- Email: [email protected]
Noncoding roX RNA Remodeling Triggers Fly Dosage Compensation Complex Assembly
Molecular Cell
Previews Noncoding roX RNA Remodeling Triggers Fly Dosage Compensation Complex Assembly Anton Wutz1,* 1Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, ETH Zu ¨ rich, Schafmattstrasse 22, 8049 Zurich, Switzerland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.07.007
Spreading of dosage compensation over the X chromosome in Drosophila males requires the noncoding roX1 and roX2 RNAs. In this issue, Ilik et al. (2013) and Maenner et al. (2013) show that these RNAs contain discrete binding sites that are remodeled during assembly of the dosage compensation complex. Drosophila melanogaster males enhance expression of their single X chromosome to approximate the two X chromosomes in females. This is achieved, at least in part, by X chromosome-wide acetylating histone H4 lysine 16 through a dosage compensation complex (DCC) containing the male-specific lethal proteins MSL1– MSL3 and the histone acetyltransferase males absent on the first (MOF). For spreading over the X chromosome, two noncoding RNAs, roX1 and roX2 (Amrein and Axel, 1997; Meller et al., 1997), and the maleless (MLE) helicase are required (Meller et al., 2000; Morra et al., 2011). Interaction of roX RNAs with MLE during DCC assembly is revealed in two studies by Ilik et al. (2013) and Maenner et al. (2013) in this issue. Ilik et al. (2013) identify RNA-binding sites of MLE and MSL2 using an individual-nucleotide resolution crosslinking and imunoprecipitation (iCLIP) protocol. Following UV crosslinking and precipitation with antibodies specific for MLE and MSL2 the site of crosslink can then be identified by sequencing reverse-transcribed fragments. This analysis shows that MLE occupies discrete binding sites on roX RNAs. Since UV crosslinks require close contact, iCLIP identifies sites of direct binding. Notably, analysis of the top 1% of iCLIP contacts identifies a consensus sequence that resembles a roX box motif that was identified as a sequence of similarity between roX1 and roX2 and is important for their function (Kelley et al., 2008; Park et al., 2007, 2008). In vitro MLE protein binds to a 50 fragment of roX2. Binding to a second binding site in the 30 half requires ATP. The fact that binding to the 30 site also requires the helicase activity of MLE
suggests that this is an active process. Notably, genetic tests in transgenic flies indicate that the ATP-dependent binding site in roX2 is functionally more important than the ATP-independent bound site in the 50 region. For this analysis, the authors use an elegant transgenic system for genetic complementation of roX1- and roX2-deficient males. The 50 region of roX2 appears far less efficient in rescuing male viability than the 30 region, showing that MLE binding alone is not sufficient. This aspect is independently investigated by Maenner et al. (2013), who use an RNA affinity purification strategy to study MLE binding. A conserved stemloop structure in roX1 and roX2 that contains roX boxes (SLroX1 and SLroX2) mediates MLE binding, which is further stimulated by the addition of ATP. This result is consistent with observations on the roX2 30 fragment by Ilik et al. (2013). Using oligonucleotide-mediated precipitation, Maenner et al. (2013) can further show that MLE binds a large stem loop in roX2 that is remodeled into two smaller loops in the presence of ATP. This observation suggests a role for the MLE helicase for remodeling roX2. Maenner et al. (2013) go on to investigate the significance for assembly of DCC by studying binding of MSL2. MSL2 is a critical component of DCC and closely associates with MLE at several high-affinity sites (HAS) on the male X chromosome (Straub et al., 2013 and Ilik et al., 2013). Using in vitro transcribed roX2 RNA immobilized through an MS2 tag, binding of MSL2 and MLE from nuclear extracts can be observed specifically in the presence of ATP. This is a remarkable result suggesting that the first steps of DCC assembly have been recapitulated in vitro. The
helicase function of MLE might therefore be important for remodeling critical binding sites for MSL2 on roX2 RNA (Figure 1). Analysis of the in vivo relevance of roX RNA remodeling remains an important aim for future work. One way to test this idea could be through rescuing MLEdeficient males by providing a remodeled transgenic version of roX2 that would not fold into a large stem loop, but instead adopt the remodeled conformation. It needs to be determined if structural mutations in the stem that maintain critical sequence motifs can be identified. However, the experiment could provide unambiguous evidence for blocked binding sites in roX2. Why roX RNAs should fold into an apparently inactive conformation in the first place appears unclear at the moment. roX RNAs and MSL2 protein are unstable in the absence of MLE, and stabilization requires DCC assembly. Jointly, the studies by Ilik et al. (2013) and Maenner et al. (2013) identify roX boxes as binding sites for MLE and implicate ATP-dependent processes in DCC assembly. Remodeling of roX RNAs could represent a rate-limiting step that could titrate the amount of DCC. Where DCC assembly occurs is another interesting question. MLE and MSL2 proteins are produced in the cytoplasm and could bind roX RNAs before or after nuclear import. Interestingly, an earlier study has implicated nuclear pore components in dosage compensation (Mendjan et al., 2006). One candidate location for DCC assembly is chromosomal HAS sites. These sites can be occupied by members of the DCC in the absence of MLE and might be entry sites for spreading over the X chromosome. A zinc finger protein that has been recently
Molecular Cell 51, July 25, 2013 ª2013 Elsevier Inc. 131
Molecular Cell
Previews cytoplasm
roX
ML
MSL2
E DCC MSL1 MSL2 MOF
H4K16Ac Figure 1. MLE Helicase Activity Remodels Binding Sites in roX RNAs for DCC Assembly roX RNAs are transcribed from the X chromosome and adopt a folded conformation. Remodeling by MLE helicase unmasks binding sites for MSL2 and triggers assembly of DCC. Incorporation of the histone acetyltransferase MOF catalyzes X chromosome-wide acetylation of histone H4 lysine 16 to mediate upregulation of X-linked genes in male flies.
plexes interact with chromosomal binding partners in future work. roX RNAs highlight remarkable features of RNA protein complexes. Several binding sites are embedded into long stretches of sequence that are less structured and might be functionally irrelevant. Depending on which conformation the RNA adopts, certain bases become accessible, revealing binding sites. Conceivably, composition of RNA protein complexes can be changed by refolding hidden conditional binding sites that would otherwise be buried in RNA secondary structure. Binding of MSL2 to roX2 provides a stunning demonstration of this feature. Certainly, this mechanism has regulatory potential for other noncoding RNA-containing complexes. The observations on roX RNAs in flies will certainly inspire similar investigations into other noncoding RNAs. REFERENCES
identified could act as a chromosomal tether for MSL proteins at HAS sites (Larschan et al., 2012). It will be interesting to see how roX RNA-containing com-
R.C., Luscombe, N., Backofen, R., et al. (2013). Mol. Cell 51, this issue, 156–173. Kelley, R.L., Lee, O.K., and Shim, Y.K. (2008). Mech. Dev. 125, 1009–1019. Larschan, E., Soruco, M.M., Lee, O.K., Peng, S., Bishop, E., Chery, J., Goebel, K., Feng, J., Park, P.J., and Kuroda, M.I. (2012). PLoS Genet. 8, e1002830. Maenner, S., Mu¨ller, M., Fro¨hlich, J., Langer, D., and Becker, P.B. (2013). Mol. Cell 51, this issue, 174–184. Meller, V.H., Wu, K.H., Roman, G., Kuroda, M.I., and Davis, R.L. (1997). Cell 88, 445–457. Meller, V.H., Gordadze, P.R., Park, Y., Chu, X., Stuckenholz, C., Kelley, R.L., and Kuroda, M.I. (2000). Curr. Biol. 10, 136–143. Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., et al. (2006). Mol. Cell 21, 811–823. Morra, R., Yokoyama, R., Ling, H., and Lucchesi, J.C. (2011). Epigenetics Chromatin 4, 6. Park, S.W., Kang, Y.Ie., Sypula, J.G., Choi, J., Oh, H., and Park, Y. (2007). Genetics 177, 1429–1437.
Amrein, H., and Axel, R. (1997). Cell 88, 459–469.
Park, S.W., Kuroda, M.I., and Park, Y. (2008). Mol. Cell. Biol. 28, 4952–4962.
Ilik, I.A., Quinn, J.J., Georgiev, P., Tavares-Cadete, F., Maticzka, D., Toscano, S., Wan, Y., Spitale,
Straub, T., Zabel, A., Gilfillan, G.D., Feller, C., and Becker, P.B. (2013). Genome Res. 23, 473–485.
Acetylphosphate: A Novel Link between Lysine Acetylation and Intermediary Metabolism in Bacteria Eric Verdin1,* and Melanie Ott1,* 1Gladstone Institutes, University of California, San Francisco, 1650 Owens Street, San Francisco, CA 94158, USA *Correspondence: [email protected] (E.V.), [email protected] (M.O.) http://dx.doi.org/10.1016/j.molcel.2013.07.006
In this issue of Molecular Cell, Weinert et al. (2013) demonstrate that the intermediary metabolite acetyl-phosphate is an important acetyl donor that contributes to global protein acetylation in growth-arrested E. coli. Protein acetylation is an emerging posttranslational modification of growing biological significance. Acetylation takes place on the ε-amino group of lysine residues and, like other posttranslational modifications, regulates diverse protein properties, including DNA-protein interactions, subcellular localization, protein
stability, protein-protein interaction, and enzymatic activity. Acetylation is particularly abundant within the mitochondrial matrix, and acetylated mitochondrial proteins are involved in energy metabolism, including key enzymes in the TCA cycle, oxidative phosphorylation, b-oxidation of lipids, amino acid metabolism, and the
132 Molecular Cell 51, July 25, 2013 ª2013 Elsevier Inc.
urea cycle (Rardin et al., 2013; Hebert et al., 2013). Acetylation at distinct lysine residues is thought to occur either enzymatically via the activity of a lysine acetyltransferase (KAT) or nonenzymatically (reviewed in He et al., 2012) and depends on the intermediary metabolite acetylcoenzyme A as the exclusive acetyl donor.
Previews Noncoding roX RNA Remodeling Triggers Fly Dosage Compensation Complex Assembly Anton Wutz1,* 1Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, ETH Zu ¨ rich, Schafmattstrasse 22, 8049 Zurich, Switzerland *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.07.007
Spreading of dosage compensation over the X chromosome in Drosophila males requires the noncoding roX1 and roX2 RNAs. In this issue, Ilik et al. (2013) and Maenner et al. (2013) show that these RNAs contain discrete binding sites that are remodeled during assembly of the dosage compensation complex. Drosophila melanogaster males enhance expression of their single X chromosome to approximate the two X chromosomes in females. This is achieved, at least in part, by X chromosome-wide acetylating histone H4 lysine 16 through a dosage compensation complex (DCC) containing the male-specific lethal proteins MSL1– MSL3 and the histone acetyltransferase males absent on the first (MOF). For spreading over the X chromosome, two noncoding RNAs, roX1 and roX2 (Amrein and Axel, 1997; Meller et al., 1997), and the maleless (MLE) helicase are required (Meller et al., 2000; Morra et al., 2011). Interaction of roX RNAs with MLE during DCC assembly is revealed in two studies by Ilik et al. (2013) and Maenner et al. (2013) in this issue. Ilik et al. (2013) identify RNA-binding sites of MLE and MSL2 using an individual-nucleotide resolution crosslinking and imunoprecipitation (iCLIP) protocol. Following UV crosslinking and precipitation with antibodies specific for MLE and MSL2 the site of crosslink can then be identified by sequencing reverse-transcribed fragments. This analysis shows that MLE occupies discrete binding sites on roX RNAs. Since UV crosslinks require close contact, iCLIP identifies sites of direct binding. Notably, analysis of the top 1% of iCLIP contacts identifies a consensus sequence that resembles a roX box motif that was identified as a sequence of similarity between roX1 and roX2 and is important for their function (Kelley et al., 2008; Park et al., 2007, 2008). In vitro MLE protein binds to a 50 fragment of roX2. Binding to a second binding site in the 30 half requires ATP. The fact that binding to the 30 site also requires the helicase activity of MLE
suggests that this is an active process. Notably, genetic tests in transgenic flies indicate that the ATP-dependent binding site in roX2 is functionally more important than the ATP-independent bound site in the 50 region. For this analysis, the authors use an elegant transgenic system for genetic complementation of roX1- and roX2-deficient males. The 50 region of roX2 appears far less efficient in rescuing male viability than the 30 region, showing that MLE binding alone is not sufficient. This aspect is independently investigated by Maenner et al. (2013), who use an RNA affinity purification strategy to study MLE binding. A conserved stemloop structure in roX1 and roX2 that contains roX boxes (SLroX1 and SLroX2) mediates MLE binding, which is further stimulated by the addition of ATP. This result is consistent with observations on the roX2 30 fragment by Ilik et al. (2013). Using oligonucleotide-mediated precipitation, Maenner et al. (2013) can further show that MLE binds a large stem loop in roX2 that is remodeled into two smaller loops in the presence of ATP. This observation suggests a role for the MLE helicase for remodeling roX2. Maenner et al. (2013) go on to investigate the significance for assembly of DCC by studying binding of MSL2. MSL2 is a critical component of DCC and closely associates with MLE at several high-affinity sites (HAS) on the male X chromosome (Straub et al., 2013 and Ilik et al., 2013). Using in vitro transcribed roX2 RNA immobilized through an MS2 tag, binding of MSL2 and MLE from nuclear extracts can be observed specifically in the presence of ATP. This is a remarkable result suggesting that the first steps of DCC assembly have been recapitulated in vitro. The
helicase function of MLE might therefore be important for remodeling critical binding sites for MSL2 on roX2 RNA (Figure 1). Analysis of the in vivo relevance of roX RNA remodeling remains an important aim for future work. One way to test this idea could be through rescuing MLEdeficient males by providing a remodeled transgenic version of roX2 that would not fold into a large stem loop, but instead adopt the remodeled conformation. It needs to be determined if structural mutations in the stem that maintain critical sequence motifs can be identified. However, the experiment could provide unambiguous evidence for blocked binding sites in roX2. Why roX RNAs should fold into an apparently inactive conformation in the first place appears unclear at the moment. roX RNAs and MSL2 protein are unstable in the absence of MLE, and stabilization requires DCC assembly. Jointly, the studies by Ilik et al. (2013) and Maenner et al. (2013) identify roX boxes as binding sites for MLE and implicate ATP-dependent processes in DCC assembly. Remodeling of roX RNAs could represent a rate-limiting step that could titrate the amount of DCC. Where DCC assembly occurs is another interesting question. MLE and MSL2 proteins are produced in the cytoplasm and could bind roX RNAs before or after nuclear import. Interestingly, an earlier study has implicated nuclear pore components in dosage compensation (Mendjan et al., 2006). One candidate location for DCC assembly is chromosomal HAS sites. These sites can be occupied by members of the DCC in the absence of MLE and might be entry sites for spreading over the X chromosome. A zinc finger protein that has been recently
Molecular Cell 51, July 25, 2013 ª2013 Elsevier Inc. 131
Molecular Cell
Previews cytoplasm
roX
ML
MSL2
E DCC MSL1 MSL2 MOF
H4K16Ac Figure 1. MLE Helicase Activity Remodels Binding Sites in roX RNAs for DCC Assembly roX RNAs are transcribed from the X chromosome and adopt a folded conformation. Remodeling by MLE helicase unmasks binding sites for MSL2 and triggers assembly of DCC. Incorporation of the histone acetyltransferase MOF catalyzes X chromosome-wide acetylation of histone H4 lysine 16 to mediate upregulation of X-linked genes in male flies.
plexes interact with chromosomal binding partners in future work. roX RNAs highlight remarkable features of RNA protein complexes. Several binding sites are embedded into long stretches of sequence that are less structured and might be functionally irrelevant. Depending on which conformation the RNA adopts, certain bases become accessible, revealing binding sites. Conceivably, composition of RNA protein complexes can be changed by refolding hidden conditional binding sites that would otherwise be buried in RNA secondary structure. Binding of MSL2 to roX2 provides a stunning demonstration of this feature. Certainly, this mechanism has regulatory potential for other noncoding RNA-containing complexes. The observations on roX RNAs in flies will certainly inspire similar investigations into other noncoding RNAs. REFERENCES
identified could act as a chromosomal tether for MSL proteins at HAS sites (Larschan et al., 2012). It will be interesting to see how roX RNA-containing com-
R.C., Luscombe, N., Backofen, R., et al. (2013). Mol. Cell 51, this issue, 156–173. Kelley, R.L., Lee, O.K., and Shim, Y.K. (2008). Mech. Dev. 125, 1009–1019. Larschan, E., Soruco, M.M., Lee, O.K., Peng, S., Bishop, E., Chery, J., Goebel, K., Feng, J., Park, P.J., and Kuroda, M.I. (2012). PLoS Genet. 8, e1002830. Maenner, S., Mu¨ller, M., Fro¨hlich, J., Langer, D., and Becker, P.B. (2013). Mol. Cell 51, this issue, 174–184. Meller, V.H., Wu, K.H., Roman, G., Kuroda, M.I., and Davis, R.L. (1997). Cell 88, 445–457. Meller, V.H., Gordadze, P.R., Park, Y., Chu, X., Stuckenholz, C., Kelley, R.L., and Kuroda, M.I. (2000). Curr. Biol. 10, 136–143. Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., et al. (2006). Mol. Cell 21, 811–823. Morra, R., Yokoyama, R., Ling, H., and Lucchesi, J.C. (2011). Epigenetics Chromatin 4, 6. Park, S.W., Kang, Y.Ie., Sypula, J.G., Choi, J., Oh, H., and Park, Y. (2007). Genetics 177, 1429–1437.
Amrein, H., and Axel, R. (1997). Cell 88, 459–469.
Park, S.W., Kuroda, M.I., and Park, Y. (2008). Mol. Cell. Biol. 28, 4952–4962.
Ilik, I.A., Quinn, J.J., Georgiev, P., Tavares-Cadete, F., Maticzka, D., Toscano, S., Wan, Y., Spitale,
Straub, T., Zabel, A., Gilfillan, G.D., Feller, C., and Becker, P.B. (2013). Genome Res. 23, 473–485.
Acetylphosphate: A Novel Link between Lysine Acetylation and Intermediary Metabolism in Bacteria Eric Verdin1,* and Melanie Ott1,* 1Gladstone Institutes, University of California, San Francisco, 1650 Owens Street, San Francisco, CA 94158, USA *Correspondence: [email protected] (E.V.), [email protected] (M.O.) http://dx.doi.org/10.1016/j.molcel.2013.07.006
In this issue of Molecular Cell, Weinert et al. (2013) demonstrate that the intermediary metabolite acetyl-phosphate is an important acetyl donor that contributes to global protein acetylation in growth-arrested E. coli. Protein acetylation is an emerging posttranslational modification of growing biological significance. Acetylation takes place on the ε-amino group of lysine residues and, like other posttranslational modifications, regulates diverse protein properties, including DNA-protein interactions, subcellular localization, protein
stability, protein-protein interaction, and enzymatic activity. Acetylation is particularly abundant within the mitochondrial matrix, and acetylated mitochondrial proteins are involved in energy metabolism, including key enzymes in the TCA cycle, oxidative phosphorylation, b-oxidation of lipids, amino acid metabolism, and the
132 Molecular Cell 51, July 25, 2013 ª2013 Elsevier Inc.
urea cycle (Rardin et al., 2013; Hebert et al., 2013). Acetylation at distinct lysine residues is thought to occur either enzymatically via the activity of a lysine acetyltransferase (KAT) or nonenzymatically (reviewed in He et al., 2012) and depends on the intermediary metabolite acetylcoenzyme A as the exclusive acetyl donor.