Moonlighting proteins mediate cross talk between different pathways and cellular control networks. Sometimes, they even coordinate subsequent steps in the same pathway. For this Outlook paper we asked the question, which cellular processes employ multifunctional proteins (MFPs) and what makes them so attractive to cells and organisms. After reviewing their widespread occurrence, we will focus on higher eukaryotic model systems and on few examples that are linked to ongoing work in our laboratory. We will discuss the activities of transcription factor IIH (TFIIH), and its subcomplexes containing Xpd and Cdk7, and we will cover an aminoacyl-tRNA synthetase (LysRS) and DEAD box RNA helicases. Furthermore, we will analyze how cells are able to properly regulate the different biological activities of multifunctional proteins and which advantages such proteins offer to cells and organisms. Finally we also note that the proteins we discuss are linked to tumor formation or recruited by viruses that coopt the multifunctional protein for yet another purpose. 1. Introduction For decades one hypothesis ruled most scientist’s mind: one gene gives rise to one protein, which performs one specific role in the cell. Nowadays, this hypothesis gets severely challenged as there is increasing evidence that many proteins perform multiple functions and many cells seem to exploit the opportunities offered by these multitask proteins. Numerous cellular control processes act simultaneously in the same cell and many proteins function in more than one control process. This realization has spurred our interest in finding out how cells take advantage of such dual- or even multifunctional proteins (MFPs). There are examples of dual roles that appear to be simple evolutionary “duplication steps” (Figure 1). Such MFPs can then be found in different protein complexes, which account for their different biological functions. In other cases, however, MFPs regulate parallel or subsequent cellular processes in a coordinated fashion (Figure 2). This points to a higher level of cellular control associated with the reuse of this protein. In yet other cases two alternative pathways share a component that is present only in limited amounts in the cell and this low abundance allows it to only function in one process at the time (Figure 3). In this case cells may use such a protein as a switch between two alternative processes, regulating them in an inverse manner. Figure 1: Evolutionary duplication steps. MFPs can be found in different complexes, promoting different activities. For example, RPN5
G. C. Atkinson, T. Tenson, and V. Hauryliuk, “The RelA/SpoT Homolog (RSH) superfamily: distribution and functional evolution of ppgpp synthetases and hydrolases across the tree of life,” PLoS ONE, vol. 6, no. 8, Article ID e23479, 2011.
T. Hogg, U. Mechold, H. Malke, M. Cashel, and R. Hilgenfeld, “Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response,” Cell, vol. 117, no. 1, pp. 57–68, 2004.
V. Jain, R. Saleem-Batcha, and D. Chatterji, “Synthesis and hydrolysis of pppGpp in mycobacteria: a ligand mediated conformational switch in Rel,” Biophysical Chemistry, vol. 127, no. 1-2, pp. 41–50, 2007.
D. Hoepfner, M. van den Berg, P. Philippsen, H. F. Tabak, and E. H. Hettema, “A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae,” Journal of Cell Biology, vol. 155, no. 6, pp. 979–990, 2001.
M. Fagarasanu, A. Fagarasanu, Y. Y. C. Tam, J. D. Aitchison, and R. A. Rachubinski, “Inp1p is a peroxisomal membrane protein required for peroxisome inheritance in Saccharomyces cerevisiae,” Journal of Cell Biology, vol. 169, no. 5, pp. 765–775, 2005.
A. H. Sarker, S. E. Tsutakawa, S. Kostek et al., “Recognition of RNA polymerase II and transcription bubbles by XPG, CSB, and TFIIH: insights for transcription-coupled repair and Cockayne syndrome,” Molecular Cell, vol. 20, no. 2, pp. 187–198, 2005.
S. Larochelle, J. Chen, R. Knights et al., “T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity,” EMBO Journal, vol. 20, no. 14, pp. 3749–3759, 2001.
S. Larochelle, J. Batliner, M. J. Gamble et al., “Dichotomous but stringent substrate selection by the dual-function Cdk7 complex revealed by chemical genetics,” Nature Structural and Molecular Biology, vol. 13, no. 1, pp. 55–62, 2006.
J. Bartkova, M. Zemanova, and J. Bartek, “Expression of CDK7/CAK in normal and tumor cells of diverse histogenesis, cell-cycle position and differentiation,” International Journal of Cancer, vol. 66, no. 6, pp. 732–737, 1996.
K. Glover-Cutter, S. Larochelle, B. Erickson et al., “TFIIH-associated Cdk7 kinase functions in phosphorylation of C-terminal domain Ser7 residues, promoter-proximal pausing, and termination by RNA polymerase II,” Molecular and Cellular Biology, vol. 29, no. 20, pp. 5455–5464, 2009.
S. Larochelle, R. Amat, K. Glover-Cutter et al., “Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II,” Nature Structural and Molecular Biology, vol. 19, no. 11, pp. 1108–1115, 2012.
W. Vermeulen, E. Bergmann, J. Auriol et al., “Sublimiting concentration of TFIIH transcription/DNA repair factor causes TTD-A trichothiodystrophy disorder,” Nature Genetics, vol. 26, no. 3, pp. 307–313, 2000.
F. Coin, J.-C. Marinoni, C. Rodolfo, S. Fribourg, A. M. Pedrini, and J.-M. Egly, “Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH,” Nature Genetics, vol. 20, no. 2, pp. 184–188, 1998.
S. Dubaele, L. P. de Santis, R. J. Bienstock et al., “Basal transcription defect discriminates between xeroderma pigmentosum and trichothiodystrophy in XPD patients,” Molecular Cell, vol. 11, no. 6, pp. 1635–1646, 2003.
F. Coin, V. Oksenych, and J.-M. Egly, “Distinct roles for the XPB/p52 and XPD/p44 subcomplexes of TFIIH in damaged DNA opening during nucleotide excision repair,” Molecular Cell, vol. 26, no. 2, pp. 245–256, 2007.
K. Gari, A. M. León Ortiz, V. Borel, H. Flynn, J. M. Skehel, and S. J. Boulton, “MMS19 links cytoplasmic iron-sulfur cluster assembly to DNA metabolism,” Science, vol. 337, no. 6091, pp. 243–245, 2012.
N. van Wietmarschen, A. Moradian, G. B. Morin, P. M. Lansdorp, and E.-J. Uringa, “The mammalian proteins MMS19, MIP18, and ANT2 are involved in cytoplasmic iron-sulfur cluster protein assembly,” The Journal of Biological Chemistry, vol. 287, no. 52, pp. 43351–43358, 2012.
M. Guo, M. Ignatov, K. Musier-Forsyth, P. Schimmel, and X.-L. Yang, “Crystal structure of tetrameric form of human lysyl-tRNA synthetase: implications for multisynthetase complex formation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 7, pp. 2331–2336, 2008.
O. Kellermann, H. Tonetti, A. Brevet, M. Mirande, J. P. Pailliez, and J. P. Waller, “Macromolecular complexes from sheep and rabbit containing seven aminoacyl-tRNA synthetases. I. Species specificity of the polypeptide composition,” The Journal of Biological Chemistry, vol. 257, no. 18, pp. 11041–11048, 1982.
D. L. Johnson and D. C. H. Yang, “Stoichiometry and composition of an aminoacyl-tRNA synthetase complex from rat liver,” Proceedings of the National Academy of Sciences of the United States of America, vol. 78, no. 7, pp. 4059–4062, 1981.
J.-C. Robinson, P. Kerjan, and M. Mirande, “Macromolecular assemblage of aminoacyl-tRNA synthetases: quantitative analysis of protein-protein interactions and mechanism of complex assembly,” Journal of Molecular Biology, vol. 304, no. 5, pp. 983–994, 2000.
M. Mirande, B. Cirako？lu, and J. P. Waller, “Seven mammalian aminoacyl-tRNA synthetases associated within the same complex are functionally independent,” European Journal of Biochemistry, vol. 131, no. 1, pp. 163–170, 1983.
S. G. Park, K. L. Ewalt, and S. Kim, “Functional expansion of aminoacyl-tRNA synthetases and their interacting factors: new perspectives on housekeepers,” Trends in Biochemical Sciences, vol. 30, no. 10, pp. 569–574, 2005.
J. M. Han, M. J. Lee, S. G. Park et al., “Hierarchical network between the components of the multi-tRNA synthetase complex: implications for complex formation,” The Journal of Biological Chemistry, vol. 281, no. 50, pp. 38663–38667, 2006.
J. Y. Kim, Y.-S. Kang, J.-W. Lee et al., “p38 is essential for the assembly and stability of macromolecular tRNA synthetase complex: implications for its physiological significance,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 12, pp. 7912–7916, 2002.
M. J. Kim, B.-J. Park, Y.-S. Kang et al., “Downregulation of FUSE-binding protein and c-myc by tRNA synthetase cofactor p38 is required for lung cell differentiation,” Nature Genetics, vol. 34, no. 3, pp. 330–336, 2003.
S. V. Kyriacou and M. P. Deutscher, “An important role for the multienzyme aminoacyl-tRNA synthetase complex in mammalian translation and cell growth,” Molecular Cell, vol. 29, no. 4, pp. 419–427, 2008.
P. G. Zamecnik, M. L. Stephenson, C. M. Janeway, and K. Randerath, “Enzymatic synthesis of diadenosine tetraphosphate and diadenosine triphosphate with a purified lysyl-sRNA synthetase,” Biochemical and Biophysical Research Communications, vol. 24, no. 1, pp. 91–97, 1966.
A. Brevet, P. Plateau, B. Cirako？lu, J. P. Pailliez, and S. Blanquet, “Zinc-dependent synthesis of 5′,5′-diadenosine tetraphosphate by sheep liver lysyl- and phenylalanyl-tRNA synthetases,” The Journal of Biological Chemistry, vol. 257, no. 24, pp. 14613–14615, 1982.
E. Razin, Z. C. Zhang, H. Nechushtan et al., “Suppression of microphthalmia transcriptional activity by its association with protein kinase C-interacting protein 1 in mast cells,” The Journal of Biological Chemistry, vol. 274, no. 48, pp. 34272–34276, 1999.
Y.-N. Lee, H. Nechushtan, N. Figov, and E. Razin, “The function of lysyl-tRNA synthetase and Ap4A as signaling regulators of MITF activity in FcεRI-activated mast cells,” Immunity, vol. 20, no. 2, pp. 145–151, 2004.
Y.-N. Lee and E. Razin, “Nonconventional involvement of LysRS in the molecular mechanism of USF2 transcriptional activity in FcεRI-activated mast cells,” Molecular and Cellular Biology, vol. 25, no. 20, pp. 8904–8912, 2005.
H. Javanbakht, R. Halwani, S. Cen et al., “The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly,” The Journal of Biological Chemistry, vol. 278, no. 30, pp. 27644–27651, 2003.
M. Guo, R. Shapiro, G. M. Morris, X.-L. Yang, and P. Schimmel, “Packaging HIV virion components through dynamic equilibria of a human tRNA synthetase,” Journal of Physical Chemistry B, vol. 114, no. 49, pp. 16273–16279, 2010.
J. Mak, M. Jiang, M. A. Wainberg, M.-L. Hammarskj？ld, D. Rekosh, and L. Kleiman, “Role of Pr160gag-pol in mediating the selective incorporation of tRNALys into human immunodeficiency virus type 1 particles,” Journal of Virology, vol. 68, no. 4, pp. 2065–2072, 1994.
R. Halwani, S. Cen, H. Javanbakht et al., “Cellular distribution of Lysyl-tRNA synthetase and its interaction with gag during human immunodeficiency virus type 1 assembly,” Journal of Virology, vol. 78, no. 14, pp. 7553–7564, 2004.
S. Cen, H. Javanbakht, M. Niu, and L. Kleiman, “Ability of wild-type and mutant lysyl-tRNA synthetase to facilitate tRNALys) incorporation into human immunodeficiency virus type 1,” Journal of Virology, vol. 78, no. 3, pp. 1595–1601, 2004.
F. Guo, J. Gabor, S. Cen, K. Hu, A. J. Mouland, and L. Kleiman, “Inhibition of cellular HIV-1 protease activity by lysyl-tRNA synthetase,” The Journal of Biological Chemistry, vol. 280, no. 28, pp. 26018–26023, 2005.
S. G. Park, H. J. Kim, Y. H. Min et al., “Human lysyl-tRNA synthetase is secreted to trigger proinflammatory response,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 18, pp. 6356–6361, 2005.
D. G. Kim, J. W. Choi, J. Y. Lee et al., “Interaction of two translational components, lysyl-tRNA synthetase and p40/37LRP, in plasma membrane promotes laminin-dependent cell migration,” The FASEB Journal, vol. 26, no. 10, pp. 4142–4159, 2012.
D. Soulat, T. Bürckstümmer, S. Westermayer et al., “The DEAD-box helicase DDX3X is a critical component of the TANK-binding kinase 1-dependent innate immune response,” EMBO Journal, vol. 27, no. 15, pp. 2135–2146, 2008.
H. Endoh, K. Maruyama, Y. Masuhiro et al., “Purification and identification of p68 RNA helicase acting as a transcriptional coactivator specific for the activation function 1 of human estrogen receptor α,” Molecular and Cellular Biology, vol. 19, no. 8, pp. 5363–5372, 1999.
B. J. Wilson, G. J. Bates, S. M. Nicol, D. J. Gregory, N. D. Perkins, and F. V. Fuller-Pace, “The p68 and p72 DEAD box RNA helicases interact with HDAC1 and repress transcription in a promoter-specific manner,” BMC Molecular Biology, vol. 5, article 11, 2004.
C. Merz, H. Urlaub, C. L. Will, and R. Lührmann, “Protein composition of human mRNPs spliced in vitro and differential requirements for mRNP protein recruitment,” RNA, vol. 13, no. 1, pp. 116–128, 2007.
T. Burckin, R. Nagel, Y. Mandel-Gutfreund, et al., “Exploring functional relationships between components of the gene expression machinery,” Nature Structural & Molecular Biology, vol. 12, no. 2, pp. 175–182, 2005.
C.-S. Lee, A. P. Dias, M. Jedrychowski, A. H. Patel, J. L. Hsu, and R. Reed, “Human DDX3 functions in translation and interacts with the translation initiation factor eIF3,” Nucleic Acids Research, vol. 36, no. 14, pp. 4708–4718, 2008.
M. Schr？der, “Human DEAD-box protein 3 has multiple functions in gene regulation and cell cycle control and is a prime target for viral manipulation,” Biochemical Pharmacology, vol. 79, no. 3, pp. 297–306, 2010.
H. S. Chahar, S. Chen, and N. Manjunath, “P-body components LSM1, GW182, DDX3, DDX6 and XRN1 are recruited to WNV replication sites and positively regulate viral replication,” Virology, vol. 436, no. 1, pp. 1–7, 2013.
S. C. Cloutier, W. K. Ma, L. T. Nguyen, and E. J. Tran, “The DEAD-box RNA helicase Dbp2 connects RNA quality control with repression of aberrant transcription,” The Journal of Biological Chemistry, vol. 287, no. 31, pp. 26155–26166, 2012.
A. Hilliker, Z. Gao, E. Jankowsky, and R. Parker, “The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex,” Molecular Cell, vol. 43, no. 6, pp. 962–972, 2011.
J. Liu, J. Henao-Mejia, H. Liu, Y. Zhao, and J. J. He, “Translational regulation of HIV-1 replication by HIV-1 rev cellular cofactors Sam68, eIF5A, hRIP, and DDX3,” Journal of Neuroimmune Pharmacology, vol. 6, no. 2, pp. 308–321, 2011.
C. Lin, L. Yang, J. J. Yang, Y. Huang, and Z.-R. Liu, “ATPase/helicase activities of p68 RNA helicase are required for pre-mRNA splicing but not for assembly of the spliceosome,” Molecular and Cellular Biology, vol. 25, no. 17, pp. 7484–7493, 2005.
Y.-J. Choi and S.-G. Lee, “The DEAD-box RNA helicase DDX3 interacts with DDX5, co-localizes with it in the cytoplasm during the G2/M phase of the cycle, and affects its shuttling during mrnp export,” Journal of Cellular Biochemistry, vol. 113, no. 3, pp. 985–996, 2012.
B. Prud'homme, N. Lartillot, G. Balavoine, A. Adoutte, and M. Vervoort, “Phylogenetic analysis of the Wnt gene family: insights from lophotrochozoan members,” Current Biology, vol. 12, no. 16, pp. 1395–1400, 2002.