Identification of Synaptic Targets of Drosophila Pumilio
Gengxin ChenWanhe LiQing‐Shuo ZhangMichael RegulskiNishi SinhaJody BarditchTim TullyAdrian R. KrainerMichael Q. ZhangJosh Dubnau
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Abstract:
Drosophila Pumilio (Pum) protein is a translational regulator involved in embryonic patterning and germline development. Recent findings demonstrate that Pum also plays an important role in the nervous system, both at the neuromuscular junction (NMJ) and in long-term memory formation. In neurons, Pum appears to play a role in homeostatic control of excitability via down regulation of para, a voltage gated sodium channel, and may more generally modulate local protein synthesis in neurons via translational repression of eIF-4E. Aside from these, the biologically relevant targets of Pum in the nervous system remain largely unknown. We hypothesized that Pum might play a role in regulating the local translation underlying synapse-specific modifications during memory formation. To identify relevant translational targets, we used an informatics approach to predict Pum targets among mRNAs whose products have synaptic localization. We then used both in vitro binding and two in vivo assays to functionally confirm the fidelity of this informatics screening method. We find that Pum strongly and specifically binds to RNA sequences in the 3′UTR of four of the predicted target genes, demonstrating the validity of our method. We then demonstrate that one of these predicted target sequences, in the 3′UTR of discs large (dlg1), the Drosophila PSD95 ortholog, can functionally substitute for a canonical NRE (Nanos response element) in vivo in a heterologous functional assay. Finally, we show that the endogenous dlg1 mRNA can be regulated by Pumilio in a neuronal context, the adult mushroom bodies (MB), which is an anatomical site of memory storage.Keywords:
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Translational regulation
Translational regulation plays an essential role in many phases of the Drosophila life cycle. During embryogenesis, specification of the developing body pattern requires co-ordination of the translation of oskar, gurken and nanos mRNAs with their subcellular localization. In addition, dosage compensation is controlled by Sex-lethal-mediated translational regulation while dFMR1 (the Drosophila homologue of the fragile X mental retardation protein) controls translation of various mRNAs which function in the nervous system. Here we describe some of the mechanisms that are utilized to regulate these various processes. Our review highlights the complexity that can be involved with multiple factors employing different mechanisms to control the translation of a single mRNA.
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The 5′-untranslated region (5′UTR) of mRNAs often affects the translational efficiency of the downstream open reading frames (ORFs), and some of its regulatory elements are involved in the initiation of translation. We found that the 5′UTR of the rice OsMac1 mRNA, which consisted of more than 500 nucleotides, yielded a significant enhancement of the translational efficiency of the downstream ORF. In the rice genome, OsMac1 represents a conserved gene family with two homologues, OsMac2 and OsMac3, which contain DUF300 (domain of unknown function 300) domains with predicted transmembrane regions. Similarly to the OsMac1 mRNA, the OsMac2 and OsMac3 mRNAs possess long 5′UTRs consisting of 312 and 318 nucleotides, respectively, that precede the main ORFs, which allow the elevation of the translational efficiency of the downstream ORF. The estimation of the translational efficiency of the GUS gene, which is located after the 5′UTRs, in suspension cultures of rice protoplasts showed that it was significantly greater than that of the control. These results suggest that 5′UTRs of OsMac2 and OsMac3 enhance the translation of the downstream ORF. Our results indicate that these 5′UTRs play a role of novel translational enhancer elements that enable the efficient translation of the downstream ORF.
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Variation in protein output across the genome is controlled at several levels, but the relative contributions of different regulatory mechanisms remain poorly understood. Here, we obtained global measurements of decay and translation rates for mRNAs with alternative 3′ untranslated regions (3′ UTRs) in murine 3T3 cells. Distal tandem isoforms had slightly but significantly lower mRNA stability and greater translational efficiency than proximal isoforms on average. The diversity of alternative 3′ UTRs also enabled inference and evaluation of both positively and negatively acting cis -regulatory elements. The 3′ UTR elements with the greatest implied influence were microRNA complementary sites, which were associated with repression of 32% and 4% at the stability and translational levels, respectively. Nonetheless, both the decay and translation rates were highly correlated for proximal and distal 3′ UTR isoforms from the same genes, implying that in 3T3 cells, alternative 3′ UTR sequences play a surprisingly small regulatory role compared to other mRNA regions.
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An important regulatory mechanism affecting mRNA translation involves various covalent modifications of RNA, which establish distinct epitranscriptomic signatures that actively influence various physiological processes. Dendritic translation in mammalian neurons is a potent target for RNA modification-based regulation. In this mini-review, we focus on the effect of potential RNA modifications on the spatiotemporal regulation of the dendritic translation of mRNAs, which are targeted by two important neuronal translational co-regulators, namely TDP-43 and Fragile X Mental Retardation Protein (FMRP).
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MicroRNAs (miRs) commonly regulate translation from target mRNA 3′ untranslated regions (UTRs). While effective miR-binding sites have also been identified in 5′ untranslated regions (UTRs) or open reading frames (ORFs), the mechanism(s) of miR-mediated regulation from these sites has not been defined. Here, we systematically investigate how the position of miR-binding sites influences translational regulation and characterize their mechanistic basis. We show that specific translational regulation is elicited in vitro and in vivo not only from the 3′UTR, but equally effectively from six Drosophila miR-2-binding sites in the 5′UTR or the ORF. In all cases, miR-2 triggers mRNA deadenylation and inhibits translation initiation in a cap-dependent fashion. In contrast, single or dual miR-2-binding sites in the 5′UTR or the ORF yield rather inefficient or no regulation. This work represents the first demonstration that 5′UTR and ORF miR-binding sites can function mechanistically similarly to the intensively investigated 3′UTR sites. Using single or dual binding sites, it also reveals a biological rationale for the high prevalence of miR regulatory sites in the 3′UTR.
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