involved in the progression of HCC and investigate its clinical and prognostic significance. METHOD: First, the dataset GSE76427 was used to construct a co-expression network. Weighted gene co-expression network analysis (WGCNA) was used to investigate the meaningful module. Then protein-protein interaction (PPI) network analysis and Gene Set Enrichment Analysis (GSEA) were applied to study hub genes correlated with the HCC progression. The hub gene expression and their prognostic correlation were further analyzed by a series of database. Paraffin-embedded HCC tissues obtained by biopsy from 225 patients were subjected to immunohistochemistry. RESULT: Twelve co-expressed gene modules were identified using WGCNA. The pink module showed a higher correlation with overall survival years (r = 0.69, P = 0.02). Bioinformatics analysis show the real hub gene was PABPC1 and the PABPC1 mRNA expression was higher in HCC tissues compared with normal tissues. GSEA analysis indicated that PABPC1 expression was associated with P53 signaling pathway. High expression of PABPC1 was correlated with TNM stage (P = 0.004) and serum AFP (P = 0.001). High expression of PABPC1 was correlated with worse overall survival for HCC. Multivariate analysis showed that PABPC1 was an independent prognostic factor for HCC (HR = 4.137, 95%CI: 2.454-6.974, P = 0.001). CONCLUSION: In general, PABPC1 may contribute to the progression of HCC. Moreover, PABPC1 has potential to be used as prognostic markers in HCC.

Impact of poly(A)-tail G-content on Arabidopsis PAB binding and their role in enhancing translational efficiency.

BACKGROUND: Polyadenylation plays a key role in producing mature mRNAs in eukaryotes. It is widely believed that the poly(A)-binding proteins (PABs) uniformly bind to poly(A)-tailed mRNAs, regulating their stability and translational efficiency. RESULTS: We observe that the homozygous triple mutant of broadly expressed Arabidopsis thaliana PABs, AtPAB2, AtPAB4, and AtPAB8, is embryonic lethal. To understand the molecular basis, we characterize the RNA-binding landscape of these PABs. The AtPAB-binding efficiency varies over one order of magnitude among genes. To identify the sequences accounting for the variation, we perform poly(A)-seq that directly sequences the full-length poly(A) tails. More than 10% of poly(A) tails contain at least one guanosine (G); among them, the G-content varies from 0.8 to 28%. These guanosines frequently divide poly(A) tails into interspersed A-tracts and therefore cause the variation in the AtPAB-binding efficiency among genes. Ribo-seq and genome-wide RNA stability assays show that AtPAB-binding efficiency of a gene is positively correlated with translational efficiency rather than mRNA stability. Consistently, genes with stronger AtPAB binding exhibit a greater reduction in translational efficiency when AtPAB is depleted. CONCLUSIONS: Our study provides a new mechanism that translational efficiency of a gene can be regulated through the G-content-dependent PAB binding, paving the way for a better understanding of poly(A) tail-associated regulation of gene expression.

Translational activation of maternally derived mRNAs in oocytes and early embryos and the role of embryonic poly(A) binding protein (EPAB).

Transcription ceases upon stimulation of oocyte maturation and gene expression during oocyte maturation, fertilization, and early cleavage relies on translational activation of maternally derived mRNAs. Two key mechanisms that mediate translation of mRNAs in oocytes have been described in detail: cytoplasmic polyadenylation-dependent and -independent. Both of these mechanisms utilize specific protein complexes that interact with cis-acting sequences located on 3'-untranslated region (3'-UTR), and both involve embryonic poly(A) binding protein (EPAB), the predominant poly(A) binding protein during early development. While mechanistic details of these pathways have primarily been elucidated using the Xenopus model, their roles are conserved in mammals and targeted disruption of key regulators in mouse results in female infertility. Here, we provide a detailed account of the molecular mechanisms involved in translational activation during oocyte and early embryo development, and the role of EPAB in this process.

Functional compensation for the loss of testis-specific poly(A)-binding protein, PABPC2, during mouse spermatogenesis.

Mouse testes contain several isoforms of cytoplasmic poly(A)-binding proteins (PABPCs), including ubiquitous PABPC1 and testis-specific PABPC2/PABPt. PABPC2 is characterized by its absence from translationally active polyribosomes and elongating spermatids. To elucidate the function of PABPC2 in spermatogenesis, we produced mutant mice lacking PABPC2. The PABPC2-null mice showed normal fertility. The processes of spermatogenesis and sperm migration in the testes and epididymides, respectively, were normal in the mutant mice. When the involvement of PABPC2 in translational regulation of haploid-specific mRNAs was examined, these mRNAs were correctly transcribed in round spermatids and translated in elongating spermatids. Moreover, immunoblot analysis revealed low abundance of PABPC2 relative to PABPC1 in spermatogenic cells. These results suggest that PABPC2 may be either functionally redundant with other PABPCs (including PABPC1) or largely dispensable for translational regulation during spermiogenesis.

TIA-1 and TIAR interact with 5'-UTR of enterovirus 71 genome and facilitate viral replication.

Enterovirus 71 is one of the major causative pathogens of HFMD in children. Upon infection, the viral RNA is translated in an IRES-dependent manner and requires several host factors for effective replication. Here, we found that T-cell-restricted intracellular antigen 1 (TIA-1), and TIA-1 related protein (TIAR) were translocated from nucleus to cytoplasm after EV71 infection and localized to the sites of viral replication. We found that TIA-1 and TIAR can facilitate EV71 replication by enhancing the viral genome synthesis in host cells. We demonstrated that both proteins bound to the stem-loop I of 5'-UTR of viral genome and improved the stability of viral genomic RNA. Our results suggest that TIA-1 and TIAR are two new host factors that interact with 5-UTR of EV71 genome and positively regulate viral replication.

Embryonic Poly(A)-Binding Protein Is Required During Early Stages of Mouse Oocyte Development for Chromatin Organization, Transcriptional Silencing, and Meiotic Competence.

During oocyte maturation, fertilization, and early embryo development until zygotic genome activation (ZGA), transcription is suppressed, and gene expression is dependent upon the timely activation of stored mRNAs. Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse, and human oocytes and early embryos and is important for regulating translational activation of maternally stored mRNAs. EPAB is critical for early development because Epab(-/-) female mice do not produce mature eggs and are infertile. In this study, we further characterize morphological and molecular aspects of Epab(-/-) oocytes. We demonstrated that Epab(-/-) oocytes are smaller in size, contain peripheral germinal vesicles, and are loosely associated with cumulus cells. The chromatin reorganization of the surrounded nucleolus (SN) configuration and transcriptional silencing that normally occurs during oocyte growth does not occur in Epab(-/-) oocytes. Interestingly, microinjection of Epab mRNA into Epab(-/-) preantral follicle-enclosed oocytes rescues reorganization of chromatin and oocyte maturation to metaphase II. Overall, these results demonstrate an important role for EPAB during oocyte growth and the acquisition of meiotic competence.

Genome-wide profiling reveals a role for T-cell intracellular antigens TIA1 and TIAR in the control of translational specificity in HeLa cells.

TIA (T-cell intracellular antigens)-knockdown HeLa cells show an increase in ribosomes and translational machinery components. This increase correlates with specific changes in translationally up-regulated mRNAs involved in cell-cycle progression and DNA repair, as shown in polysomal profiling analysis. Our data support the hypothesis that a concerted activation of both global and selective translational rates leads to the transition to a more proliferative status in TIA-knockdown HeLa cells.

The structure of the Pan2-Pan3 core complex reveals cross-talk between deadenylase and pseudokinase.

Pan2-Pan3 is a conserved complex involved in the shortening of mRNA poly(A) tails, the initial step in eukaryotic mRNA turnover. We show that recombinant Saccharomyces cerevisiae Pan2-Pan3 can deadenylate RNAs in vitro without needing the poly(A)-binding protein Pab1. The crystal structure of an active ~200-kDa core complex reveals that Pan2 and Pan3 interact with an unusual 1:2 stoichiometry imparted by the asymmetric nature of the Pan3 homodimer. An extended region of Pan2 wraps around Pan3 and provides a major anchoring point for complex assembly. A Pan2 module formed by the pseudoubiquitin-hydrolase and RNase domains latches onto the Pan3 pseudokinase with intertwined interactions that orient the deadenylase active site toward the A-binding site of the interacting Pan3. The molecular architecture of Pan2-Pan3 suggests how the nuclease and its pseudokinase regulator act in synergy to promote deadenylation.

The role of HuR in the post-transcriptional regulation of interleukin-3 in T cells.

Human Interleukin-3 (IL-3) is a lymphokine member of a class of transiently expressed mRNAs harboring Adenosine/Uridine-Rich Elements (ARE) in their 3' untranslated regions (3'-UTRs). The regulatory effects of AREs are often mediated by specific ARE-binding proteins (ARE-BPs). In this report, we show that the human IL-3 3'-UTR plays a post-transcriptional regulation role in two human transformed cell lines. More specifically, we demonstrate that the hIL-3 3'-UTR represses the translation of a luciferase reporter both in HeLa and Jurkat T-cells. These results also revealed that the hIL-3 3'-UTR-mediated translational repression is exerted by an 83 nt region comprised mainly by AREs and some non-ARE sequences. Moreover, electrophoretic mobility shift assays (EMSAs) and UV-crosslinking analysis show that this hIL-3 ARE-rich region recruits five specific protein complexes, including the ARE-BPs HuR and TIA-1. HuR binding to this ARE-rich region appears to be spatially modulated during T-cell activation. Together, these results suggest that HuR recognizes the ARE-rich region and plays a role in the IL-3 3'-UTR-mediated post-transcriptional control in T-cells.

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.

miRNA repression of translation in vitro takes place during 43S ribosomal scanning.

microRNAs (miRNAs) regulate gene expression at multiple levels by repressing translation, stimulating deadenylation and inducing the premature decay of target messenger RNAs (mRNAs). Although the mechanism by which miRNAs repress translation has been widely studied, the precise step targeted and the molecular insights of such repression are still evasive. Here, we have used our newly designed in vitro system, which allows to study miRNA effect on translation independently of deadenylation. By using specific inhibitors of various stages of protein synthesis, we first show that miRNAs target exclusively the early steps of translation with no effect on 60S ribosomal subunit joining, elongation or termination. Then, by using viral proteases and IRES-driven mRNA constructs, we found that translational inhibition takes place during 43S ribosomal scanning and requires both the poly(A) binding protein and eIF4G independently from their physical interaction.

Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1.

T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. Small-angle X-ray scattering analyses demonstrated a "V" shape for a TIA-1 construct comprising the three RRMs and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed.

Knockdown of T-cell intracellular antigens triggers cell proliferation, invasion and tumour growth.

TIA (T-cell intracellular antigen) proteins function as DNA/RNA trans-acting regulators to expand transcriptome and proteome diversity in mammals. In the present paper we report that the stable silencing of TIA1 and/or TIAR/TIAL1 (TIA1-related/like protein 1) expression in HeLa cells enhances cell proliferation, anchorage-dependent and -independent growth and invasion. HeLa cells lacking TIA1 and/or TIAR generate larger and faster-growing epithelial tumours with high rates of proliferation and angiogenesis in nude mice xenografts. Protein array analysis of a collection of human tumours shows that TIA1 and TIAR protein expression is down-regulated in a subset of epithelial tumours relative to normal tissues. Our results suggest a link between the epigenetic control exerted by TIA proteins and the transcriptional and post-transcriptional regulation of a subset of specific genes involved in tumour progression. Taken together, these results are consistent with a role for TIA proteins as growth/tumour-suppressor genes.

TIA-1 cytotoxic granule-associated RNA binding protein improves the prognostic performance of CD8 in mismatch repair-proficient colorectal cancer.

BACKGROUND: Evidence suggests a confounding effect of mismatch repair (MMR) status on immune response in colorectal cancer. The identification of innate and adaptive immune cells, that can complement the established prognostic effect of CD8 in MMR-proficient colorectal cancers patients, representing 85% of all cases, has not been performed. METHODOLOGY/PRINCIPAL FINDINGS: Colorectal cancers from a test (n=1197) and external validation (n=209) cohort of MMR-proficient colorectal cancers were mounted onto single and multiple punch tissue microarrays. Immunohistochemical quantification (score 0-3) was performed for CD3, CD4, CD8, CD45RO, CD68, CD163, FoxP3, GranzymeB, iNOS, mast cell tryptase, MUM1, PD1 and TIA-1 tumor-infiltrating (TILs) reactive cells. Coexpression experiments on fresh colorectal cancer specimens using specific cell population markers were performed. In the test group, higher numbers of CD3+ (p<0.001), CD4+ (p=0.029), CD8+ (p<0.001), CD45RO+ (p=0.048), FoxP3+ (p<0.001), GranzymeB+ (p<0.001), iNOS+ (p=0.035), MUM1+ (p=0.014), PD1+ (p=0.034) and TIA-1+ TILs (p<0.001) were linked to favourable outcome. Adjusting for age, gender, TNM stage and post-operative therapy, higher CD8+ (p<0.001; HR (95%CI): 0.66 (0.64-0.68)) and TIA-1+ (p<0.001; HR (95%CI): 0.56 (0.5-0.6)) were independent prognostic factors. Moreover, among patients with CD8+ infiltrates, TIA-1 further stratified 355 (35.6%) patients into prognostic subgroups (p<0.001; HR (95%CI): 0.89 (95%CI: 0.8-0.9)). Results were confirmed on the validation cohort (p=0.006). TIA-1+ cells were mostly CD8+ (57%), but also stained for TCRγδ (22%), CD66b (13%) and only rarely for CD4+, macrophage and NK cell markers. CONCLUSIONS: TIA-1 adds prognostic information to TNM stage and adjuvant therapy in MMR-proficient colorectal cancer patients. The prognostic effect of CD8+ TILs is confounded by the presence of TIA-1+ which translates into improved risk stratification for approximately 35% of all patients with MMR-proficient colorectal cancers.

Crossing the borders: poly(A)-binding proteins working on both sides of the fence.

The addition of a 3' poly(A) tail is a pre-requisite for the maturation of the majority of eukaryotic transcripts. In most eukaryotic species, RNA poly(A) tails are bound by two important poly(A)-binding proteins (PABPs): PABPC1 and PABPN1 that localize to the cytoplasm and the nucleus, respectively. Such steady state localization for PABPN1 and PABPC1 led to a model whereby PABPN1-bound nuclear mRNAs are remodeled during or after nuclear export so that PABPN1 is replaced by PABPC1 to allow robust cap-dependent translation in the cytoplasm. Here we discuss evidence that challenge the view in which PABPN1 and PABPC1 function solely in the nucleus and cytoplasm, respectively. We discuss accumulating evidence that support nuclear roles for PABPC1 in mRNA biogenesis as well as cytoplasmic roles for PABPN1 in translational control. Because 3' poly(A) tails can also act as a degradation mark via the exosome complex of 3'-5' exonucleases, we also discuss recent results that involve the nuclear PABP in posttranscriptional gene regulation.

Localization to, and effects of Pbp1, Pbp4, Lsm12, Dhh1, and Pab1 on stress granules in Saccharomyces cerevisiae.

The regulation of translation and mRNA degradation in eukaryotic cells involves the formation of cytoplasmic mRNP granules referred to as P-bodies and stress granules. The yeast Pbp1 protein and its mammalian ortholog, Ataxin-2, localize to stress granules and promote their formation. In Saccharomyces cerevisiae, Pbp1 also interacts with the Pab1, Lsm12, Pbp4, and Dhh1 proteins. In this work, we determined whether these Pbp1 interacting proteins also accumulated in stress granules and/or could affect their formation. These experiments revealed the following observations. First, the Lsm12, Pbp4, and Dhh1 proteins all accumulate in stress granules, whereas only the Dhh1 protein is a constitutive P-body component. Second, deletion or over-expression of the Pbp4 and Lsm12 proteins did not dramatically affect the formation of stress granules or P-bodies. In contrast, Pbp1 and Dhh1 over-expression inhibits cell growth, and for Dhh1, leads to the accumulation of stress granules. Finally, a strain lacking the Pab1 protein was reduced at forming stress granules, although they could still be detected. This indicates that Pab1 affects, but is not absolutely required for, stress granule formation. These observations offer new insight into the function of stress granule components with roles in stress granule assembly and mRNP regulation.

Nuclear poly(a)-binding proteins and nuclear degradation: take the mRNA and run?

Recent work from Lemay et al. (2010) in this issue of Molecular Cell reveals a role for a nuclear poly(A)-binding protein in promoting degradation of small nucleolar RNAs (snoRNAs) by the nuclear exosome.

Stable formation of compositionally unique stress granules in virus-infected cells.

Stress granules are sites of mRNA storage formed in response to a variety of stresses, including viral infections. Here, the mechanisms and consequences of stress granule formation during poliovirus infection were examined. The results indicate that stress granules containing T-cell-restricted intracellular antigen 1 (TIA-1) and mRNA are stably constituted in infected cells despite lacking intact RasGAP SH3-domain binding protein 1 (G3BP) and eukaryotic initiation factor 4G. Fluorescent in situ hybridization revealed that stress granules in infected cells do not contain significant amounts of viral positive-strand RNA. Infection does not prevent stress granule formation in response to heat shock, indicating that poliovirus does not block de novo stress granule formation. A mutant TIA-1 protein that prevents stress granule formation during oxidative stress also prevents formation in infected cells. However, stress granule formation during infection is more dependent upon ongoing transcription than is formation during oxidative stress or heat shock. Furthermore, Sam68 is recruited to stress granules in infected cells but not to stress granules formed in response to oxidative stress or heat shock. These results demonstrate that stress granule formation in poliovirus-infected cells utilizes a transcription-dependent pathway that results in the appearance of stable, compositionally unique stress granules.

Poly(A)-binding protein (PABP): a common viral target.

Cytoplasmic PABP [poly(A)-binding protein] is a multifunctional protein with well-studied roles in mRNA translation and stability. In the present review, we examine recent evidence that the activity of PABP is altered during infection with a wide range of viruses, bringing about changes in its stability, complex formation and intracellular localization. Targeting of PABP by both RNA and DNA viruses highlights the role of PABP as a central regulator of gene expression.

The DAZL and PABP families: RNA-binding proteins with interrelated roles in translational control in oocytes.

Gametogenesis is a highly complex process that requires the exquisite temporal, spatial and amplitudinal regulation of gene expression at multiple levels. Translational regulation is important in a wide variety of cell types but may be even more prevalent in germ cells, where periods of transcriptional quiescence necessitate the use of post-transcriptional mechanisms to effect changes in gene expression. Consistent with this, studies in multiple animal models have revealed an essential role for mRNA translation in the establishment and maintenance of reproductive competence. While studies in humans are less advanced, emerging evidence suggests that translational regulation plays a similarly important role in human germ cells and fertility. This review highlights specific mechanisms of translational regulation that play critical roles in oogenesis by activating subsets of mRNAs. These mRNAs are activated in a strictly determined temporal manner via elements located within their 3'UTR, which serve as binding sites for trans-acting factors. While we concentrate on oogenesis, these regulatory events also play important roles during spermatogenesis. In particular, we focus on the deleted in azoospermia-like (DAZL) family of proteins, recently implicated in the translational control of specific mRNAs in germ cells; their relationship with the general translation initiation factor poly(A)-binding protein (PABP) and the process of cytoplasmic mRNA polyadenylation.