A Multiprotein Complex That Mediates Translational Enhancement in Drosophila

Modulating the efficiency of translation plays an important role in a wide variety of cellular processes and is often mediated by trans-acting factors that interact with cis-acting sequences within the mRNA. Here we show that a cis-acting element, the Hsp83 degradation element (HDE), within the 3′-untranslated region of the Drosophila Hsp83 mRNA functions as a translational enhancer. We show that this element is bound by a multiprotein complex, and we identify components using a novel affinity-based method called tandem RNA affinity purification tagging. Three proteins (DDP1, Hrp48, and poly(A)-binding protein) are components of the HDE-binding complex and function in translational enhancement. Our data support a model whereby the HDE is composed of several cis-acting subelements that represent binding sites for trans-acting factors, and the combined action of these trans-acting factors underlies the ability of the HDE to stimulate translation. Modulating the efficiency of translation plays an important role in a wide variety of cellular processes and is often mediated by trans-acting factors that interact with cis-acting sequences within the mRNA. Here we show that a cis-acting element, the Hsp83 degradation element (HDE), within the 3′-untranslated region of the Drosophila Hsp83 mRNA functions as a translational enhancer. We show that this element is bound by a multiprotein complex, and we identify components using a novel affinity-based method called tandem RNA affinity purification tagging. Three proteins (DDP1, Hrp48, and poly(A)-binding protein) are components of the HDE-binding complex and function in translational enhancement. Our data support a model whereby the HDE is composed of several cis-acting subelements that represent binding sites for trans-acting factors, and the combined action of these trans-acting factors underlies the ability of the HDE to stimulate translation. Regulated translation plays an essential role in a wide variety of cellular processes. Although translational regulation is likely to function in virtually all eukaryotic cell types, these controls are particularly important in cells where transcriptional regulation is not an option. For example, maturation of mammalian red blood cells occurs after the nucleus is extruded and thus is driven by previously synthesized mRNAs. Similarly, in early metazoan embryos, the zygotic genome is transcriptionally silent, and maternally deposited mRNAs control early development. Translational regulation is also very important in large cells, such as neurons, where correct spatial and temporal expression of proteins cannot be achieved through transcriptional controls alone. Regulation of specific transcripts is often mediated by cisacting elements within the 5′-or3′-untranslated region (UTR) 4The abbreviations used are: UTR, untranslated region; PABP, poly(A)-binding protein; HDE, Hsp83 degradation element; TRAP, tandem RNA affinity purification; hnRNP, heterogeneous nuclear ribonucleoprotein. of an mRNA (1Hentze M.W. Gebauer F. Preiss T. Mathews M.B. Sonenberg N. Hershey J.W. Translational Control in Biology and Medicine. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2007: 269-295Google Scholar). These elements can act as binding sites for trans-acting factors that either directly or indirectly contact the translational machinery. Some of the best characterized mechanisms serve to repress protein expression, but mechanisms that stimulate protein production also exist. In principle, these positively acting events can be divided into two different classes. The first acts on transcripts that are translationally repressed. Translational stimulation is achieved by blocking the repressive mechanism (i.e. enhancement results from relief of repression). The second class of stimulatory events acts on mRNAs that are not repressed. In these cases, an mRNA is better able to recruit the basic translation machinery and is, therefore, expressed at a higher level. This latter type of mechanism is likely to be particularly important when a component of the translation machinery is limiting and, consequently, transcripts must compete for access to the translational apparatus. Many viral RNAs contain elements that aid in preferential expression in infected cells. For example, the 5′-UTR of the tobacco mosaic virus RNA contains a cis-acting element, Ω, that is bound by Hsp101, which in turn recruits the eIF4F translation initiation complex to the RNA to enhance its translation (2Wells D.R. Tanguay R.L. Le H. Gallie D.R. Genes Dev. 1998; 12: 3236-3251Crossref PubMed Scopus (116) Google Scholar, 3Gallie D.R. Nucleic Acids Res. 2002; 30: 3401-3411Crossref PubMed Google Scholar). Examples of cellular mRNAs that carry cis-acting translational enhancers have also been described, but, for the most part, the molecular mechanisms involved are poorly understood. One well characterized example involves metazoan histone mRNAs, which terminate in a stem-loop structure and do not carry a poly(A) tail. The stem-loop is bound by the stem-loop-binding protein which interacts directly with the translation initiation factors, eIF4G and eIF3 (4Jaeger S. Barends S. Giege R. Eriani G. Martin F. Biochimie (Paris). 2005; 87: 827-834Crossref PubMed Scopus (50) Google Scholar). Stem-loop-binding protein has an analogous function to poly(A)-binding protein (PABP); both bind to the 3′ end of the transcript and interact with the translation initiation machinery to facilitate protein synthesis (1Hentze M.W. Gebauer F. Preiss T. Mathews M.B. Sonenberg N. Hershey J.W. Translational Control in Biology and Medicine. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2007: 269-295Google Scholar). PABP can also bind to cis-acting sequences that are outside the poly(A) tail. The mammalian YB-1 RNA, for example, carries a PABP binding sequence within its 3′-UTR (5Skabkina O.V. Skabkin M.A. Popova N.V. Lyabin D.N. Penalva L.O. Ovchinnikov L.P. J. Biol. Chem. 2003; 278: 18191-18198Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). By binding to this element, PABP enhances translation of YB-1 RNA in a poly(A) tail-independent manner. Previous work provided indirect evidence that the Drosophila Hsp83 mRNA contains a translational enhancer in its 3′-UTR that functions in the early embryo (6Bashirullah A. Halsell S.R. Cooperstock R.L. Kloc M. Karaiskakis A. Fisher W.W. Fu W. Hamilton J.K. Etkin L.D. Lipshitz H.D. EMBO J. 1999; 18: 2610-2620Crossref PubMed Scopus (177) Google Scholar). The Hsp83 transcript is regulated through multiple mechanisms. The RNA is localized to the posterior of the embryo by selective degradation of the transcript in the bulk cytoplasm of the embryo, whereas transcripts present in the germ plasm at the posterior are protected. A region of the 3′-UTR, termed the Hsp83 degradation element (HDE), was originally identified through its ability to direct the degradation of transgenic mRNAs. Expression of one of the mRNAs results in embryonic defects, which were enhanced when the HDE is present in the reporter mRNA 3′-UTR, suggesting that the HDE might stimulate translation. To obtain direct evidence that the HDE functions as a translational enhancer and to dissect the molecular mechanisms involved, we developed a microinjection-based assay that recapitulates HDE-mediated translational enhancement in Drosophila embryos. Deletion analysis demonstrated that the HDE contains multiple subelements capable of mediating translational enhancement. To identify trans-acting factors that act through the HDE, we developed a widely applicable method to identify proteins that bind to RNA sequences of interest, which we call tandem RNA affinity purification (TRAP) tagging. Using several approaches, including TRAP tagging, we identified three proteins (DDP1, Hrp48, and PABP) that bind to the HDE as part of a protein complex. We show that both DDP1 and Hrp48 function in translational enhancement. Although previous work indicated that Hrp48 functions as a translational repressor, our data suggest that Hrp48 is a bifunctional modulator of translation and that the ability of Hrp48 to act as a repressor or activator is regulated by other proteins that are recruited to the target mRNA. We propose that the HDE is a modular element composed of several cis-acting sequences representing binding sites for various factors that regulate translation. Thus, HDE-mediated translational enhancement is a read-out of the combined action of its associated trans-acting factors. RNA Injection Assay—Luciferase RNAs were generated as described previously (7Smibert C.A. Lie Y.S. Shillinglaw W. Henzel W.J. Macdonald P.M. RNA. 1999; 5: 1535-1547Crossref PubMed Scopus (113) Google Scholar). Injection experiments were performed as described by Nelson et al. (8Nelson M.R. Leidal A.M. Smibert C.A. EMBO J. 2004; 23: 150-159Crossref PubMed Scopus (198) Google Scholar) with the following modifications. Wild-type embryos were collected from w1118 mothers, and hrp48 germ line clones were generated for hrp4802647 and hrp4810280 mutant alleles (9Hammond L.E. Rudner D.Z. Kanaar R. Rio D.C. Mol. Cell. Biol. 1997; 17: 7260-7267Crossref PubMed Scopus (59) Google Scholar) using established techniques (10Chou T.-B. Perrimon N. Genetics. 1992; 131: 643-653Crossref PubMed Google Scholar, 11Chou T.B. Perrimon N. Genetics. 1996; 144: 1673-1679Crossref PubMed Google Scholar). Firefly luciferase and Renilla luciferase RNAs were injected at a concentration of 200 and 50 ng/μl, respectively. Injected embryos were harvested 3.0–3.5 h after egg laying. Translational enhancement for RNAs bearing 3′-UTR inserts were expressed as its corrected value divided by the corrected value from firefly RNA carrying no insert. For example, the translational enhancement of luc-HDE-(1–97) RNA = (firefly activity of luc-HDE-(1–97)/Renilla activity)/(firefly activity of luciferase RNA carrying no insert/Renilla activity). Embryo Extract Preparation—All experiments employed extract prepared by collecting embryos 0–2 h after egg laying from w1118 animals. Embryos were disrupted in a minimal volume of 150 mm KCl, 20 mm Hepes-KOH (pH 7.4), 1 mm MgCl2, 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride, 2 mm 2 2 and 1 mm the was with to a concentration of and at of of 1 of of 150 mm KCl, 20 mm 1 mm MgCl2, mm 1 RNA, and 1 of embryo were for at and on a in at employed of RNA, of embryo of of of mm KCl, 20 mm all in a volume of were for at and as previously described C.A. Macdonald P.M. Genes Dev. 1996; PubMed Scopus Google by the of of A and for at were on an and proteins were by were with which was in by of the DDP1 G. M. Sonenberg N. Mol. Cell. Biol. 2004; PubMed Scopus Google (9Hammond L.E. Rudner D.Z. Kanaar R. Rio D.C. Mol. Cell. Biol. 1997; 17: 7260-7267Crossref PubMed Scopus (59) Google or (7Smibert C.A. Lie Y.S. Shillinglaw W. Henzel W.J. Macdonald P.M. RNA. 1999; 5: 1535-1547Crossref PubMed Scopus (113) Google Scholar). For that were to be by were and the complex was as described were at for in 150 50 mm A and were to of and and at a D.R. RNA. PubMed Scopus Google Scholar, D.R. 2002; PubMed Scopus Google by two protein binding sites M. Mol. 1998; PubMed Scopus (50) Google Scholar) and the sequence of interest, were generated in using RNA of RNA was with of in mm KCl, 20 mm 1 mm MgCl2, and for 1 h at The was with Embryo extract was with to and of of protein and on for 20 The extract was and the was and with for 2 h at with RNA was with of mm The was for 2 h at with 20 carrying 2 The was with and proteins were in of of and for 1 h at were by and with were from the and identified The HDE as a Translational in Drosophila that the HDE functions as a translational enhancer and to the mechanism involved, we developed a microinjection-based assay that recapitulates translational enhancement in a to previously developed to translational (8Nelson M.R. Leidal A.M. Smibert C.A. EMBO J. 2004; 23: 150-159Crossref PubMed Scopus (198) Google Scholar, R. S. A. PubMed Scopus Google Scholar). We that of the HDE into a reporter RNA enhance its translation to that of the reporter the The reporter RNAs the firefly luciferase and a poly(A) tail. To control for such as the of RNA injected and the efficiency of embryo an RNA Renilla luciferase was with firefly luciferase reporter RNA. The two luciferase have different to and the of in the The of firefly activity was thus to that of the Renilla the reporter RNA an HDE a in luciferase expression to reporter RNA carrying no and of the Hsp83 3′-UTR, which for the of the to enhance luciferase expression, translational enhancer function to the To that the in luciferase expression at the of was performed on RNA from injected embryos to the of firefly reporter RNA to the Renilla RNA. The of firefly RNA from experiments was whereas the for luc-HDE-(1–97)/Renilla RNA for three experiments was These are not different as by = and thus the HDE not have a on the of the reporter RNA. We that the luciferase expression for the luc-HDE-(1–97) RNA occurs at the of translation. our assay recapitulates the translational enhancer function of the HDE, we were to translational enhancement. The HDE into Translational within the HDE for translational the of various on the ability of the HDE to stimulate translation were using the assay Deletion of the first of the HDE a enhancement of luciferase that generated by the This the of a repressor element in the first of the Deletion of the of the HDE, on the other translational enhancement by to suggesting the of positively acting sequences within this of the first and the whereas of the first directly to the a enhancement. the of a repressive element within the first was that the on might stimulate translation for the of the first to the This is the the enhanced expression the reporter by In the HDE contains multiple subelements that are able to including one that can HDE-mediated enhancement and several that The of the HDE thus the combined action of all of these The HDE by a of multiple subelements within the HDE that several trans-acting factors interact with the HDE and that the combined action of all of these factors, both stimulatory and in is for the of translational To identify these trans-acting factors, we performed an assay to embryonic proteins that are capable of with the We a suggesting that a protein or a complex of proteins interacts with the HDE This complex HDE RNA for whereas an RNA the of different of specific This that the complex in the of multiple proteins with different for the HDE and different that the HDE is bound by a multiprotein complex. We employed a assay to proteins that directly contact the HDE of the including at and and three at bound HDE RNA binding an RNA. These HDE-binding proteins are of translational enhancement in early embryos. DDP1 with the purification of the HDE-binding protein its as the Drosophila protein 1 DDP1 contains A. S. F. EMBO J. 1999; 18: PubMed Scopus Google Scholar) and to a of proteins called several of which are to function as M.A. D.R. S. A. PubMed Scopus Google Scholar, Nucleic Acids Res. PubMed Scopus Google Scholar, A.M. A. Nucleic Acids Res. 2003; PubMed Scopus Google Scholar). To the of DDP1 as the HDE-binding we performed a using embryo extract and HDE RNA, by with either an or The protein that at is by the but not by These results that DDP1 is an HDE-binding protein and that DDP1 the RNA Hrp48 with the facilitate the of HDE-binding we to binding sites within the HDE, that RNAs be in the method described To carry this we used of the HDE as in the at was able to bind to these RNA Although the bound to the two bound to sequences within the and 3′ of the To identify the HDE-binding we developed an affinity-based purification method that we to as The TRAP involves in of an RNA that carries the sequence of with two RNA affinity The RNA is with and the RNA with bound proteins is the The first used in this purification is an that and be by D.R. RNA. PubMed Scopus Google Scholar, D.R. 2002; PubMed Scopus Google Scholar). This binding of protein to the RNA, for a second of purification the second affinity which is composed of two that bind to the protein M. Mol. 1998; PubMed Scopus (50) Google Scholar). The from are on protein on proteins are by with Our RNA for TRAP three of HDE Three of HDE were used as the this region binding to the proteins the TRAP method were and by A that with of the HDE and at was from the and identified as the Drosophila heterogeneous nuclear Hrp48, using Hrp48 is a of the of proteins that has previously been to function in RNA RNA and translational (9Hammond L.E. Rudner D.Z. Kanaar R. Rio D.C. Mol. Cell. Biol. 1997; 17: 7260-7267Crossref PubMed Scopus (59) Google Scholar, Kanaar R. Rio D.C. Genes Dev. PubMed Scopus Google Scholar, T. 2004; 131: PubMed Scopus Google Scholar, Dev. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, T. A. A. Dev. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). To that Hrp48 is the HDE-binding we performed a with HDE RNA by with an The at was by the but not by that the HDE binding activity is PABP an HDE-binding experiments indicated that proteins to DDP1 and Hrp48 bind directly to the HDE To identify HDE-binding we used TRAP tagging. with of the HDE indicated that most of the HDE-binding proteins sequences to bind to the RNA. we used HDE as in this We that several proteins with HDE but not with an control RNA of One of these proteins was identified as but not the protein present in the We that PABP with the HDE, that Hrp48 with the but not HDE, and that DDP1 with but in by One of these data is that the of DDP1 and Hrp48 proteins is not for to with the HDE TRAP our results the of using both subelements and the element as Hrp48, and PABP of the HDE-binding that Hrp48, and PABP bind directly to the HDE and that is a HDE-binding complex we all three proteins are present in the complex and function in translational enhancement. To do we performed a assay with HDE the complex, and the to to HDE-binding proteins to the were from the in the of the was to with the or and protein was following Hrp48, and PABP were in the from the complex, whereas which not interact with the HDE not was not These results suggest that that Hrp48, and PABP interact directly and with the HDE to a complex. DDP1 in HDE-mediated Translational a role for PABP in translational stimulation is well the cannot be for DDP1 or We to our on for DDP1 and Hrp48 in HDE We were to obtain in the HDE that binding of one of these proteins and not of binding sites within the HDE to For a was not we that of DDP1 that are not for an to the role of DDP1 in translational we of DDP1 to a reporter mRNA have on its translation. The DDP1 has been to bind to RNAs carrying of the sequence H. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google and a to this was within the A RNA carrying three tandem of this was used as a in a by with an or The the DDP1 whereas not of the RNA carrying these three was able to compete for binding to DDP1 in a In of the to a within an RNA from for DDP1 binding but no on binding of other that in the function of the and mutant sequences a specific in DDP1 Firefly luciferase reporter RNAs were generated carrying either the or mutant sequence into the firefly luciferase 3′-UTR and injected into embryos. The element enhanced translation to firefly luciferase RNA carrying no and the mutant element translational enhancement by These results suggest that DDP1 by translational enhancement and that HDE sequences and binding proteins as Hrp48 and PABP) are for enhancement. is also that proteins interact with and to the ability of the sequence to stimulate translation. Hrp48 in HDE-mediated Translational we the role of Hrp48 in HDE-mediated translational enhancement using our assay in embryos that of hrp48 at the (9Hammond L.E. Rudner D.Z. Kanaar R. Rio D.C. Mol. Cell. Biol. 1997; 17: 7260-7267Crossref PubMed Scopus (59) Google we used (10Chou T.-B. Perrimon N. Genetics. 1992; 131: 643-653Crossref PubMed Google Scholar, 11Chou T.B. Perrimon N. Genetics. 1996; 144: 1673-1679Crossref PubMed Google Scholar) to clones of germ line cells with of such clones are for hrp48 no are the of alleles to embryos for experiments that the HDE translation in these mutant embryos to the as in type not We that this might have from the that the activity of the HDE the combined action of several proteins that interact with the HDE and that a of Hrp48 thus not have a This is with the that within the HDE were able to of translational enhancement We that a mutant of the HDE that is in its ability to stimulate translation might be to of We the ability of the HDE in the contains both of the sites and is for translational enhancement. mutant hrp48 alleles mediated translational enhancement at a that was by to that in embryos activity was not in these mutant embryos was to the Hrp48 of TRAP described is a novel method for of proteins that interact with a RNA The TRAP is to a wide variety of we have employed RNAs generated in to proteins from a In principle, RNAs also be expressed in for the purification of RNA This be particularly important when the of a complex to an RNA is by Although techniques for analysis of and are well to the (i.e. the of all RNAs and proteins within the The TRAP a method to in such Translational in our previous work provided evidence that the HDE is a cis-acting element capable of both translation and transcript we have experiments that that the HDE is a translational enhancer. These experiments identified Hrp48, and PABP as HDE-binding and our assay provided evidence that both DDP1 and Hrp48 to the ability of HDE to stimulate translation. in the of the translational stimulation occurs in the of transcript these two processes are likely to be mediated by mechanisms. This is with our which show that the RNA element in the Hsp83 whereas the HDE functions as an degradation A. and H. in of the Translational with data of suggest that all three of the HDE-binding proteins identified in translational enhancement. In PABP enhances translation of YB-1 mRNA in a poly(A) tail-independent by binding to an element in the 3′-UTR (5Skabkina O.V. Skabkin M.A. Popova N.V. Lyabin D.N. Penalva L.O. Ovchinnikov L.P. J. Biol. Chem. 2003; 278: 18191-18198Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). our data that PABP interacts with the HDE that of PABP to elements to a 3′-UTR be a mechanism for translational enhancement. We that we do not translational enhancement the HDE is poly(A) DDP1 is a of the of These proteins have been to function in including and control of mRNA and translation. The of RNA, is present on and with specific mRNAs in Nucleic Acids Res. PubMed Scopus Google Scholar, A.M. A. Nucleic Acids Res. 2003; PubMed Scopus Google Scholar). One of these mRNAs, in an indirect evidence that enhance translation. Here we have that DDP1 in translational enhancement by binding to the HDE, suggesting a function for this of proteins in translational Hrp48 is a of the G. S. PubMed Scopus Google Scholar). A mammalian translation of basic protein mRNA in through an element in the 3′-UTR S. J. Biol. 1999; PubMed Scopus Google Scholar). the can also translational when recruits to the transcript L.D. G. J.W. Mol. Biol. Cell. 17: PubMed Scopus Google Scholar). can both translational and enhancement the Drosophila Hrp48 was first identified on its role in (9Hammond L.E. Rudner D.Z. Kanaar R. Rio D.C. Mol. Cell. Biol. 1997; 17: 7260-7267Crossref PubMed Scopus (59) Google Scholar, Kanaar R. Rio D.C. Genes Dev. PubMed Scopus Google Scholar). has been a role in the translational and of mRNAs in Drosophila T. 2004; 131: PubMed Scopus Google Scholar, Dev. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, T. A. A. Dev. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Here we have that Hrp48 the HDE and translational enhancement. The ability of Hrp48 to function in both translational and stimulation that its function is by other trans-acting factors that are to be bound to that element DDP1 and PABP) as well as proteins present in the HDE-binding complex. A for to a role in translational our results show that Hrp48, and PABP function in this of the complex on the HDE either or after are bound to the mRNA. with this we were to identify that the binding of one of these proteins to the HDE binding of the these proteins can a complex in the of the HDE and bind RNA or on the HDE in a proteins within the complex for example, serve different such as transcript complex and with the translational apparatus. to the structure and function of that translational our results support a model whereby the HDE is composed of binding sites for several of which functions in translational Thus, the ability of the HDE to stimulate translation of the action of of its bound with this we that no within the HDE its ability to stimulate translation. these enhance or the of translational of the of the HDE be achieved in different cell and at different by the of HDE-binding proteins that are By the expression of mRNAs other Hsp83 is likely to be cis-acting elements to the HDE, which are composed of different of binding sites for proteins that translational enhancement and We and T. for Drosophila and with

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