Post-Transcriptional Regulation of Plant Gene Expression
Plant translation initiation factors: it is not easy to be green K.S. Browning1 Department of Chemistry and Biochemistry, and the Institute of Cell and Molecular Biology, University of Texas at Austin, 1 University Station, A5300 Austin, TX 78712, U.S.A.
Abstract Plants have significant differences in some of the ‘parts’ of the translational machinery. There are two forms of eukaryotic initiation factor (eIF) 4F, eIF3 has two novel subunits, eIF4B is poorly conserved, and eIF2 kinases and eIF4E binding proteins (4E-BP) are yet to be discovered. These differences suggest that plants may regulate their translation in unique ways.
Selecting a eukaryotic mRNA for translation into a polypeptide is a complicated process and requires 12 or more initiation factors (for a recent review and schematic on eukaryotic translation, see [1]; for previous reviews on plant translation, see [2,3]). The fundamental process is similar in all eukaryotes; however, there are differences in mammalian, plant and yeast translation initiation factors. The most striking differences occur between plant initiation factors and those from other eukaryotes, particularly with respect to eIF4F/eIF(iso)4F, eIF3, eIF4B and eIF2 (where eIF stands for eukaryotic initiation factor).
eIF4F and eIF(iso)4F: the twin cap-binding complexes eIF4F is a cap-binding complex and consists of eIF4E, a small cap-binding protein that binds the m7 G group on the 5 ends of mRNAs, and eIF4G, a large protein that interacts with other initiation factors. The interaction of eIF4F with mRNA is supposed to be the first step in the initiation of translation. eIF4A, the original DEAD box helicase, is loosely associated with this complex and participates in ATPdependent unwinding of the mRNA [4]. Plant eIF4A was recently reported to be associated with a cyclin-dependent kinase during active cell proliferation [5]. This finding opens a whole new arena for the regulation of translation during plant cell growth/development. Plants have a second form of eIF4F, termed eIF(iso)4F, which is not present in other eukaryotes. eIF(iso)4F, similar to eIF4F, consists of two subunits, a small cap-binding protein (eIF(iso)4E) and a large subunit eIF(iso)4G, and has in vitro activities similar to eIF4F [3]. The cap-binding subunits eIF4E and eIF(iso)4E show approx. 50% similarity in their amino acid sequences, and the molecular mass of Key words: cap-binding complex, eukaryotic initiation factor 4F (eIF4F), eIF(iso)4F, eukaryote, translation initiation factor. Abbreviations used: 4E-BP, 4E-binding protein; eIF, eukaryotic initiation factor; GCN, positive general control of transcription; PABP, poly A-binding protein; PKR, double-stranded-RNAdependent protein kinase. 1 email
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both is approx. 24 kDa. However, the eIF4G and eIF(iso)4G subunits are substantially different in molecular mass, 180 kDa versus 86 kDa [3]. eIF(iso)4G lacks a significant amount of sequence in the N-terminal domain when compared with eIF4G. This substantial difference in the mass of eIF4G and eIF(iso)4G suggests that the role and/or regulation of these complexes may be very different. The functional significance of having two forms of eIF4F has not been elucidated, but evidence suggests that these two forms may discriminate between mRNAs that have internal initiation elements or structure [6]. We have biochemical evidence that shows that the subunits of eIF4F and eIF(iso)4F are functionally interchangeable and they are capable of discriminating between different mRNAs in vitro (L. Allen, P. Murphy, L. Campbell, K. Ruud and K.S. Browning, unpublished work). The cap-binding subunits eIF4E and eIF(iso)4E have been shown to have a variety of roles during plant viral infection, primarily through interaction with the viral Vpg [7–11]. eIF4E and eIF(iso)4E have been shown to interact directly with RNA elements in the 3 -untranslated region of satellite tobacco necrosis virus RNA [12] and barley yellow dwarf virus (W.A. Miller, personal communication). An Arabidopsis knockout for the single eIF(iso)4E gene is ‘normal’, although the amount of eIF4E is increased in these mutants [8], consistent with our biochemical observation that the subunits of eIF4F and eIF(iso)4F are functionally interchangeable. In addition, the eIF(iso)4E knockout plants are resistant to infection by potyviruses [8]. These observations suggest that viruses have figured out how to manipulate the eIF4F/ eIF(iso)4F cellular machinery to their benefit. There are several other players in the cap recognition machinery. A ‘novel’ cap-binding protein is present in plants and other eukaryotes; however, the function of this form of cap-binding protein has not been determined [13]. A subunit of the nuclear cap-binding complex CBP80 (ABH1) was shown to be involved in abscisic acid signalling events [14]. A ‘missing in action’ factor is 4E-BP (4E-binding protein). This factor plays an important role in the regulation of mammalian protein synthesis by sequestration of eIF4E. The interaction C 2004
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of 4E-BP with eIF4E is affected by phosphorylation via signal transduction pathways [15]. However, 4E-BP has not been identified so far in plants or in a plant genome. It is also interesting to note that plant eIF4G/eIF(iso)4G lacks the mammalian C-terminal motif for binding the MNKI kinase. The MNKI kinase phosphorylates mammalian eIF4E bound to the N-terminal domain of mammalian eIF4G [16]. The apparent lack of 4E-BP and the lack of an MNKI-binding domain suggest that plants may regulate protein synthesis by different mechanisms.
eIF3: the largest initiation-factor complex The most complex initiation factor is eIF3. The subunit composition varies from five non-identical subunits for Saccharomyces cerevisiae eIF3 to 11 non-identical subunits for mammalian eIF3 and 12 non-identical subunits for plant eIF3 [17]. We have found two subunits that are unique to plant eIF3 and have no analogous proteins in the mammalian complex [18]. The five subunits in S. cerevisiae eIF3 are supposed to constitute a functional ‘core’; the additional subunits in plant and mammalian eIF3 are probably regulatory. Interestingly, plant eIF3 subunits have been found to be associated with the COP9 signalosome [19] and as a brassinosteriod regulated protein [20]. These observations suggest that eIF3 may be very important in intracellular communication between various pathways as a method to regulate translation, in addition to its role in forming protein– protein contacts with eIF4F, eIF5, eIF2, eIF1, eIF1A and the ribosome during initiation [1]. Roles for the two plantspecific subunits are yet to be discovered, but they are probable targets for regulation.
eIF2: the most recalcitrant initiation factor eIF2 from plants does not differ significantly in composition or amino acid sequence from eIF2 from other eukaryotes, but there seem to be significant differences in the way eIF2 and protein synthesis are regulated in plants. The phosphorylation of eIF2α in mammals and yeast is a significant regulatory pathway that prevents the interaction of eIF2–GDP with eIF2B to exchange GDP for GTP and shuts down protein synthesis [26]. Plants have been reported to have a similar kinase activity, PKR (double-stranded-RNAdependent protein kinase), which phosphorylates eIF2 [27]; however, a PKR gene sequence has not come forth from the Arabidopsis or other plant genomes/expressed sequence tags. An orthologue of the PKR inhibitor has been identified in plants and appears to have a role in viral pathogenesis, but the PKR itself has not been identified yet [28]. Another eIF2α kinase in yeast, GCN2 (positive general control of transcription-2), regulates translation during amino acid starvation [26]. A GCN2 homologue has been reported in plants; however, the absence of a plant GCN4 that is the target of translational control by GCN2 implies that there may be differences in how plants regulate translation during amino acid starvation [29]. Although there is evidence that plants have eIF2α kinases, there is still uncertainty about how similar they are to other better characterized mammalian and yeast eIF2α kinase pathways. One may speculate that either plants do not use this pathway for general regulation of protein synthesis or it may be a minor pathway in plants. It is probable that plants have other, as yet unidentified, ways of regulating protein synthesis. The recent discovery of a cyclindependent kinase that is associated with and phosphorylates eIF4A may represent such a new plant-specific regulatory pathway [5].
eIF4B: the least conserved initiation factor Every initiation factor, except eIF4B, shows a high degree of conservation among eukaryotes. There is essentially no primary amino acid sequence conservation among yeast, mammalian and plant eIF4Bs [21]. The function of eIF4B is conserved as an RNA-binding protein that stimulates eIF4F and eIF4A RNA-unwinding activities. mRNAs are able to discriminate and show preferences for one or more forms of plant eIF4B and also show distinct preferences for eIF4F over eIF(iso)4F in the presence of different forms of eIF4B (L. Mayberry and K.S. Browning, unpublished work). Plant eIF4B, similar to mammalian and yeast eIF4Bs, interacts directly with eIF3 [22]. Interestingly, eIF4B acts as a competitor for the cauliflower mosaic virus re-initiation factor (TAV) binding to eIF3 [22]. Plant eIF4B also interacts with PABP (poly A-binding protein) and stimulates cap-binding and RNA-unwinding activities [23,24]. eIF4Bs from mammals and plants are known to be highly phosphorylated, suggesting that phosphorylation plays a role in the regulation of translation initiation. The interaction of plant eIF4B with PABP depends on the phosphorylation states of both eIF4B and PABP [25]. Regulation of protein synthesis by eIF4B and its kinase(s) will surely yield some interesting facts. C 2004
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Conclusions There are several significant differences in the translational machinery of plants when compared with other eukaryotes, such as the presence of two forms of eIF4F, the apparent absence of (or undiscovered) 4E-BP equivalent, two novel eIF3 subunits, divergent eIF4B and the apparent absence of (or undiscovered) eIF2α PKR kinase. These differences suggest that plants regulate the initiation of translation by alternative pathways. Plants carry out several unique biochemical processes that other eukaryotes do not, e.g. photosynthesis, cellulose biosynthesis, plant hormone signalling and many secondary metabolic pathways. It is therefore not surprising that plants manage their protein synthesis machinery in various ways to regulate and communicate with these unique processes and vice versa. It is not easy to be green.
This work is supported by grants from the National Science Foundation (MCB-0214996), Department of Energy (DE-FG03-97ER20283) and the Welch Foundation (F-1339). A database, Factors in
Post-Transcriptional Regulation of Plant Gene Expression
Arabidopsis Translation (FIAT), is available at http://www.cm. utexas.edu/browning/db. Thanks are due to Dr J. Ravel and Dr D. Gallie for a critical reading of this paper.
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Received 23 March 2004
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