mRNA Processing Bodies Research Articles

Expression of maize OXS2a in Arabidopsis stunts plant growth but enhances heat tolerance

As plants encounter various environmental stresses, judicial allocation of resources to stress response is crucial for plant fitness. The plant OXS2 (OXIDATIVE STRESS 2) family has been reported to play important roles in growth regulation and stress response. Here, we report that the maize OXS2 family member ZmOXS2a when expressed in Arabidopsis retards growth including delayed flowering, but improves heat tolerance. ZmOXS2a can be found in the cytoplasm, nucleus and PBs/P bodies (mRNA processing bodies), but heat treatment induces higher accumulation in the PBs. Deletion of ARR (arginine rich region) and TZF (tandem zinc finger) domains for high-affinity RNA-binding reduced PBs accumulation of ZmOXS2a; and unlike ZmOXS2a, expression of this deletion mutant gene affected neither Arabidopsis growth nor heat tolerance. This suggests that ZmOXS2a might be involved in RNA degradation, which would also account for the larger amount of down-regulated genes found in ZmOXS2a expressing lines. Furthermore, 240 of 890 down-regulated genes contain ARE (AU-rich elements) in the mRNA 3′UTR that might be potential targets of ZmOXS2a. Expression of ZmOXS2a also disturbs the response to ABA (abscisic acid) and cytokinin, as GO (gene ontology) analysis shows that 50 and 15 DEGs (differentially expressed genes) are enriched in the GO term for ABA and cytokinin responses, respectively. ZmOXS2a expression lines are more sensitive to ABA, but less sensitive to cytokinin. It is likely that ZmOXS2a promotes the degradation of the mRNA of down-regulated genes containing ARE, which consequently perturbs the hormone pathways that affect stress response-related plant growth.

Assembly and regulation of the mammalian mRNA processing body.

Messenger RNA processing bodies (P-bodies) are cytoplasmic membrane-free organelles that contain proteins involved in mRNA silencing, storage and decay. The mechanism by which P-body components interact and the factors that regulate the stability of these structures are incompletely understood. In this study, we used a fluorescence-based, two-hybrid assay to investigate interactions between P-body components that occur inside the cell. LSm14a, PATL1, XRN1, and NBDY were found to interact with the N-terminal, WD40-domain-containing portion of EDC4. The N-terminus of full-length PATL1 was required to mediate the interaction between EDC4 and DDX6. The C-terminal, alpha helix-domain- containing portion of EDC4 was sufficient to mediate interaction with DCP1a and CCHCR1. In the absence of endogenous P-bodies, caused by depletion of LSm14a or DDX6, expression of the portion of EDC4 that lacked the N-terminus retained the ability to form cytoplasmic dots that were indistinguishable from P-bodies at the level of UV light microscopy. Despite the absence of endogenous P-bodies, this portion of EDC4 was able to recruit DCP1a, CCHCR1 and EDC3 to cytoplasmic dots. The results of this study permit the development of a new model of P-body formation and suggest that the N-terminus of EDC4 regulates the stability of these structures.

Non-synonymous, synonymous, and non-coding nucleotide variants contribute to recurrently altered biological processes during retinoblastoma progression.

Retinoblastomas form in response to biallelic RB1 mutations or MYCN amplification and progress to more aggressive and therapy-resistant phenotypes through accumulation of secondary genomic changes. Progression-related changes include recurrent somatic copy number alterations and typically non-recurrent nucleotide variants, including synonymous and non-coding variants, whose significance has been unclear. To determine if nucleotide variants recurrently affect specific biological processes, we identified altered genes and over-represented variant gene ontologies in 168 exome or whole-genome-sequenced retinoblastomas and 12 tumor-matched cell lines. In addition to RB1 mutations, MYCN amplification, and established retinoblastoma somatic copy number alterations, the analyses revealed enrichment of variant genes related to diverse biological processes including histone monoubiquitination, mRNA processing (P) body assembly, and mitotic sister chromatid segregation and cytokinesis. Importantly, non-coding and synonymous variants increased the enrichment significance of each over-represented biological process term. To assess the effects of such mutations, we examined the consequences of a 3' UTR variant of PCGF3 (a BCOR-binding component of Polycomb repressive complex I), dual 3' UTR variants of CDC14B (a regulator of sister chromatid segregation), and a synonymous variant of DYNC1H1 (a regulator of P-body assembly). One PCGF3 and one of two CDC14B 3' UTR variants impaired gene expression whereas a base-edited DYNC1H1 synonymous variant altered protease sensitivity and stability. Retinoblastoma cell lines retained only ~50% of variants detected in tumors and enriched for new variants affecting p53 signaling. These findings reveal potentially important differences in retinoblastoma cell lines and tumors and implicate synonymous and non-coding variants, along with non-synonymous variants, in retinoblastoma oncogenesis.

QKI degradation in macrophage by RNF6 protects mice from MRSA infection via enhancing PI3K p110β dependent autophagy

BackgroundSepsis is a fatal condition commonly caused by Methicillin-resistant Staphylococcus aureus (MRSA) with a high death rate. Macrophages can protect the host from various microbial pathogens by recognizing and eliminating them. Earlier we found that Quaking (QKI), an RNA binding protein (RBP), was involved in differentiation and polarization of macrophages. However, the role of QKI in sepsis caused by pathogenic microbes, specifically MRSA, is unclear. This study aimed to investigate the role of QKI in regulation of host–pathogen interaction in MRSA-induced sepsis and explored the underlying mechanisms.MethodsTransmission electron microscope and immunofluorescence were used to observe the autophagy level in macrophages. Real-time PCR and western blot were used to analyzed the expression of mRNA and protein respectively. The potential protein interaction was analyzed by iTRAQ mass spectrometry and Immunoprecipitation. RNA fluorescence in situ hybridization, dual-luciferase reporter assay and RNA immunoprecipitation were used to explore the mechanism of QKI regulating mRNA of PI3K-p110β.ResultsThe mRNA level of QKI was aberrantly decreased in monocytes and PBMCs of septic patients with the increasing level of plasma procalcitonin (PCT). Then the mice with myeloid specific knockout of QKI was challenged with MRSA or Cecal Ligation and Puncture (CLP). Mice in these two models displayed higher survival rates and lower bacterial loads. Mechanistically, QKI deletion promoted phagocytosis and autophagic degradation of MRSA via activating p110β (a member of Class IA phosphoinositide 3-kinases) mediated autophagic response. QKI expression in macrophages led to the sequestration of p110β in mRNA processing (P) bodies and translational repression. Upon infection, the direct interaction of RNF6, a RING-type E3 ligase, mediated QKI ubiquitination degradation and facilitated PI3K-p110β related autophagic removal of pathogen. The administration of nanoparticles with QKI specific siRNA significantly protected mice from MRSA infection.ConclusionsThis study disclosed the novel function of QKI in the P body mRNA regulation during infection. QKI degradation in macrophage by RNF6 protects mice from MRSA infection via enhancing PI3K-p110β dependent autophagy. It suggested that QKI may serve as a potential theranostic marker in MRSA-induced sepsis.Graphical

RNA Binding Protein OsTZF7 Traffics Between the Nucleus and Processing Bodies/Stress Granules and Positively Regulates Drought Stress in Rice.

Tandem CCCH zinc finger (TZF) proteins are the essential components of processing bodies (PBs) and stress granules (SGs), which play critical roles in growth development and stress response in both animals and plants through posttranscriptional regulation of target mRNA. In this study, we characterized the biological and molecular functions of a novel tandem zinc finger protein, OsTZF7. The expression of OsTZF7 was upregulated by abiotic stresses, including polyethylene glycol (PEG) 4000, NaCl, and abscisic acid (ABA) in rice. Accordingly, the overexpression of OsTZF7 increased drought tolerance and enhanced sensitivity to exogenous ABA in rice, whereas the knockdown of OsTZF7 resulted in the opposite phenotype. RNA-seq analysis revealed that genes related to “response to stress,” “abscisic acid signaling,” “methylated histone binding,” and “cytoplasmic mRNA processing body” are regulated by OsTZF7. We demonstrated that OsTZF7 can traffic between the nucleus and PBs/SGs, and the leucine-rich nuclear export signal (NES) mediates the nuclear export of OsTZF7. Additionally, we revealed that OsTZF7 can bind adenine- and uridine-rich (AU-rich) element (ARE) or ARE-like motifs within the 3′ untranslated region of downregulated mRNAs, and interact with PWWP family proteins in vitro. Together, these results indicate that OsTZF7 positively regulates drought response in rice via ABA signaling and may be involved in mRNA turnover.

Atomic-level insight into mRNA processing bodies by combining solid and solution-state NMR spectroscopy

Liquid–liquid phase separation is increasingly recognized as a process involved in cellular organization. Thus far, a detailed structural characterization of this intrinsically heterogeneous process has been challenging. Here we combine solid- and solution-state NMR spectroscopy to obtain atomic-level insights into the assembly and maturation of cytoplasmic processing bodies that contain mRNA as well as enzymes involved in mRNA degradation. In detail, we have studied the enhancer of decapping 3 (Edc3) protein that is a central hub for processing body formation in yeast. Our results reveal that Edc3 domains exhibit diverse levels of structural organization and dynamics after liquid–liquid phase separation. In addition, we find that interactions between the different Edc3 domains and between Edc3 and RNA in solution are largely preserved in the condensed protein state, allowing processing bodies to rapidly form and dissociate upon small alterations in the cellular environment.

TFL Deletion Induces Incredible CXCL13 Secretion and Cachexia in Vavp-Bcl2Transgenic Mice

Diffuse large B-cell lymphomas (DLBCL) are heterogeneous diseases caused by several genetic aberrations. The novel post-transcriptional regulator gene called transformed follicular lymphoma (TFL) was first identified from t(2;6)(p12;q23), which appeared during the transformation of FL to DLBCL (Minagawa et al. Br J Haematol 2007). Normal human lymphocytes generally express TFL, but it is defective in some leukemia/lymphoma cell lines. TFL overexpression in such cell lines inhibited cell growth, suggesting that TFL functions as a tumor suppressor (Minagawa et al. Mol Cancer Res 2009). TFL locates in mRNA processing body in the cytoplasm and has the unique CCCH-type zinc finger motif functioning as RNase. TFL regulates several cytokines, including IL-2, IL-6, IL-10, TNF-α, and IL-17a, via mRNA degradation. In an experimental autoimmune encephalitis model, TFL null mice (TFL-/-) demonstrated persistent paralysis, resulting from more infiltration of Th17 cells into CNS with markedly increased IL-17a mRNA levels. Therefore, a TFL-driven feedback mechanism for excessive inflammation is indispensable to suppress T-cell-mediated autoimmune diseases (Minagawa et al. J Immunol 2014).TFL deletion examined by FISH using a 110kbp DNA probe containing an entireTFL locus was found in 12.8% of mature B-cell lymphomas (n=86, FL=30, DLBCL=40). However, the pathological significance of TFL deletion has not yet been clarified. To investigate how TFL loss affects lymphoma biology, we developed VavP-bcl2 transgenic (Bcl2-Tg)/TFL-/-mice. Although the survival of TFL-/- was comparable to the wild-type, Bcl2-Tg/TFL-/- died about 19 weeks earlier than Bcl2-Tg (Fig. 1). Both strains developed lymphadenopathy and splenomegaly similarly. No different microscopic finding was noted in lymph nodes, spleen, or bone marrow (BM). No additional malignancy was found in Bcl2-Tg/TFL-/- on autopsy. However, significant body weight loss appeared by 30 weeks in Bcl2-Tg/TFL-/- but not in Bcl2-Tg (Fig. 3).To identify what causes earlier death in Bcl2-Tg/TFL-/-, we carefully examined the phenotypic change of BM lymphocytes. We found a unique B220-IgM+ population in Bcl2-Tg BM, which was not found in wild-type. We speculated that TFL deficiency in this population might drive the deterioration in Bcl2-Tg/TFL-/-. To identify which mRNA was dysregulated by TFL deficiency, we comprehensively analyzed mRNA expression profiles in B220-IgM+ cells in both strains using cDNA microarray chip. Among several genes upregulated at least threefold in Bcl2-Tg/TFL-/- than Bcl2-Tg, we paid attention to CXCL13, the mRNA expression of which in Bcl2-Tg/TFL-/- was 4.19-fold higher than that in Bcl2-Tg (p=0.03). In fact, CXCL13 concentration in BM extracellular fluid as well as plasma in Bcl2-Tg/TFL-/- showed incredible increase in a logarithmic scale (Fig. 2). As a noteworthy event, body weight loss in Bcl2-Tg/TFL-/- followed the increase of CXCL13 in plasma by 30 weeks (Fig. 3). To confirm that TFL post-transcriptionally regulates CXCL13 mRNA through the degradation of its 3′UTR, we performed a reporter assay with a plasmid vector containing 3′UTR of CXCL13 mRNA. Co-transfection with a TFL expression vector showed decreased luciferase activity compared to the control. This suggests that TFL directly regulates CXCL13 mRNA via its 3′UTR degradation. This regulation occurs more prominently in B-cell lineage rather than myeloid or T-cell lineage, whereas IL-2 mRNA regulation occurs promiscuously.CXCL13 secretion was significantly increased in the culture supernatant of BM cells but not spleen cells derived from Bcl2-Tg/TFL-/-. We further sorted several cell populations, including B220-IgM+ in BM, and cultured them for 96 h. CXCL13 secretion from B220-IgM+ population was increased significantly compared to other populations. Thus, we concluded that B220-IgM+ cells in BM are the main producer of CXCL13 in Bcl2-Tg/TFL-/-. Loss of TFL-driven attenuation for excessive inflammation in lymphoma-bearing mice could contribute to the short survival. It is of interest whether high plasma CXCL13 directly affects cachexia and early death in Bcl2-Tg/TFL-/-.TFL deletion in human lymphoma might contribute not only to malignant transformation but also to a major B symptom, i.e., weight loss. Our findings may open a new window for the predictive factor on the prognosis of B-cell lymphoma and/or new therapeutic intervention by targeting CXCL13. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Maintenance of Proteostasis by P Body-Mediated Regulation of eIF4E Availability during Aging in Caenorhabditis elegans

SummaryAging is accompanied by a pervasive collapse of proteostasis, while reducing general protein synthesis promotes longevity across taxa. Here, we show that the eIF4E isoform IFE-2 is increasingly sequestered in mRNA processing (P) bodies during aging and upon stress in Caenorhabditis elegans. Loss of the enhancer of mRNA decapping EDC-3 causes further entrapment of IFE-2 in P bodies and lowers protein synthesis rates in somatic tissues. Animals lacking EDC-3 are long lived and stress resistant, congruent with IFE-2-deficient mutants. Notably, neuron-specific expression of EDC-3 is sufficient to reverse lifespan extension, while sequestration of IFE-2 in neuronal P bodies counteracts age-related neuronal decline. The effects of mRNA decapping deficiency on stress resistance and longevity are orchestrated by a multimodal stress response involving the transcription factor SKN-1, which mediates lifespan extension upon reduced protein synthesis. Our findings elucidate a mechanism of proteostasis control during aging through P body-mediated regulation of protein synthesis in the soma.

P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance

mRNA-processing (P-) bodies are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete complement of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the abundance of HHT1, ACF4, ARL3, TMA16, RRS1 and YOX1 mRNAs to prevent their toxic accumulation during replication stress. Accumulation of YOX1 mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and leads to toxic acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response.

Cytoplasmic overexpression of RNA-binding protein HuR is a marker of poor prognosis in meningioma, and HuR knockdown decreases meningioma cell growth and resistance to hypoxia.

HuR regulates cytoplasmic mRNA stability and translatability, and the HuR expression level has been shown to correlate with poor disease outcome in several cancer types; however, the prognostic value and potential pro-oncogenic properties of HuR in meningioma remain unclear. Thus, in the present study, we analysed 85 meningioma tissue samples to establish the relationship between HuR expression, tumour cell proliferation, and/or patient survival. In addition, we examined the anti-proliferative effects of HuR knockdown in two meningioma cell lines (IOMM-Lee and Ben-Men-1) and conducted transcriptome-wide analyses (IOMM-Lee cells) to elucidate the molecular consequences of HuR knockdown. The results of the present study showed HuR cytoplasmic expression to correlate positively with tumour grade (p = 1.2 × 10-8 ) and negatively with progression-free and overall survival (p = 0.01) time in human meningioma tissues. In vitro, siHuR-induced HuR knockdown was shown to reduce the growth of both Ben-Men-1 (p = 2 × 10-8 ) and IOMM-Lee (p = 4 × 10-9 ) cells. Transcriptome analyses revealed HuR knockdown in IOMM-Lee cells to deregulate the HIF1A signalling pathway (p = 1.5 × 10-6 ) and to up-regulate the expression of genes essential for the assembly of the cytoplasmic mRNA processing body, global genome nucleotide-excision repair, poly(A)-specific ribonuclease activity, the positive regulation of apoptosis and of cell cycle arrest, and the negative regulation of RNA splicing [p(FDR) < 0.001]. Interestingly, HuR knockdown under hypoxic culture conditions further potentiated the effects of HuR knockdown on cell growth, apoptosis, and HIF1A expression. We thus conclude that cytoplasmic HuR expression is a marker of poor prognosis in meningioma and that HuR is a promising potential therapeutic target for use in tumours refractory to standard therapies. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Ribonucleoprotein bodies are phased in.

Intracellular compartments are necessary for the regulation of many biochemical processes that ensure cell survival, growth and proliferation. Compartmentalisation is commonly achieved in organelles with defined lipid membranes, such as mitochondria, endoplasmic reticulum or the Golgi apparatus. While these organelles are responsible for many localised biochemical processes, recent evidence points to another class of compartments that lack membrane boundaries. The structure and content of these bodies depend on their function and subcellular localisation, but they mainly incorporate proteins and RNA. Examples of these ribonucleoprotein bodies (RNPBs) include eukaryotic mRNA processing bodies (P-bodies) and stress granules (SGs). While most of these structures have been widely studied for their capacity to bind, store and process mRNAs under different conditions, their biological functions and physical properties are poorly understood. Recent intriguing data suggest that liquid-liquid phase separation (LLPS) represents an important mechanism seeding the formation and defining the function of RNPBs. In this review, we discuss how LLPS is transforming our ideas about the biological functions of SGs and P-bodies and their link to diseases.

Identification of novel nesprin-1 binding partners and cytoplasmic matrin-3 in processing bodies.

Nesprins are highly conserved spectrin repeat-containing scaffold proteins predominantly known to function at the nuclear envelope (NE). However, nesprin isoforms are emerging with localizations and scaffolding functions at sites away from the NE, suggesting their functions are more diverse than originally thought. In this study, we combined nesprin-1 coimmunoprecipitations with mass spectrometry to identify novel nesprin-1 binding partners for isoforms that localize to subcellular compartments beyond the NE. We show that one of these interactors, matrin-3 (matr3), localizes to mRNA processing bodies (PBs), where we have previously shown a nesprin-1 isoform to localize. Furthermore, we show that Matr3 is part of PB mRNP complexes, is a regulator of miRNA-mediated gene silencing, and possibly shuttles to stress granules in stressed cells. More importantly, we identify a new C-terminally truncated Matr3 isoform that is likely to be involved in these functions and PB localization. This study highlights several novel nesprin-1 binding partners and a new function and localization for Matr3 in cytoplasmic RNA granules.

The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization

Members of the highly conserved class of BEACH domain containing proteins (BDCPs) have been established as broad facilitators of protein–protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI) is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1), associates to mRNA processing bodies (P-bodies), and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation) interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

AtCCR4a and AtCCR4b are Involved in Determining the Poly(A) Length of Granule-bound starch synthase 1 Transcript and Modulating Sucrose and Starch Metabolism in Arabidopsis thaliana.

Removing the poly(A) tail is the first and rate-limiting step of mRNA degradation and apparently an effective step not only for modulating mRNA stability but also for translation of many eukaryotic transcripts. Carbon catabolite repressor 4 (CCR4) has been identified as a major cytoplasmic deadenylase in Saccharomyces cerevisiae. The Arabidopsis thaliana homologs of the yeast CCR4, AtCCR4a and AtCCR4b, were identified by sequence-based analysis; however, their role and physiological significance in plants remain to be elucidated. In this study, we revealed that AtCCR4a and AtCCR4b are localized to cytoplasmic mRNA processing bodies, which are specific granules consisting of many enzymes involved in mRNA turnover. Double mutants of AtCCR4a and AtCCR4b exhibited tolerance to sucrose application but not to glucose. The levels of sucrose in the seedlings of the atccr4a/4b double mutants were reduced, whereas no difference was observed in glucose levels. Further, amylose levels were slightly but significantly increased in the atccr4a/4b double mutants. Consistent with this observation, we found that the transcript encoding granule-bound starch synthase 1 (GBSS1), which is responsible for amylose synthesis, is accumulated to a higher level in the atccr4a/4b double mutant plants than in the control plants. Moreover, we revealed that GBSS1 has a longer poly(A) tail in the double mutant than in the control plant, suggesting that AtCCR4a and AtCCR4b can influence the poly(A) length of transcripts related to starch metabolism. Our results collectively suggested that AtCCR4a and AtCCR4b are involved in sucrose and starch metabolism in A. thaliana.

LMKB/MARF1 localizes to mRNA processing bodies, interacts with Ge-1, and regulates IFI44L gene expression.

The mRNA processing body (P-body) is a cellular structure that regulates the stability of cytoplasmic mRNA. MARF1 is a murine oocyte RNA-binding protein that is associated with maintenance of mRNA homeostasis and genomic stability. In this study, autoantibodies were used to identify Limkain B (LMKB), the human orthologue of MARF1, as a P-body component. Indirect immunofluorescence demonstrated that Ge-1 (a central component of the mammalian core-decapping complex) co-localized with LMKB in P-bodies. Two-hybrid and co-immunoprecipitation assays were used to demonstrate interaction between Ge-1 and LMKB. The C-terminal 120 amino acids of LMKB mediated interaction with Ge-1 and the N-terminal 1094 amino acids of Ge-1 were required for interaction with LMKB. LMKB is the first protein identified to date that interacts with this portion of Ge-1. LMKB was expressed in human B and T lymphocyte cell lines; depletion of LMKB increased expression of IFI44L, a gene that has been implicated in the cellular response to Type I interferons. The interaction between LMKB/MARF1, a protein that contains RNA-binding domains, and Ge-1, which interacts with core-decapping proteins, suggests that LMKB has a role in the regulation of mRNA stability. LMKB appears to have different functions in different cell types: maintenance of genomic stability in developing oocytes and possible dampening of the inflammatory response in B and T cells.

MRNA localization to P-bodies in yeast is bi-phasic with many mRNAs captured in a late Bfr1p-dependent wave

ABSTRACTThe relocalization of translationally repressed mRNAs to mRNA processing bodies Pbodies is a key consequence of cellular stress across many systems. Pbodies harbor mRNA degradation components and are implicated in mRNA decay, but the relative timing and control of mRNA relocalization to Pbodies is poorly understood. We used the MS2GFP system to follow the movement of specific endogenous mRNAs in live Saccharomyces cerevisiae cells after nutritional stress. It appears that the relocalization of mRNA to Pbodies after stress is biphasic some mRNAs are present early, whereas others are recruited much later concomitant with recruitment of translation initiation factors, such as eIF4E. We also find that Bfr1p is a latephaselocalizing Pbody protein that is important for the delayed entry of certain mRNAS to Pbodies. Therefore, for the mRNAs tested, relocalization to Pbodies varies both in terms of the kinetics and factor requirements. This work highlights a potential new regulatory juncture in gene expression that would facilitate the overall rationalization of protein content required for adaptation to stress.

APOBEC3G and APOBEC3F rarely co-mutate the same HIV genome

BackgroundThe human immune proteins APOBEC3G and APOBEC3F (hA3G and hA3F) induce destructive G-to-A changes in the HIV genome, referred to as ‘hypermutation’. These two proteins co-express in human cells, co-localize to mRNA processing bodies and might co-package into HIV virions. Therefore they are expected to also co-mutate the HIV genome. Here we investigate the mutational footprints of hA3G and hA3F in a large population of full genome HIV-1 sequences from naturally infected patients to uniquely identify sequences hypermutated by either or both of these proteins. We develop a method of identification based on the representation of hA3G and hA3F target and product motifs that does not require an alignment to a parental/consensus sequence.ResultsOut of nearly 100 hypermutated HIV-1 sequences only one sequence from the HIV-1 outlier group showed clear signatures of co-mutation by both proteins. The remaining sequences were affected by either hA3G or hA3F.ConclusionUsing a novel method of identification of HIV sequences hypermutated by the hA3G and hA3F enzymes, we report a very low rate of co-mutation of full-length HIV sequences, and discuss the potential mechanisms underlying this.

The RNA-binding protein Y14 inhibits mRNA decapping and modulates processing body formation.

The exon-junction complex (EJC) deposited on a newly spliced mRNA plays an important role in subsequent mRNA metabolic events. Here we show that an EJC core heterodimer, Y14/Magoh, specifically associates with mRNA-degradation factors, including the mRNA-decapping complex and exoribonucleases, whereas another core factor, eIF4AIII/MLN51, does not. We also demonstrate that Y14 interacts directly with the decapping factor Dcp2 and the 5' cap structure of mRNAs via different but overlapping domains and that Y14 inhibits the mRNA-decapping activity of Dcp2 in vitro. Accordingly, overexpression of Y14 prolongs the half-life of a reporter mRNA. Therefore Y14 may function independently of the EJC in preventing mRNA decapping and decay. Furthermore, we observe that depletion of Y14 disrupts the formation of processing bodies, whereas overexpression of a phosphomimetic Y14 considerably increases the number of processing bodies, perhaps by sequestering the mRNA-degradation factors. In conclusion, this report provides unprecedented evidence for a role of Y14 in regulating mRNA degradation and processing body formation and reinforces the influence of phosphorylation of Y14 on its activity in postsplicing mRNA metabolism.

HIV-1 Replication and APOBEC3 Antiviral Activity Are Not Regulated by P Bodies

The APOBEC3 cytidine deaminases play a critical role in host-mediated defense against exogenous viruses, most notably, human immunodeficiency virus type-1 (HIV-1) and endogenous transposable elements. APOBEC3G and APOBEC3F interact with numerous proteins that regulate cellular RNA metabolism, including components of the RNA-induced silencing complex (RISC), and colocalize with a subset of these proteins to mRNA processing bodies (P bodies), which are sites of mRNA translational repression and decay. We sought to determine the role of P bodies and associated proteins in HIV-1 replication and APOBEC3 antiviral activity. While we established a positive correlation between APOBEC3 protein incorporation into virions and localization to P bodies, depletion of the P-body components DDX6 or Lsm1 did not affect HIV-1 replication, APOBEC3 packaging into virions or APOBEC3 protein mediated inhibition of HIV-1 infectivity. In addition, neither HIV-1 genomic RNA nor Gag colocalized with P-body proteins. However, simultaneous depletion of multiple Argonaute family members, the effector proteins of RISC, could modestly increase viral infectivity. Because some APOBEC3 proteins interact with several Argonaute proteins, we also tested whether they could modulate microRNA (miRNA) activity. We found no evidence for the specific regulation of miRNA function by the APOBEC3 proteins, though more general effects on transfected gene expression were observed. In sum, our results indicate that P bodies and certain associated proteins do not regulate HIV-1 replication or APOBEC3 protein antiviral activity. Localization to P bodies may therefore provide a means of sequestering APOBEC3 enzymatic activity away from cellular DNA or may be linked to as yet unidentified cellular functions.

Moloney Leukemia Virus Type 10 Inhibits Reverse Transcription and Retrotransposition of Intracisternal A Particles

Moloney leukemia virus type 10 protein (MOV10) is an RNA helicase that is induced by type I interferon. It inhibits HIV replication at several steps of its replicative cycle. Of interest, MOV10 is a component of mRNA processing (P) bodies, which inhibit retrotransposition (RTP) of intracisternal A particles (IAP). In this report, we studied the effects of MOV10 on IAP RTP and its dependence on P bodies. Indeed, MOV10 inhibited IAP RTP. It decreased significantly not only the products of reverse transcriptase but also its endogenous activity. MOV10 also associated with IAP RNA. Furthermore, although it was found in IAP virus-like particles, it did not affect their incorporation of IAP RNA, primer tRNAPhe (phenylalanine tRNA), or IAP Gag. Concerning P bodies, the exogenously expressed MOV10 had no effect on their size and number, and the inhibition of IAP RTP persisted despite the depletion of their RCK subunit. Thus, by interfering with reverse transcription, MOV10 inhibits IAP RTP, and this inhibition is independent of P bodies.