Human polypyrimidine tract-binding protein interacts with mitochondrial tRNAThr in the cytosol

Human polypyrimidine tract-binding protein PTB is a multifunctional RNA-binding protein with four RNA recognition motifs (RRM1 to RRM4). PTB is a nucleocytoplasmic shuttle protein that functions as a key regulator of alternative pre-mRNA splicing in the nucleoplasm and promotes internal ribosome entry site-mediated translation initiation of viral and cellular mRNAs in the cytoplasm. Here, we demonstrate that PTB and its paralogs, nPTB and ROD1, specifically interact with mitochondrial (mt) tRNA^Thr both in human and mouse cells. In vivo and in vitro RNA-binding experiments demonstrate that PTB forms a direct interaction with the T-loop and the D-stem-loop of mt tRNA^Thr using its N-terminal RRM1 and RRM2 motifs. RNA sequencing and cell fractionation experiments show that PTB associates with correctly processed and internally modified, mature mt tRNA^Thr in the cytoplasm outside of mitochondria. Consistent with this, PTB activity is not required for mt tRNA^Thr biogenesis or for correct mitochondrial protein synthesis. PTB association with mt tRNA^Thr is largely increased upon induction of apoptosis, arguing for a potential role of the mt tRNA^Thr/PTB complex in apoptosis. Our results lend strong support to the recently emerging conception that human mt tRNAs can participate in novel cytoplasmic processes independent from mitochondrial protein synthesis.     

Interplay between microRNAs and RNA-binding proteins determines developmental processes

MicroRNAs (miRNAs) are genes involved in normal development and cancer. They inhibit gene expression by associating with 3'-Untranslated regions (3' UTRs) of messenger RNAs (mRNAs), and are thought to regulate a large proportion of protein coding genes. However, it is becoming apparent that miRNA activity is not necessarily always determined by its expression in the cell. MiRNA activity can be affected by RNA-binding proteins (RBPs). For example, the RNA-binding protein HuR associates with the 3'UTR of the CAT1 mRNA after stress, counteracting the effect of miR-122. Second, we found that the expression of an RNA-binding protein called Dead end (Dnd1) prohibits the function of several miRNAs by blocking the accessibility of target mRNAs. Dnd1 function is essential for proper development of primordial germ cells (PGCs) in zebrafish and mammals, indicating a crucial role for RBP/miRNA interplay on 3'UTRs of mRNAs in developmental decisions. In this perspective we discuss the interplay between RBPs and miRNAs in the context of germ cells and review current observations implicating RBPs in miRNA function.     

Polypyrimidine tract binding protein (hnRNP I) is possibly a conserved modulator of miRNA-mediated gene regulation

MiRNAs can regulate gene expression through versatile mechanisms that result in increased or decreased expression of the targeted mRNA and it could effect the expression of thousands of protein in a particular cell. An increasing body of evidence suggest that miRNAs action can be modulated by proteins that bind to the same 3'UTRs that are targeted by miRNAs, suggesting that other factors apart from miRNAs and their target sites determine miRNA-modulation of gene expression. We applied an affinity purification protocol using biotinylated let-7 miRNA inhibitor to isolate proteins that are involved in let-7 mediated gene regulation that resulted in an affinity purification of Polypyrimidine Tract Binding protein (PTB). Here we show that PTB interacts with miRNAs and human Argonaute 2 (hAgo2) through RNA as well as identified potential mammalian cellular targets that are co-regulated by PTB and hAgo2. In addition, using genetic approach, we have demonstrated that PTB genetically interacts with Caenorhabditis elegans let-7 indicating a conserved role for PTB in miRNA-mediated gene regulation.     

Polypyrimidine tract-binding proteins are cleaved by caspase-3 during apoptosis

The polypyrimidine tract-binding protein (PTB), an RNA-binding protein, is required for efficient translation of some mRNAs containing internal ribosomal entry sites (IRESs). Here we provide evidence that the addition of apoptosis-inducing agents to cells results in the cleavage of PTB isoforms 1, 2, and 4 by caspase-3. This cleavage of PTB separated the N-terminal region, containing NLS-RRM1, from the C-terminal region, containing RRM2-3-4. Our data indicate that there are three noncanonical caspase-3 target sites in PTBs, namely Ile-Val-Pro-Asp(7)Ile, Leu-Tyr-Thr-Asp(139)Ser, and Ala-Ala-Val-Asp(172)Ala. The C-terminal PTB fragments localized to the cytoplasm, as opposed to the nucleus where most intact PTBs are found. Moreover, these C-terminal PTB fragments inhibited translation of polioviral mRNA, which contains an IRES element requiring PTB for its activation. This suggests that translation of some IRES-containing mRNAs is regulated by proteolytic cleavage of PTB during apoptosis.     

PTB蛋白:一种调控肿瘤代谢的RNA可变剪接调节因子

多聚嘧啶区结合蛋白(polypyrimidine tract binding protein,PTB或hnRNP I)是一种在细胞内部参与mRNA代谢过程的蛋白质。PTB蛋白可结合于核酸分子上富含嘧啶碱基的序列,对mRNA前体的剪接进行调控。如在部分肿瘤细胞中,PTB的表达量升高可对肿瘤代谢过程中关键的丙酮酸激酶M(pyruvate kinase M,PKM)基因的表达进行调控,通过抑制PKM基因的可变剪接的方式上调PKM2(pyruvate kinase M2,PKM2)的表达,进而强化肿瘤细胞的有氧糖酵解过程并促进肿瘤的发展。本文结合PTB蛋白的结构及其在PKM可变剪接过程中的调节机制,简要综述了PTB蛋白对肿瘤代谢的调控作用。     

Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a nucleocytoplasmic shuttling protein that regulates gene expression through its action on mRNA metabolism and translation. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here, we show that hnRNP A1 is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5' untranslated region of both the human rhinovirus-2 and the human apoptotic peptidase activating factor 1 (apaf-1) mRNAs, thereby regulating their translation. Furthermore, the cytoplasmic redistribution of hnRNP A1 after rhinovirus infection leads to enhanced rhinovirus IRES-mediated translation, whereas the cytoplasmic relocalization of hnRNP A1 after UVC irradiation limits the UVC-triggered translational activation of the apaf-1 IRES. Therefore, this study provides a direct demonstration that IRESs behave as translational enhancer elements regulated by specific trans-acting mRNA binding proteins in given physiological conditions. Our data highlight a new way to regulate protein synthesis in eukaryotes through the subcellular relocalization of a nuclear mRNA-binding protein.     

TDP-43 and FUS RNA-binding proteins bind distinct sets of cytoplasmic messenger RNAs and differently regulate their post-transcriptional fate in motoneuron-like cells

The RNA-binding proteins TDP-43 and FUS form abnormal cytoplasmic aggregates in affected tissues of patients with amyotrophic lateral sclerosis and frontotemporal lobar dementia. TDP-43 and FUS localize mainly in the nucleus where they regulate pre-mRNA splicing, but they are also involved in mRNA transport, stability, and translation. To better investigate their cytoplasmic activities, we applied an RNA immunoprecipitation and chip analysis to define the mRNAs associated to TDP-43 and FUS in the cytoplasmic ribonucleoprotein complexes from motoneuronal NSC-34 cells. We found that they bind different sets of mRNAs although converging on common cellular pathways. Bioinformatics analyses identified the (UG)(n) consensus motif in 80% of 3'-UTR sequences of TDP-43 targets, whereas for FUS the binding motif was less evident. By in vitro assays we validated binding to selected target 3'-UTRs, including Vegfa and Grn for TDP-43, and Vps54, Nvl, and Taf15 for FUS. We showed that TDP-43 has a destabilizing activity on Vegfa and Grn mRNAs and may ultimately affect progranulin protein content, whereas FUS does not affect mRNA stability/translation of its targets. We also demonstrated that three different point mutations in TDP-43 did not change the binding affinity for Vegfa and Grn mRNAs or their protein level. Our data indicate that TDP-43 and FUS recognize distinct sets of mRNAs and differently regulate their fate in the cytoplasm of motoneuron-like cells, therefore suggesting complementary roles in neuronal RNA metabolism and neurodegeneration.     

Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3′-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing

In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5′-splice site SD3632; produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5′-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression.     

eIF2α Phosphorylation Tips the Balance to Apoptosis during Osmotic Stress

Regulation of cell volume is of great importance because persistent swelling or shrinkage leads to cell death. Tissues experience hypertonicity in both physiological (kidney medullar cells) and pathological states (hypernatremia). Hypertonicity induces an adaptive gene expression program that leads to cell volume recovery or apoptosis under persistent stress. We show that the commitment to apoptosis is controlled by phosphorylation of the translation initiation factor eIF2α, the master regulator of the stress response. Studies with cultured mouse fibroblasts and cortical neurons show that mutants deficient in eIF2α phosphorylation are protected from hypertonicity-induced apoptosis. A novel link is revealed between eIF2α phosphorylation and the subcellular distribution of the RNA-binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Stress-induced phosphorylation of eIF2α promotes apoptosis by inducing the cytoplasmic accumulation of hnRNP A1, which attenuates internal ribosome entry site-mediated translation of anti-apoptotic mRNAs, including Bcl-xL that was studied here. Hypertonic stress induced the eIF2α phosphorylation-independent formation of cytoplasmic stress granules (SGs, structures that harbor translationally arrested mRNAs) and the eIF2α phosphorylation-dependent accumulation of hnRNP A1 in SGs. The importance of hnRNP A1 was demonstrated by induction of apoptosis in eIF2α phosphorylation-deficient cells that express exogenous cytoplasmic hnRNP A1. We propose that eIF2α phosphorylation during hypertonic stress promotes apoptosis by sequestration of specific mRNAs in SGs in a process mediated by the cytoplasmic accumulation of hnRNP A1.     

Requirement of adaptor protein GULP during stabilin-2-mediated cell corpse engulfment

The prompt clearance of cells undergoing apoptosis is critical during embryonic development and normal tissue turnover, as well as during inflammation and autoimmune responses. We recently demonstrated that stabilin-2 is a phosphatidylserine receptor that mediates the clearance of apoptotic cells, thereby releasing the anti-inflammatory cytokine, transforming growth factor-beta. However, the downstream signaling components of stabilin-2-mediated phagocytosis are not known. Here, we provide evidence that the adaptor protein, GULP, physically and functionally interacts with the stabilin-2 cytoplasmic tail. Using fluorescent resonance energy transfer analysis and biochemical approaches, we show that GULP directly binds to the cytoplasmic tail of stabilin-2. Knockdown of endogenous GULP expression significantly decreased stabilin-2-mediated phagocytosis. Conversely, overexpression of GULP caused an increase in aged cell engulfment. The phosphotyrosine binding (PTB) domain of GULP was sufficient for the interaction with stabilin-2; therefore, transduction of TAT fusion PTB domain acts as a dominant negative, resulting in impaired engulfment of aged red blood cells in stabilin-2 expressing cells. In addition, the PTB domain of GULP was able to specifically interact with the NPXY motif of the stabilin-2 cytoplasmic tail. Taken together, these results indicate that GULP is a likely downstream molecule in the stabilin-2-mediated signaling pathway and plays an important role in stabilin-2-mediated phagocytosis.