Novel mRNA-containing cytoplasmic granules in ALK-transformed cells

In mammalian cells, nontranslating messenger RNAs (mRNAs) are concentrated in different cytoplasmic foci, such as processing bodies (PBs) and stress granules (SGs), where they are either degraded or stored. In the present study, we have thoroughly characterized cytoplasmic foci, hereafter called AGs for ALK granules that form in transformed cells expressing the constitutively active anaplastic lymphoma kinase (ALK). AGs contain polyadenylated mRNAs and a unique combination of several RNA binding proteins that so far has not been described in mammalian foci, including AUF1, HuR, and the poly (A(+)) binding protein PABP. AGs shelter neither components of the mRNA degradation machinery present in PBs nor known markers of SGs, such as translation initiation factors or TIA/TIAR, showing that they are distinct from PBs or SGs. AGs and PBs, however, both move on microtubules with similar dynamics and frequently establish close contacts. In addition, in conditions in which mRNA metabolism is perturbed, AGs concentrate PB components with the noticeable exception of the 5' to 3' exonuclease XRN1. Altogether, we show that AGs constitute novel mRNA-containing cytoplasmic foci and we propose that they could protect translatable mRNAs from degradation, contributing thus to ALK-mediated oncogenicity.     

Identification of FUSE-binding proteins as interacting partners of TIA proteins

TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm.     

Poliovirus-mediated disruption of cytoplasmic processing bodies

Metazoan cells form cytoplasmic mRNA granules such as stress granules (SG) and processing bodies (P bodies) that are proposed to be sites of aggregated, translationally silenced mRNAs and mRNA degradation. Poliovirus (PV) is a plus-strand RNA virus containing a genome that is a functional mRNA; thus, we investigated if PV antagonizes the processes that lead to formation of these structures. We have previously shown that PV infection inhibits the ability of cells to form stress granules by cleaving RasGAP-SH3-binding protein (G3BP). Here, we show that P bodies are also disrupted during PV infection in cells by 4 h postinfection. The disruption of P bodies is more rapid and more complete than disruption of stress granules. The kinetics of P body disruption correlated with production of viral proteinases and required substantial viral gene product expression. The organizing mechanism that forms P body foci in cells is unknown; however, potential scaffolding, aggregating, or other regulatory proteins found in P bodies were investigated for degradation. Two factors involved in 5'-end mRNA decapping and degradation, Xrn1 and Dcp1a, and the 3' deadenylase complex component Pan3 underwent accelerated degradation during infection, and Dcp1a may be a direct substrate of PV 3C proteinase. Several other key factors proposed to be essential for P body formation, GW182, Edc3, and Edc4, were unaffected by poliovirus infection. Since deadenylation has been reported to be required for P body formation, viral inhibition of deadenylation, through Pan3 degradation, is a potential mechanism of P body disruption.     

A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies

mRNP remodeling events required for the transition of an mRNA from active translation to degradation are currently poorly understood. We identified protein factors potentially involved in this transition, which are present in mammalian P bodies, cytoplasmic foci enriched in 5' --> 3' mRNA degrading enzymes. We demonstrate that human P bodies contain the cap-binding protein eIF4E and the related factor eIF4E-transporter (eIF4E-T), suggesting novel roles for these proteins in targeting mRNAs for 5' --> 3' degradation. Furthermore, fluorescence resonance energy transfer (FRET) studies indicate that eIF4E interacts with eIF4E-T and the putative DEAD box helicase rck/p54 in the P bodies in vivo. RNAi-mediated knockdowns revealed that a subset of P body factors, including eIF4E-T, LSm1, rck/p54, and Ccr4 are required for the accumulation of each other and eIF4E in P bodies. In addition, treatment of HeLa cells with cycloheximide, which inhibits translation, revealed that mRNA is also required for accumulation of mRNA degradation factors in P bodies. In contrast, knockdown of the decapping enzyme Dcp2, which initiates the actual 5' --> 3' mRNA degradation did not abolish P body formation, indicating it first functions after mRNPs have been targeted to these cytoplasmic foci. These data support a model in which mRNPs undergo several successive steps of remodeling and/or 3' trimming until their composition or structural organization promotes their accumulation in P bodies.     

Processing bodies and germ granules are distinct RNA granules that interact in C. elegans embryos

In somatic cells, untranslated mRNAs accumulate in cytoplasmic foci called processing bodies or P-bodies. P-bodies contain complexes that inhibit translation and stimulate mRNA deadenylation, decapping, and decay. Recently, certain P-body proteins have been found in germ granules, RNA granules specific to germ cells. We have investigated a possible connection between P-bodies and germ granules in Caenorhabditis elegans. We identify PATR-1, the C. elegans homolog of the yeast decapping activator Pat1p, as a unique marker for P-bodies in C. elegans embryos. We find that P-bodies are inherited maternally as core granules that mature differently in somatic and germline blastomeres. In somatic blastomeres, P-bodies recruit the decapping activators LSM-1 and LSM-3. This recruitment requires the LET-711/Not1 subunit of the CCR4-NOT deadenylase and correlates spatially and temporally with the onset of maternal mRNA degradation. In germline blastomeres, P-bodies are maintained as core granules lacking LSM-1 and LSM-3. P-bodies interact with germ granules, but maintain distinct dynamics and components. The maternal mRNA nos-2 is maintained in germ granules, but not in P-bodies. We conclude that P-bodies are distinct from germ granules, and represent a second class of RNA granules that behaves differently in somatic and germline cells.     

Stress granules: RNP-containing cytoplasmic bodies arising in stress: Structure and mechanism of organization

The review considers recent data on stress granules, which are dense RNP-containing cytoplasmic bodies that arise under stress conditions, e.g., in heat shock, UV irradiation, energy depletion, and oxidative stress. There is evidence that stress granules accumulate incomplete initiation complexes containing mRNA associated with proteins, small ribosomal subunits, and some translation initiation factors, and that stress granules are formed when cells are depleted of the ternary complex (eIF2-tRNA^Met-GTP), in particular, upon eIF2A phosphorylation or a decrease in GTP. Large ribosomal subunits and the ternary complex are absent from stress granules. The structural basis of stress granules is known. It is probable, however, that RNA-binding protein TIA-1, which normally occurs in the nucleus, forms prion-like aggregates that serve as scaffolds for other components of stress granules. The cytoskeleton facilitates the accumulation of stress granule components in local cytoplasmic sites. Studies of the formation and composition of stress granules are important for a better understanding of the regulation of translation initiation in vivo and the mechanisms of the cell response to stress factors.     

ARE-mRNA degradation requires the 5'-3' decay pathway

As an important mode of suppressing gene expression, messenger RNAs containing an AU-rich element (ARE) in the 3' untranslated region are rapidly degraded in the cytoplasm. ARE-mediated mRNA decay (AMD) is initiated by deadenylation, and in vitro studies have indicated that subsequent degradation occurs in the 3'-5' direction through a complex of exonucleases termed the exosome. An alternative pathway of mRNA degradation occurs at processing bodies, cytoplasmic foci that contain decapping enzymes, the 5'-3' exonuclease Xrn1 and the Lsm1-7 heptamer. To determine which of the two pathways is important for AMD in live cells, we targeted components of both pathways using short interfering RNA in human HT1080 cells. We show that Xrn1 and Lsm1 are essential for AMD. On the other side, out of three exosome components tested, only knockdown of PmScl-75 caused a strong inhibition of AMD. Our results show that mammalian cells, similar to yeast, require the 5'-3' Xrn1 pathway to degrade ARE-mRNAs.     

Gawky is a component of cytoplasmic mRNA processing bodies required for early Drosophila development

In mammalian cells, the GW182 protein localizes to cytoplasmic bodies implicated in the regulation of messenger RNA (mRNA) stability, translation, and the RNA interference pathway. Many of these functions have also been assigned to analogous yeast cytoplasmic mRNA processing bodies. We have characterized the single Drosophila melanogaster homologue of the human GW182 protein family, which we have named Gawky (GW). Drosophila GW localizes to punctate, cytoplasmic foci in an RNA-dependent manner. Drosophila GW bodies (GWBs) appear to function analogously to human GWBs, as human GW182 colocalizes with GW when expressed in Drosophila cells. The RNA-induced silencing complex component Argonaute2 and orthologues of LSm4 and Xrn1 (Pacman) associated with 5'-3' mRNA degradation localize to some GWBs. Reducing GW activity by mutation or antibody injection during syncytial embryo development leads to abnormal nuclear divisions, demonstrating an early requirement for GWB-mediated cytoplasmic mRNA regulation. This suggests that gw represents a previously unknown member of a small group of genes that need to be expressed zygotically during early embryo development.     

GW body disassembly triggered by siRNAs independently of their silencing activity

GW bodies (or P-bodies) are cytoplasmic granules containing proteins involved in both mRNA degradation and storage, including the RNA interference machinery. Their mechanism of assembly and function are still poorly known although their number depends upon the flux of mRNA to be stored or degraded. We show here that silencing of the translational regulator CPEB1 leads to their disappearance, as reported for other GW body components. Surprisingly, the same results were obtained with several siRNAs targeting genes encoding proteins unrelated to mRNA metabolism. The disappearance of GW bodies did not correlate with the silencing activity of the siRNA and did not inhibit further silencing by siRNA. Importantly, in most cases, GW bodies were rapidly reinduced by arsenite, indicating that their assembly was not prevented by the inhibition of the targeted or off-target genes. We therefore propose that some siRNA sequences affect mRNA metabolism so as to diminish the amount of mRNA directed to the GW bodies. As an exception, GW bodies were not reinduced following Rck/p54 depletion by interference, indicating that this component is truly required for the GW body assembly. Noteworthy, Rck/p54 was dispensable for the assembly of stress granules, in spite of their close relationship with the GW bodies.     

Insulin silences apolipoprotein B mRNA translation by inducing intracellular traffic into cytoplasmic RNA granules

Insulin is a potent inducer of global mRNA translation and protein synthesis, yet it negatively regulates apolipoprotein B (apoB) mRNA translation, via an unknown mechanism. ApoB mRNA has a long half-life of 16 h, suggesting intracellular storage as mRNPs likely in the form of RNA granules. The availability of apoB mRNA for translation may be regulated by the rate of release from translationally silenced mRNPs within cytoplasmic foci called processing bodies (P bodies). In this report, we directly imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by entering cytoplasmic P bodies. We assessed the colocalization of apoB mRNA and β-globin mRNA (as a control) with P body (PB) markers using a strong interaction between the bacteriophage capsid protein MS2 and a sequence specific RNA stem-loop structure. We observed statistically significant increases in the localization of apoB mRNA into P bodies 4-16 h after insulin treatment (by 72-89%). The movement of apoB mRNA into cytoplasmic P bodies correlated with reduced translational efficiency as assessed by polysomal profiling and measurement of apoB mRNA abundance. PB localization of β-globin mRNA was insensitive to insulin treatment, suggesting selective regulation of apoB mRNA by insulin. Overall, our data suggest that insulin may specifically silence apoB mRNA translation by reprogramming its mRNA into P bodies and reducing the size of translationally competent mRNA pools. Translational control via traffic into cytoplasmic RNA granules may be an important mechanism for controlling the rate of apoB synthesis and hepatic lipoprotein production.     

Poliovirus unlinks TIA1 aggregation and mRNA stress granule formation

In response to environmental stress and viral infection, mammalian cells form foci containing translationally silenced mRNPs termed stress granules (SGs). As aggregates of stalled initiation complexes, SGs are defined by the presence of translation initiation machinery in addition to mRNA binding proteins. Here, we report that cells infected with poliovirus (PV) can form SGs early that contain T-cell-restricted intracellular antigen 1 (TIA1), translation initiation factors, RNA binding proteins, and mRNA. However, this response is blocked as infection progresses, and a type of pseudo-stress granule remains at late times postinfection and contains TIA but lacks translation initiation factors, mRNA binding proteins, and most polyadenylated mRNA. This result was observed using multiple stressors, including viral infection, oxidative stress, heat shock, and endoplasmic reticulum stress. Multiple proteins required for efficient viral internal ribosome entry site-dependent translation are localized to SGs under stress conditions, providing a potential rationale for the evolution and maintenance of the SG inhibition phenotype. Further, the expression of a noncleavable form of the RasGAP-SH3 domain binding protein in PV-infected cells enables SGs whose constituents are consistent with the presence of stalled 48S translation preinitiation complexes to persist throughout infection. These results indicate that in poliovirus-infected cells, the functions of TIA self-aggregation and aggregation of stalled translation initiation complexes into stress granules are severed, leading to novel foci that contain TIA1 but lack other stress granule-defining components.