General translational repression by activators of mRNA decapping

Translation and mRNA degradation are affected by a key transition where eukaryotic mRNAs exit translation and assemble an mRNP state that accumulates into processing bodies (P bodies), cytoplasmic sites of mRNA degradation containing non-translating mRNAs, and mRNA degradation machinery. We identify the decapping activators Dhh1p and Pat1p as functioning as translational repressors and facilitators of P body formation. Strains lacking both Dhh1p and Pat1p show strong defects in mRNA decapping and P body formation and are blocked in translational repression. Contrastingly, overexpression of Dhh1p or Pat1p causes translational repression, P body formation, and arrests cell growth. Dhh1p, and its human homolog, RCK/p54, repress translation in vitro, and Dhh1p function is bypassed in vivo by inhibition of translational initiation. These results identify a broadly acting mechanism of translational repression that targets mRNAs for decapping and functions in translational control. We propose this mechanism is competitively balanced with translation, and shifting this balance is an important basis of translational control.     

Role of p54 RNA Helicase Activity and Its C-terminal Domain in Translational Repression, P-body Localization and Assembly

The RNA helicase p54 (DDX6, Dhh1, Me31B, Cgh-1, RCK) is a prototypic component of P-(rocessing) bodies in cells ranging from yeast to human. Previously, we have shown that it is also a component of the large cytoplasmic polyadenylation element-binding protein translation repressor complex in Xenopus oocytes and that when tethered to the 3′ untranslated region, Xp54 represses reporter mRNA translation. Here, we examine the role of the p54 helicase activity in translational repression and in P-body formation. Mutagenesis of conserved p54 helicase motifs activates translation in the tethered function assay, reduces accumulation of p54 in P-bodies in HeLa cells, and inhibits its capacity to assemble P-bodies in p54-depleted cells. Similar results were obtained in four helicase motifs implicated in ATP binding and in coupling ATPase and RNA binding activities. This is accompanied by changes in the interaction of the mutant p54 with the oocyte repressor complex components. Surprisingly, the C-terminal D2 domain alone is sufficient for translational repression and complete accumulation in P-bodies, although it is deficient for P-body assembly. We propose a novel RNA helicase model, in which the D2 domain acts as a protein binding platform and the ATPase/helicase activity allows protein complex remodeling that dictates the balance between repressors and an activator of translation.     

Nanos and Pumilio are essential for dendrite morphogenesis in Drosophila peripheral neurons

Much attention has focused on dendritic translational regulation of neuronal signaling and plasticity. For example, long-term memory in adult Drosophila requires Pumilio (Pum), an RNA binding protein that interacts with the RNA binding protein Nanos (Nos) to form a localized translation repression complex essential for anterior-posterior body patterning in early embryogenesis. Whether dendrite morphogenesis requires similar translational regulation is unknown. Here we report that nos and pum control the elaboration of high-order dendritic branches of class III and IV, but not class I and II, dendritic arborization (da) neurons. Analogous to their function in body patterning, nos and pum require each other to control dendrite morphogenesis, a process likely to involve translational regulation of nos itself. The control of dendrite morphogenesis by Nos/Pum, however, does not require hunchback, which is essential for body patterning. Interestingly, Nos protein is localized to RNA granules in the dendrites of da neurons, raising the possibility that the Nos/Pum translation repression complex operates in dendrites. This work serves as an entry point for future studies of dendritic translational control of dendrite morphogenesis.     

Spatial regulation of nanos is required for its function in dendrite morphogenesis

Spatial control of mRNA translation can generate cellular asymmetries and functional specialization of polarized cells like neurons. A requirement for the translational repressor Nanos (Nos) in the Drosophila larval peripheral nervous system (PNS) implicates translational control in dendrite morphogenesis [1]. Nos was first identified by its requirement in the posterior of the early embryo for abdomen formation [2]. Nos synthesis is targeted to the posterior pole of the oocyte and early embryo through translational repression of unlocalized nos mRNA coupled with translational activation of nos mRNA localized at the posterior pole [3, 4]. Abolishment of nos localization prevents abdominal development, whereas translational derepression of unlocalized nos mRNA suppresses head/thorax development, emphasizing the importance of spatial regulation of nos mRNA [3, 5]. Loss and overexpression of Nos affect dendrite branching complexity in class IV dendritic arborization (da) neurons, suggesting that nos also might be regulated in these larval sensory neurons [1]. Here, we show that localization and translational control of nos mRNA are essential for da neuron morphogenesis. RNA-protein interactions that regulate nos translation in the oocyte and early embryo also regulate nos in the PNS. Live imaging of nos mRNA shows that the cis-acting signal responsible for posterior localization in the oocyte/embryo mediates localization to the processes of class IV da neurons but suggests a different transport mechanism. Targeting of nos mRNA to the processes of da neurons may reflect a local requirement for Nos protein in dendritic translational control.     

Drosophila Ge-1 promotes P body formation and oskar mRNA localization

mRNA localization coupled with translational control is a widespread and conserved strategy that allows the localized production of proteins within eukaryotic cells. In Drosophila, oskar (osk) mRNA localization and translation at the posterior pole of the oocyte are essential for proper patterning of the embryo. Several P body components are involved in osk mRNA localization and translational repression, suggesting a link between P bodies and osk RNPs. In cultured mammalian cells, Ge-1 protein is required for P body formation. Combining genetic, biochemical and immunohistochemical approaches, we show that, in vivo, Drosophila Ge-1 (dGe-1) is an essential gene encoding a P body component that promotes formation of these structures in the germline. dGe-1 partially colocalizes with osk mRNA and is required for osk RNP integrity. Our analysis reveals that although under normal conditions dGe-1 function is not essential for osk mRNA localization, it becomes critical when other components of the localization machinery, such as staufen, Drosophila decapping protein 1 and barentsz are limiting. Our findings suggest an important role of dGe-1 in optimization of the osk mRNA localization process required for patterning the Drosophila embryo.     

Drosophila Me31B is a Dual eIF4E-Interacting Protein

Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). In this study, we show that Drosophila eIF4E-1 and eIF4E-3 occur in PBs along the DEAD-box RNA helicase Me31B. We show that Me31B interacts with eIF4E-1 and eIF4E-3 by means of yeast two-hybrid system, FRET in D. melanogaster S2 cells and coimmunoprecipitation in testis. Truncation and point mutations of Me31B proteins show two eIF4E-binding sites located in different protein domains. Residues Y401-L407 (at the carboxy-terminus) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminus) are critical for interaction with eIF4E-3. The residue W117 in eIF4E-1 and the homolog position F103 in eIF4E-3 are necessary for Me31B-eIF4E interaction suggesting that the change of tryptophan to phenylalanine provides specificity. Me31B represents a novel type of eIF4E-interacting protein with dual and specific interaction domains that might be recognized by different eIF4E isoforms in different tissues, adding complexity to the control of gene expression in eukaryotes.     

Identification and characterization of the expression of the translation initiation factor 4A (eIF4A) from Drosophila melanogaster

We have identified the initiation factor 4A (eIF4A) in a two-dimensional protein database of Drosophila wing imaginal discs. eIF4A, a member of the DEAD-box family of RNA helicases, forms the active eIF4F complex that in the presence of eIF4B and eIF4H unwinds the secondary structure of the 5'-UTR of mRNAs during translational initiation. Two-dimensional gel electrophoresis and microsequencing allowed us to purify eIF4A, and generate specific polyclonal antibodies. A combination of immunoblotting and labelling with [(35)S]methionine + [(35)S]cysteine revealed the existence of a single eIF4A isoform encoded by a previously reported gene that maps to chromosome 2L at 26A7-9. Expression of this gene yields two mRNA species, generated by alternative splicing in the 3'-untranslated region. The two mRNAs contain the same open reading frame and produce the identical eIF4A protein. No expression was detected of the eIF4A-related gene CG7483. We detected eIF4A protein expression in the wing imaginal discs of several Drosophila species, and in haltere, leg 1, leg 2, leg 3, and eye-antenna imaginal discs of D. melanogaster. Examination of eIF4A in tumor suppressor mutants showed significantly increased (> 50%) expression in the wing imaginal discs of these larvae. We observed ubiquitous expression of eIF4A mRNA and protein during Drosophila embryogenesis. Yeast two-hybrid analysis demonstrated the in vivo interaction of Drosophila eIF4G with the N-terminal third of eIF4A.     

Overlapping but distinct RNA elements control repression and activation of nanos translation

Spatially restricted synthesis of Nanos protein in the Drosophila embryo is essential for anterior-posterior patterning. Nanos translation is restricted to the posterior of the embryo by translational repression of nanos mRNA throughout the bulk cytoplasm and selective activation of posteriorly localized nanos mRNA. A 90-nucleotide translational control element (TCE) mediates translational repression. We show that TCE function requires formation of a bipartite secondary structure that is recognized by Smaug repressor and at least one additional factor. We also demonstrate that translational activation requires the interaction of localization factors with sequences that overlap TCE structural motifs. The identification of separate but overlapping recognition motifs for translational repressors and localization factors provides a molecular mechanism for the switch between translational repression and activation.     

Smaug assembles an ATP-dependent stable complex repressing nanos mRNA translation at multiple levels

The nanos (nos) mRNA encodes the posterior determinant of the Drosophila embryo. Translation of the RNA is repressed throughout most of the embryo by the protein Smaug binding to Smaug recognition elements (SREs) in the 3' UTR. Translation is locally activated at the posterior pole by Oskar. This paper reports that the SREs govern the time- and ATP-dependent assembly of an exceedingly stable repressed ribonucleoprotein particle (RNP) in embryo extract. Repression can be virtually complete. Smaug and its co-repressor Cup as well as Trailer hitch and the DEAD box protein Me31B are part of the repressed RNP. The initiation factor eIF4G is specifically displaced, and 48S pre-initiation complex formation is inhibited. However, later steps in translation initiation are also sensitive to SRE-dependent inhibition. These data confirm several previously untested predictions of a current model for Cup-dependent repression but also suggest that the Cup model by itself is insufficient to explain translational repression of the nos RNA. In the embryo extract, recombinant Oskar relieves translational repression and deadenylation by preventing Smaug's binding to the SREs.     

Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis

Embryonic patterning in Drosophila is regulated by maternal factors. Many such factors become localized as mRNAs within the oocyte during oogenesis and are translated in a spatio-temporally regulated manner. These processes are controlled by trans-acting proteins, which bind to the target RNAs to form a ribonucleoprotein (RNP) complex. We report that a DEAD-box protein, Me31B, forms a cytoplasmic RNP complex with oocyte-localizing RNAs and Exuperantia, a protein involved in RNA localization. During early oogenesis, loss of Me31B causes premature translation of oocyte-localizing RNAs within nurse cells, without affecting their transport to the oocyte. These results suggest that Me31B mediates translational silencing of RNAs during their transport to the oocyte. Our data provide evidence that RNA transport and translational control are linked through the assembly of RNP complex.     

Functional domains of Drosophila UNR in translational control

Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex-lethal (SXL) is an essential regulatory step of X chromosome dosage compensation in Drosophila. Translation inhibition requires that SXL recruits the protein upstream of N-ras (UNR) to the 3' UTR of msl-2 mRNA. UNR is a conserved, ubiquitous protein that contains five cold-shock domains (CSDs). Here, we dissect the domains of UNR required for translational repression and complex formation with SXL and msl-2 mRNA. Using gel-mobility shift assays, the domain involved in interactions with SXL and msl-2 was mapped specifically to the first CSD (CSD1). Indeed, excess of a peptide containing this domain derepressed msl-2 translation in vitro. The CSD1 of human UNR can also form a complex with SXL and msl-2. Comparative analyses of the CSDs of the Drosophila and human proteins together with site-directed mutagenesis experiments revealed that three exposed residues within CSD1 are required for complex formation. Tethering assays showed that CSD1 is not sufficient for translational repression, indicating that UNR binding to SXL and msl-2 can be distinguished from translation inhibition. Repression by tethered UNR requires residues from both the amino-terminal Q-rich stretch and the two first CSDs, indicating that the translational effector domain of UNR resides within the first 397 amino acids of the protein. Our results identify domains and residues required for UNR function in translational control.     

Glorund, a Drosophila hnRNP F/H homolog, is an ovarian repressor of nanos translation

Patterning of the anterior-posterior body axis of the Drosophila embryo requires production of Nanos protein selectively in the posterior. Spatially restricted Nanos synthesis is accomplished by translational repression of unlocalized nanos mRNA together with translational activation of posteriorly localized nanos. Repression of unlocalized nanos mRNA is mediated by a bipartite translational control element (TCE) in its 3' untranslated region. TCE stem-loop II functions during embryogenesis, through its interaction with the Smaug repressor. Stem-loop III represses unlocalized nanos mRNA during oogenesis, but trans-acting factors that carry out this function have remained elusive. Here we identify a Drosophila hnRNP, Glorund, that interacts specifically with stem-loop III. We establish that the ability of the TCE to repress translation in vivo reflects its ability to bind Glorund in vitro. These data, together with the analysis of a glorund null mutant, reveal a specific role for an hnRNP in repression of nanos translation during oogenesis.     

Neuronal BC RNAs cooperate with eIF4B to mediate activity-dependent translational control

In neurons, translational regulation of gene expression has been implicated in the activity-dependent management of synapto-dendritic protein repertoires. However, the fundamentals of stimulus-modulated translational control in neurons remain poorly understood. Here we describe a mechanism in which regulatory brain cytoplasmic (BC) RNAs cooperate with eukaryotic initiation factor 4B (eIF4B) to control translation in a manner that is responsive to neuronal activity. eIF4B is required for the translation of mRNAs with structured 5′ untranslated regions (UTRs), exemplified here by neuronal protein kinase Mζ (PKMζ) mRNA. Upon neuronal stimulation, synapto-dendritic eIF4B is dephosphorylated at serine 406 in a rapid process that is mediated by protein phosphatase 2A. Such dephosphorylation causes a significant decrease in the binding affinity between eIF4B and BC RNA translational repressors, enabling the factor to engage the 40S small ribosomal subunit for translation initiation. BC RNA translational control, mediated via eIF4B phosphorylation status, couples neuronal activity to translational output, and thus provides a mechanistic basis for long-term plastic changes in nerve cells.     

miRISC and the CCR4–NOT complex silence mRNA targets independently of 43S ribosomal scanning

miRNAs associate with Argonaute (AGO) proteins to silence the expression of mRNA targets by inhibiting translation and promoting deadenylation, decapping, and mRNA degradation. A current model for silencing suggests that AGOs mediate these effects through the sequential recruitment of GW182 proteins, the CCR4–NOT deadenylase complex and the translational repressor and decapping activator DDX6. An alternative model posits that AGOs repress translation by interfering with eIF4A function during 43S ribosomal scanning and that this mechanism is independent of GW182 and the CCR4–NOT complex in Drosophila melanogaster. Here, we show that miRNAs, AGOs, GW182, the CCR4–NOT complex, and DDX6/Me31B repress and degrade polyadenylated mRNA targets that are translated via scanning‐independent mechanisms in both human and Dm cells. This and additional observations indicate a common mechanism used by these proteins and miRNAs to mediate silencing. This mechanism does not require eIF4A function during ribosomal scanning.     

The fragile X syndrome protein represses activity-dependent translation through CYFIP1, a new 4E-BP

Strong evidence indicates that regulated mRNA translation in neuronal dendrites underlies synaptic plasticity and brain development. The fragile X mental retardation protein (FMRP) is involved in this process; here, we show that it acts by inhibiting translation initiation. A binding partner of FMRP, CYFIP1/Sra1, directly binds the translation initiation factor eIF4E through a domain that is structurally related to those present in 4E-BP translational inhibitors. Brain cytoplasmic RNA 1 (BC1), another FMRP binding partner, increases the affinity of FMRP for the CYFIP1-eIF4E complex in the brain. Levels of proteins encoded by known FMRP target mRNAs are increased upon reduction of CYFIP1 in neurons. Translational repression is regulated in an activity-dependent manner because BDNF or DHPG stimulation of neurons causes CYFIP1 to dissociate from eIF4E at synapses, thereby resulting in protein synthesis. Thus, the translational repression activity of FMRP in the brain is mediated, at least in part, by CYFIP1.     

Down-regulation of RpS21, a putative translation initiation factor interacting with P40, produces viable minute imagos and larval lethality with overgrown hematopoietic organs and imaginal discs

Down-regulation of the Drosophila ribosomal protein S21 gene (rpS21) causes a dominant weak Minute phenotype and recessively produces massive hyperplasia of the hematopoietic organs and moderate overgrowth of the imaginal discs during larval development. Here, we show that the S21 protein (RpS21) is bound to native 40S ribosomal subunits in a salt-labile association and is absent from polysomes, indicating that it acts as a translation initiation factor rather than as a core ribosomal protein. RpS21 can interact strongly with P40, a ribosomal peripheral protein encoded by the stubarista (sta) gene. Genetic studies reveal that P40 underexpression drastically enhances imaginal disc overgrowth in rpS21-deficient larvae, whereas viable combinations between rpS21 and sta affect the morphology of bristles, antennae, and aristae. These data demonstrate a strong interaction between components of the translation machinery and showed that their underexpression impairs the control of cell proliferation in both hematopoietic organs and imaginal discs.