Cytoplasmic Processing Bodies Research Articles

In a tight spot: ARE-mRNAs at processing bodies: Figure 1.

Compartimentalization of proteins and nucleic acids within nuclear and cytoplasmic domains is essential for many cellular functions (Spector 2006). Although biochemical analysis of cellular lysates has provided important information about the processes of mRNA translation and decay, recent results showing that these events occur at discrete cytoplasmic RNA granules (Anderson and Kedersha 2006) makes it essential that we understand the contribution of subcellular localization to these basic mechanisms. In this issue of Genes & Development, Franks and Lykke-Andersen (2007) describe the importance of cytoplasmic processing bodies (PBs) in regulating the translation and decay of a class of mRNAs bearing an adenine/uridine-rich destabilizing element. Their studies provide important new insights into the link between mRNA translation and decay by revealing the role that RNA granules play in these processes.

Rpm2p, a protein subunit of mitochondrial RNase P, physically and genetically interacts with cytoplasmic processing bodies

The RPM2 gene of Saccharomyces cerevisiae codes for a protein subunit of mitochondrial RNase P and has another unknown essential function. We previously demonstrated that Rpm2p localizes to the nucleus and acts as a transcriptional activator. Rpm2p influences the level of mRNAs that encode components of the mitochondrial import apparatus and essential mitochondrial chaperones. Evidence is presented here that Rpm2p interacts with Dcp2p, a subunit of mRNA decapping enzyme in the two-hybrid assay, and is enriched in cytoplasmic P bodies, the sites of mRNA degradation and storage in yeast and mammalian cells. When overexpressed, GFP-Rpm2p does not impact the number and size of P bodies; however, it prevents their disappearance when translation elongation is inhibited by cycloheximide. Proteasome mutants, ump1-2 and pre4-2, that bypass essential Rpm2p function, also stabilize P bodies. The stabilization of P bodies by Rpm2p may occur through reduced protein degradation since GFP-Rpm2p expressing cells have lower levels of ubiquitin. Genetic analysis revealed that overexpression of Dhh1p (a DEAD box helicase localized to P bodies) suppresses temperature-sensitive growth of the rpm2-100 mutant. Overexpression of Pab1p (a poly (A)-binding protein) also suppresses rpm2-100, suggesting that Rpm2p functions in at least two aspects of mRNA metabolism. The results presented here, and the transcriptional activation function demonstrated earlier, implicate Rpm2p as a coordinator of transcription and mRNA storage/decay in P bodies.

Localization of the Developmental Timing Regulator Lin28 to mRNP Complexes, P-bodies and Stress Granules

Lin28 is a conserved cytoplasmic protein with an unusual pairing of RNA-binding motifs: a cold shock domain and a pair of retroviral-type CCHC zinc fingers. In the nematode C. elegans, it is a regulator of developmental timing. In mammals, it is abundant in diverse types of undifferentiated cells. However, its molecular function is unknown. In pluripotent mammalian cells, Lin28 is observed in RNase-sensitive complexes with Poly(A)-Binding Protein, and in polysomal fractions of sucrose gradients, suggesting it is associated with translating mRNAs. Upon cellular stress, Lin28 locates to stress granules, which contain non-translating mRNA complexes. However, Lin28 also localizes to cytoplasmic Processing bodies, or P-bodies, sites of mRNA degradation and microRNA regulation, consistent with it acting to regulate mRNA translation or stability. Mutational analysis shows that Lin28’s conserved RNA binding domains cooperate to put Lin28 in mRNPs, but that only the CCHC domain is required for localization to P-bodies. When both RNA-binding domains are mutated, Lin28 accumulates in the nucleus, suggesting that it normally shuttles from nucleus to cytoplasm bound to RNA. These studies are consistent with a model in which Lin28 binds mRNAs in the nucleus and accompanies them to ribosomes and P-bodies. We propose that Lin28 influences the translation or stability of specific mRNAs during differentiation.

RAP55, a Cytoplasmic mRNP Component, Represses Translation in Xenopus Oocytes

mRNAs in eukaryotic cells are presumed to always associate with a set of proteins to form mRNPs. In Xenopus oocytes, a large pool of maternal mRNAs is masked from the translational apparatus as storage mRNPs. Here we identified Xenopus RAP55 (xRAP55) as a component of RNPs that associate with FRGY2, the principal component of maternal mRNPs. RAP55 is a member of the Scd6 or Lsm14 family. RAP55 localized to cytoplasmic foci in Xenopus oocytes and the processing bodies (P-bodies) in cultured human cells: in the latter cells, RAP55 is an essential constituent of the P-bodies. We isolated xRAP55-containing complexes from Xenopus oocytes and identified xRAP55-associated proteins, including a DEAD-box protein, Xp54, and a protein arginine methyltransferase, PRMT1. Recombinant xRAP55 repressed translation, together with Xp54, in an in vitro translation system. In addition, xRAP55 repressed translation in oocytes when tethered with a reporter mRNA. Domain analyses revealed that the N-terminal region of RAP55, including the Lsm domain, is important for the localization to P-bodies and translational repression. Taken together, our results suggest that xRAP55 is involved in translational repression of mRNA as a component of storage mRNPs.

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.

Translation Repression in Human Cells by MicroRNA-Induced Gene Silencing Requires RCK/p54

RNA interference is triggered by double-stranded RNA that is processed into small interfering RNAs (siRNAs) by Dicer enzyme. Endogenously, RNA interference triggers are created from small noncoding RNAs called microRNAs (miRNAs). RNA-induced silencing complexes (RISC) in human cells can be programmed by exogenously introduced siRNA or endogenously expressed miRNA. siRNA-programmed RISC (siRISC) silences expression by cleaving a perfectly complementary target mRNA, whereas miRNA-induced silencing complexes (miRISC) inhibits translation by binding imperfectly matched sequences in the 3′ UTR of target mRNA. Both RISCs contain Argonaute2 (Ago2), which catalyzes target mRNA cleavage by siRISC and localizes to cytoplasmic mRNA processing bodies (P-bodies). Here, we show that RCK/p54, a DEAD box helicase, interacts with argonaute proteins, Ago1 and Ago2, in affinity-purified active siRISC or miRISC from human cells; directly interacts with Ago1 and Ago2 in vivo, facilitates formation of P-bodies, and is a general repressor of translation. Disrupting P-bodies by depleting Lsm1 did not affect RCK/p54 interactions with argonaute proteins and its function in miRNA-mediated translation repression. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm but did not significantly affect siRNA-mediated RNA functions of RISC. Depleting RCK/p54 released general, miRNA-induced, and let-7-mediated translational repression. Therefore, we propose that translation repression is mediated by miRISC via RCK/p54 and its specificity is dictated by the miRNA sequence binding multiple copies of miRISC to complementary 3′ UTR sites in the target mRNA. These studies also suggest that translation suppression by miRISC does not require P-body structures, and location of miRISC to P-bodies is the consequence of translation repression.

Relief of microRNA-Mediated Translational Repression in Human Cells Subjected to Stress

In metazoans, most microRNAs imperfectly base-pair with the 3' untranslated region (3'UTR) of target mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing microRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing its 3'UTR can be relieved from the microRNA miR-122-induced inhibition in human hepatocarcinoma cells subjected to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies and its recruitment to polysomes. The derepression requires binding of HuR, an AU-rich-element binding protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.

Targeting of Aberrant mRNAs to Cytoplasmic Processing Bodies

In eukaryotes, a specialized pathway of mRNA degradation termed nonsense-mediated decay (NMD) functions in mRNA quality control by recognizing and degrading mRNAs with aberrant termination codons. We demonstrate that NMD in yeast targets premature termination codon (PTC)-containing mRNA to P-bodies. Upf1p is sufficient for targeting mRNAs to P-bodies, whereas Upf2p and Upf3p act, at least in part, downstream of P-body targeting to trigger decapping. The ATPase activity of Upf1p is required for NMD after the targeting of mRNAs to P-bodies. Moreover, Upf1p can target normal mRNAs to P-bodies but not promote their degradation. These observations lead us to propose a new model for NMD wherein two successive steps are used to distinguish normal and aberrant mRNAs.

Pulling into a Rest Stop

CELL BIOLOGY The recent explosion of research on the interaction of small RNAs with messenger RNAs (mRNAs), which can lead either to cleavage of the double-stranded RNA complex or to translational repression, has intersected with studies of the life cycles of mRNAs, which in some cases spend part of their time in cytoplasmic processing bodies in a translationally dormant state, before degradation or reactivation. Beliakova-Bethell et al. have observed that the protein and RNA components of the yeast retrovirus-like element Ty3 congregate in cytoplasmic foci that also contain nascent virus-like particles. These sites turn out to be a way station for other proteins already shown to reside in processing bodies, among them Dhh1, a helicase that is involved in translational represssion and is required for Ty3 retrotransposition. Hence it appears that the demands of assembling proteins onto an RNA genome may be facilitated by the translational stasis imposed within processing bodies. — GJC RNA 12 , 94 (2006).

Stress-induced Reversal of MicroRNA Repression and mRNA P-body Localization in Human Cells

In metazoa, microRNAs (miRNAs) imperfectly base-pair with the 3'-untranslated region (3'UTR) of mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing miRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here, we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing the CAT-1 3'UTR or its fragments can be relieved from the miRNA miR-122-induced inhibition in human hepatoma cells in response to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies (P bodies) and its recruitment to polysomes, indicating that P bodies act as storage sites for mRNAs inhibited by miRNAs. The derepression requires binding of HuR, an AU-rich-element-binding ELAV family protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.

Multiple Processing Body Factors and the ARE Binding Protein TTP Activate mRNA Decapping

Decapping is a key step in mRNA turnover. However, the composition and regulation of the human decapping complex is poorly understood. Here, we identify three proteins that exist in complex with the decapping enzyme subunits hDcp2 and hDcp1: hEdc3, Rck/p54, and a protein in decapping we name Hedls. Hedls is important in decapping because it enhances the activity of the catalytic hDcp2 subunit and promotes complex formation between hDcp2 and hDcp1. Specific decapping factors interact with the mRNA decay activators hUpf1 and TTP, and TTP enhances decapping of a target AU-rich element (ARE) RNA in vitro. Each decapping protein localizes in cytoplasmic processing bodies (PBs), and overexpression of Hedls produces aberrant PBs and concomitant accumulation of a deadenylated ARE-mediated mRNA decay intermediate. These observations suggest that multiple proteins involved in human decapping are important subunits of PBs and are activated on ARE-mRNAs by the protein TTP.

MicroRNA function: Multiple mechanisms for a tiny RNA?: FIGURE 1.

MicroRNAs are sequence-specific regulators of post-transcriptional gene expression in many eukaryotes. They are believed to control the expression of thousands of target mRNAs, with each mRNA believed to be targeted by multiple microRNAs. Recent studies have uncovered various mechanisms by which microRNAs down-regulate their target mRNAs and have linked a well-known subcellular structure, the cytoplasmic processing bodies (PBs) to the microRNA pathway. The finding that microRNAs are misexpressed in cancers has reinforced the idea that their regulatory roles are very important.

Noncoding RNAs at Work

MicroRNAs (miRNAs), one type of small noncoding RNAs (ncRNAs), are about 21 nucleotides in length and are believed to regulate gene expression either through messenger RNA (mRNA) cleavage or by translational repression. Pillai et al. show that in human cells, the miRNA let-7 represses gene expression by inhibiting translation initiation of capped mRNAs rather than through a degradation mechanism. This repressive machinery appears to be localized to cytoplasmic processing (P) bodies, where mRNAs are stored or degraded. A large fraction of eukaryotic genomes are transcribed into ncRNAs, some of which, such as miRNAs or the much larger Xist ncRNA, have known functions. However, the great majority of ncRNAs are of unknown functional significance. Willingham et al. have developed a method for identifying functional ncRNAs--looking for evolutionary conservation and using a battery of cell-based RNA-interference assays--and have characterized the noncoding repressor of NFAT (NRON) that represses the transcription factor NFAT (nuclear factor of regulated T cells), probably through modulation of NFAT's cellular localization. R. S. Pillai, S. N. Bhattacharyya, C. G. Artus, T. Zoller, N. Cougot, E. Basyuk, E. Bertrand, W. Filipowicz, Inhibition of translational initiation by let-7 microRNA in human cells. Science 309 , 1573-1576 (2005). [Abstract] [Full Text] A. T. Willingham, A. P. Orth, S. Batalov, E. C. Peters, B. G. Wen, P. Aza-Blanc, J. B. Hogenesch, P. G. Schultz, A strategy for probing the function of noncoding RNAs finds a repressor of NFAT. Science 309 , 1570-1573 (2005). [Abstract] [Full Text]

Movement of Eukaryotic mRNAs Between Polysomes and Cytoplasmic Processing Bodies

Eukaryotic cells contain nontranslating messenger RNA concentrated in P-bodies, which are sites where the mRNA can be decapped and degraded. We present evidence that mRNA molecules within yeast P-bodies can also return to translation. First, inhibiting delivery of new mRNAs to P-bodies leads to their disassembly independent of mRNA decay. Second, P-bodies decline in a translation initiation-dependent manner during stress recovery. Third, reporter mRNAs concentrate in P-bodies when translation initiation is blocked and resume translation and exit P-bodies when translation is restored. Fourth, stationary phase yeast have large P-bodies containing mRNAs that reenter translation when growth resumes. The reciprocal movement of mRNAs between polysomes and P-bodies is likely to be important in the control of mRNA translation and degradation. Moreover, the presence of related proteins in P-bodies and maternal mRNA storage granules suggests this mechanism is widely adapted for mRNA storage.

The Developmental Timing Regulator AIN-1 Interacts with miRISCs and May Target the Argonaute Protein ALG-1 to Cytoplasmic P Bodies in C. elegans

In metazoans, microRNAs (miRNAs) carry out various regulatory functions through association with multiprotein miRNA-induced silencing complexes (miRISCs) that contain Dicer and Argonaute proteins. How miRNAs regulate the expression of their mRNA targets remains a major research question. We have identified the C. elegans ain-1 gene through a genetic suppressor screen and shown that it functions with the heterochronic genetic pathway that regulates developmental timing. Biochemical analysis indicates that AIN-1 interacts with protein complexes containing an Argonaute protein, Dicer, and miRNAs. AIN-1 shares homology with the candidate human neurological disease protein GW182, shown to localize in cytoplasmic processing bodies that are sites of mRNA degradation and storage. A functional AIN-1::GFP also localizes at the likely worm processing bodies. When coexpressed from transgenes, AIN-1 targets ALG-1 to the foci. These results suggest a model where AIN-1 regulates a subset of miRISCs by localization to the processing bodies, facilitating degradation or translational inhibition of mRNA targets.

RNAi and the P-body connection

Cytoplasmic processing bodies, also known as P-bodies, concentrate enzymes that are involved in mRNA turnover and sequester mRNAs away from the translational machinery. Strong evidence now connects the RNA interference machinery with P-body formation.

Decapping and decay of messenger RNA occur in cytoplasmic processing bodies.

A major pathway of eukaryotic messenger RNA (mRNA) turnover begins with deadenylation, followed by decapping and 5' to 3' exonucleolytic decay. We provide evidence that mRNA decapping and 5' to 3' degradation occur in discrete cytoplasmic foci in yeast, which we call processing bodies (P bodies). First, proteins that activate or catalyze decapping are concentrated in P bodies. Second, inhibiting mRNA turnover before decapping leads to loss of P bodies; however, inhibiting turnover at, or after, decapping, increases the abundance and size of P bodies. Finally, mRNA degradation intermediates are localized to P bodies. These results define the flux of mRNAs between polysomes and P bodies as a critical aspect of cytoplasmic mRNA metabolism and a possible site for regulation of mRNA degradation.