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American Society of Plant Biologists
The RNA World in PlantsPost-Transcriptional Control III
a Plant Pathology Department 351 Bessey Hall Iowa State University Ames, IA 5001
Post-transcriptional control of gene expression has gone from a curiosity involving a few special genes to a highly diverse and widespread set of processes that is truly pervasive in plant gene expression. Thus, Plant Cell readers interested in almost any aspect of plant gene expression in response to any environmental influence, or in development, are advised to read on. In May 2001, what has become the de facto third biennial Symposium on Post-Transcriptional Control of Gene Expression in Plants was held in Ames, Iowa. The meeting was hosted by the new Plant Sciences Institute of Iowa State University with additional funding from the National Science Foundation and the United States Department of Agriculture. In 1997, the annual University of California-Riverside Plant Physiology Symposium was devoted to this topic. This provided a wake-up call to the plant world, summarized in this journal (Gallie and Bailey-Serres, 1997
The meeting led off with perhaps the most dynamic and controversial topic of the meeting: post-transcriptional gene silencing (PTGS) and its suppression by viral proteins. PTGS is a targeted RNA degradation mechanism in plants (for a recent review, see Waterhouse et al., 2001  ). PTGS may be induced by transgenes or viral infection and causes the degradation of RNAs with homology or complementarity to the transgene transcript or viral genome. The signal to degrade the specific RNA sequence is transmitted throughout the plant. In contrast to much plant molecular biology, which derives its discoveries from animals or yeast, PTGS, a remarkable and fundamentally different (from other known mechanisms) way of signaling and controlling gene expression, was discovered first in plants (e.g., Lindbo et al., 1993). Only later was it found in other eukaryotic systems. One of the early pioneers investigating this phenomenon, Herve Vaucheret (Institut National de la Recherche Agronomique, Versailles, France), opened the meeting by describing his laboratory's use of many elegant experiments that combine traditional techniques such as grafting and classical genetics with modern gene mapping and expression assays to explore the signaling mechanisms and to identify a plethora of genes involved in PTGS. He discussed the roles and functions of some of these genes and described gene expression experiments in progress to identify additional genes involved in PTGS. Although the genetics and particularly the biochemistry of these processes has progressed with lightning speed in Caenorhabditis elegans and Drosophila, plants as PTGS models have held their own, with distinct differences from animals. Certainly, understanding of how a silencing signal is transmitted long distances through walled cells and across grafts remains a challenge to those studying plant PTGS.
Silencing involves the production of double-stranded RNA, in many cases by a viral or a host RNA–dependent RNA polymerase. The double stranded RNA is digested into 21- to 23-nucleotide fragments that act as guides for the degradation of homologous single- stranded RNA. These fragments also may direct the methylation of homologous DNA sequences in the chromosome and may be the signal for systemic silencing. However, experiments from several laboratories have thrown this simple model into disarray. Peter Waterhouse (Commonwealth Scientific and Industrial Research Organization, Plant Industry, Canberra, Australia) described how PTGS can be induced efficiently in plants by the expression of self-complementary hairpin (hp) RNA and that hpRNAs are degraded into short
Vicki Vance (University of South Carolina, Columbia) and Shou-Wei Ding (University of California, Riverside) presented work on the modes of action of the virus-encoded suppressors of gene silencing (Vance and Vaucheret, 2001 Olivier Voinnet (John Innes Centre) presented an extra level of complexity in the roles of short RNAs in PTGS. He found that these RNAs are composed of two different classes: one of 21 nucleotides and the other of 23 nucleotides. Voinnet showed that different virus suppressor proteins had different effects on the accumulation of the 21- and 23-nucleotide RNAs, which enabled him to analyze the mechanism. He concluded that the 21-nucleotide species is sufficient to mediate intracellular silencing and that the 23-nucleotide species is essential for systemic silencing. This finding sparked another controversy. Results from the Vance group initially seemed to suggest an opposite conclusion. They found that when the HC-Pro suppressor was expressed as a transgene, it inhibited the production of both species of short RNAs, but these plants still emitted a graft-transmissible silencing signal. This finding suggests that the short RNAs are not essential for systemic silencing. This controversy may again be attributable to the different ways in which the silencing was delivered. Trent Smith, from the Vance group, reported that in Arabidopsis when the HC-Pro was expressed to release transgene-mediated silencing, it correlated with the reduction of short RNAs to undetectable levels. However, HC-Pro expression that released amplicon-mediated silencing correlated with an increased level of short RNAs. Veronique Brault (Institut National de la Recherche Agronomique, Colmar, France) added still another variation to the suppression story. She showed that transgenic plants harboring a Beet western yellows virus-derived transgene that had undergone PTGS did not degrade invading viral RNA. Because this virus is confined to the phloem, the observations may reflect a phloem-specific viral suppression of silencing in which the majority of plant cells, outside of the phloem, remain silenced, preventing virus movement outside of the phloem. The numerous different observations in different laboratories emphasize the importance of studying silencing in different systems and warn against drawing broad generalizations from studies in only one system. In the end, the gene silencing session was interesting and exciting as much for the need to rationalize the apparently conflicting results as for the rapid advances being made in the area.
Recent advances in plant pre-mRNA splicing research covered two main areas: characterization of intron/exon splicing signals and the identification and function of proteins that interact with pre-mRNA and mRNA transcripts. In the absence of a plant in vitro splicing extract, detailed characterization of plant intron splicing signals has been difficult. The sensitivity to mutation of a potato invertase mini-exon splicing system (reported by John Brown of the Scottish Crop Research Institute, Dundee) has allowed the experimental analysis of a plant intron branch point and polypyrimidine tract at the single-nucleotide level (Simpson et al., 2000 ). The availability of the Arabidopsis genome sequence now provides the means to assemble splice site and internal intron signal information with the experimental characterization performed in a number of splicing laboratories during the last 10 or so years. Bioinformatics approaches are extremely important in the prediction of intron/exon borders, the identification of alternatively spliced genes, and the identification of noncanonical introns. Similarly, splicing signals found in exons (exon splicing enhancers) are a feature of some vertebrate transcripts, and exonic sequences are known to influence splicing of some plant transcripts. Computer-assisted screens for exon splicing enhancers (Steven Mount, University of Maryland, College Park) and alternatively spliced genes and AT-AC introns (Volker Brendel, Iowa State University, Ames) are identifying specific gene systems for detailed analysis. More general information from such screens will be used as a basis for genomics approaches, such as microarray analysis, to examine the regulation of gene expression by splicing.
One of the longest-standing questions in plant pre-mRNA splicing is the function of UA-rich sequences that are characteristic of plant introns. Plant intron splice sites and branch point sequences, small nuclear RNA (snRNA) sequences, and many small nuclear ribonucleoproteins (snRNPs) and spliceo- somal proteins are very similar to yeast and vertebrate intron signals and splicing components (Lorkovic et al., 2000 John Brown (Scottish Crop Research Institute) combined overexpression of such RNA binding proteins with a range of mutations in the invertase mini-exon system to further address the function of these proteins. Of particular interest is that the splicing behavior of invertase intron mutations is altered differentially upon expression of different RNA binding proteins. Thus, it is possible to distinguish different functions of RNA binding proteins on the basis of their effect on the splicing of introns with variations in U-rich sequences.
Alternative splicing is an important process for the regulated production of functionally different proteins from single genes in animal systems. Although alternative splicing does not appear to be as widespread in plant (Arabidopsis) genomes as it is in the human genome (35% of genes may be alternatively spliced), it is important to define the functions of proteins produced by alternative splicing and to understand the mechanisms by which alternative splicing is regulated in plants. One of the best studied alternatively spliced plant genes is the N gene of tobacco that confers resistance to Tobacco mosaic virus. Analysis of the splicing behavior and the associated resistance phenotype of a series of N gene constructs has identified regions essential for both alternative splicing and resistance (S.P. Dinesh-Kumar, Yale University, New Haven, CT). Infection by Tobacco mosaic virus induces changes in the splicing pattern of N transcripts (Dinesh-Kumar and Baker, 2000
Post-transcriptional control of gene expression is important when levels of a protein must change more rapidly than can be accommodated by transcription. This includes responses to biotic and abiotic stresses. Yet, like transcription, most stages of post-transcriptional control, including translation, are controlled by an enormously complex array of protein factors. Despite this appearance of a burgeoning molecular bureaucracy, the checks and balances of translation ensure finely tuned, rapid regulation of gene expression (Bailey-Serres, 1999 ). Fascinating examples of what is still clearly the nascent field of translational control were presented. Karen Browning (University of Texas, Austin) led off the translation sessions with an update on the vast population of initiation factors. Nonviral eukaryotic cellular mRNAs contain an m7GpppG "cap" group at their 5' ends. This cap is bound by a protein complex containing the cap binding protein eIF4E and the scaffold protein eIF4G. eIF4E and eIF4G together form eIF4F. eIF4G binds poly(A) binding protein (PABP), which, by binding the poly(A) tail of the mRNA, results in a closed-loop mRNA structure. eIF4G also serves as a scaffold for the assembly of other initiation factors, such as eIF4A and eIF3. This assembled complex is thought to then provide the means for removing any secondary structure from the 5' untranslated region (UTR) of the mRNA using the hydrolysis of ATP and the helicase activity of eIF4A. Thus, the closed-loop mRNA is prepared by the initiation factors for the 40S ribosome to bind and find the correct initiation site. Unlike other eukaryotes, plants have two distinct forms of the cap binding protein complex: eIF4F and eIFiso4F. In addition to a "conventional" eIF4G (180 kD), plants have a more abundant version called eIFiso4G, which behaves like eIF4G even though it is less than half the size. It is paired with its own version of eIF4E, called eIFiso4E, to form eIFiso4F. Although the function of the eIFiso4F complex in plants is not fully understood, evidence was presented that different mRNAs may discriminate between eIF4F and eIFiso4F, and that these two forms may play distinct roles in plant development.
All of the subunits of all canonical translation factors have now been cloned and sequenced. The last to be completed is the largest, least understood factor, eIF3, which plays a key role in bringing the ribosome to the mRNA via interaction with eIF4G. Plant and mammalian eIF3s each have 11 subunits, whereas that of Saccharomyces cerevisiae has only five. Ten of the different protein subunits of plant eIF3 resemble those in mammalian eIF3 (Burks et al., 2001 Viruses also shed light on translation factor function and continued to provide examples of the byzantine translational control mechanisms that they can employ. Satellite tobacco necrosis virus (STNV) RNA and Barley yellow dwarf virus (BYDV) RNA both lack a 5' cap and a poly(A) tail, yet they are translated very efficiently. STNV and BYDV RNAs each have a cap-independent translation element (3' TE) in their 3' UTRs that brings about efficient translation initiation at the AUG proximal to the 5' end of the RNA. The 3' TE of STNV RNA was reported by Karen Browning to bind eIF4E and eIFiso4E, presumably recruiting some portion of the translational apparatus to the 3' UTR.
The 3' location of the 3' TE begs the question of how it communicates with the 5' end to ensure ribosome entry on the mRNA there. By identifying phylogenetically conserved base-pairing covariations and providing direct experimental evidence with appropriately mutated mRNAs, Allen Miller (Iowa State University) showed that a stem-loop structure in the BYDV 3' TE base-pairs directly to a stem loop in the 5' UTR to form a kissing stem-loop structure that causes the mRNA to form a closed loop (Guo et al., 2001
The 35S RNA of Cauliflower mosaic virus (CaMV), made famous by its promoter, serves as a message for five genes in separate open reading frames (ORFs). To make things more complicated, these "real" ORFs are preceded by several very small ORFs on the long, structured leader sequence of the 35S mRNA. To be translated, the downstream ORFs require the CaMV trans-activator (TAV) protein encoded by ORF 6, which is translated from its own (19S) mRNA. To identify host proteins with which TAV interacts to cause ribosomes to reinitiate translation on downstream ORFs, Lyubov Ryabova of Thomas Hohn's laboratory (Friedrich Miescher Institute, Basel) used a yeast two-hybrid screen. They found that TAV interacts with subunit g of eIF3 and with large ribosomal subunit proteins L24 and L18. The latter was identified previously by another laboratory (Leh et al., 2000
Plants produce ribosome-inactivating proteins that are thought to have a defense role in response to infection by viruses or other pathogens. They were originally found to depurinate a specific adenosine residue in the highly conserved
Anthony Michael (Institute of Food Research, Norwich, UK) reported on a small upstream reading frame (uORF) that controls the expression of plant S-adenosylmethionine decarboxylase and is highly conserved evolutionarily (Franceschetti et al., 2001
The PABP that binds to the poly(A) tail of cellular mRNAs promotes 40S ribosomal subunit recruitment to an mRNA through its interaction with eIF4G and eIF4B. Dan Gallie (University of California, Riverside) reported that the phosphorylation state of PABP regulates its cooperative binding to a poly(A) tail as well as its interaction with eIF4G and eIF4B. The phosphorylation states of eIF4G and eIF4B also determine the strength of their interaction with PABP. PABP is present in multiple phosphorylated isoforms, and the greatest cooperative binding is observed between phosphorylated and nonphosphorylated isoforms. Although the distribution of PABP isoforms does not appear to change during development or after stress, eIF4B and eIF4G undergo rapid dephosphorylation after a short exposure to heat stress that downregulates their interaction with PABP. This inhibition of protein interaction may account for the thermorepression of translation that is characteristic of the plant heat stress response (Le et al., 2000 The pea ferredoxin I (Fed-1) mRNA in transgenic tobacco is released from polysomes within just 30 sec after a reduction in photosynthetic rates. Thus, Fed-1 expression responds to environmental changes as transient as a cloud passing over. Using mutagenesis, Marie Petracek (Oklahoma State University, Stillwater) identified a region of the Fed-1 ORF as the possible dark-responsive control element. In addition, screening of a subtractive library for genes responsive to photosynthetic electron transport is under way to find additional mRNAs regulated at both the level of translation and mRNA stability. Julia Bailey-Serres (University of California, Riverside) reported on the trans-lational responses to abiotic stresses (e.g., anoxia, heat shock, drought, and salt stress). Not surprisingly, there are pleiotropic effects on the translational machinery, including changes in the phosphorylation state of initiation and elongation factors and ribosomal proteins. These changes are reflected in changes in polysome profiles under various stress conditions, such as anoxia and water deficit. Under stress, many mRNAs are shifted to smaller polysomes, whereas stress protein mRNAs remain in larger polysomes. This suggests that there is a cellular response of most mRNAs to delay translation until better growth conditions return.
Yet another influence on translation is redox state, as evidenced by its effect on translational control of the
Translation in the chloroplast was covered by Stephen Mayfield (Scripps Research Institute, La Jolla, California). He reported that chloroplast ribosomes, although similar to those of bacteria, have mechanisms of translation that resemble a hybrid between those of prokaryotes and eukaryotes (Bruick and Mayfield, 1999
In contrast to the newly discovered targeted degradation of potentially any mRNA via the PTGS process, Pam Green (Michigan State University, East Lansing) presented the latest on her work on the control of mRNA stability via sequences in the 3' UTR: a more "traditional" control of mRNA degradation. In plants and animals, mRNAs that are destined to live a short life may con-tain AUUUA repeats in the 3' UTR. However, some short-lived plant mRNAs may instead contain a different destabilization (DST) motif. These mRNAs have specific physiological roles, such as the small auxin-upregulated mRNAs. Using a clever selection system, Green and colleagues identified Arabidopsis mutants with reduced ability to degrade DST-containing mRNAs (Johnson et al., 2000 ). Mutations in either of two independent genes, dst1 and dst2, caused similar increases in small auxin- upregulated mRNA half-lives and could not be functionally distinguished until the application of microarray hybridization. By probing an 11,000-gene Arabidopsis microarray with mRNA preparations from wild-type and mutant plants obtained before and after transcriptional shutoff, Green's group discovered a plethora of unpredicted mRNAs whose half-lives were affected by the dst mutations. The different dst genes affected the half-lives of different sets of genes, which were confirmed by RNA gel blot experiments. Thus, mRNA stability control is an important regulator of expression of numerous gene families. Dr. Green's talk brought an appropriate close to the meeting because it epitomized the melding of traditional, hypothesis-driven "single gene" research with the new large-scale genomics and bioinformatics approaches that were used in much of the research presented at this conference. Her leading research also foreshadowed things to come with the expansion of observations on one or a few genes to the whole genome.
And the list goes on. Light, hormones, oxidative stress, pathogens: is there any influence on a plant that does not involve post-transcriptional control? And we did not even cover such processes as premature stop codon–mediated decay, RNA editing, the specialized events in mitochondria, and post-translational events. (Again, our apologies to those whose work was not mentioned in this overview.) A take-home lesson from this meeting is that post-transcriptional control has gone from a collection of novel observations to a diverse set of phenomena that may regulate the expression of most genes. Another obvious point is that we are really just beginning. Most of the research is still at a rather rudimentary, descriptive level of understanding. We look forward to an exciting future as the mechanisms are worked out in detail. Moreover, with the widespread application of microarrays and genomics techniques, the number of known post-transcriptionally controlled genes will surely increase. In the next 10 years, as plant scientists set out to achieve the National Science Foundation's goal of complete understanding of the function and regulation of all plant genes, those investigating post-transcriptional control will have their hands full. This portends an exciting fourth symposium in 2003.
1 peterw{at}pi.csiro.au 
4 kbrowning{at}mail.utexas.edu
Bailey-Serres, J. (1999). Selective translation of cytoplasmic mRNAs in plants. Trends Plant Sci. 4, 142–148.[CrossRef][Web of Science][Medline] Bailey-Serres, J., Rochaix, J.D., Wassenegger, M., and Filipowicz, W. (1999). Plants, their organelles, viruses and transgenes reveal the mechanisms and relevance of post-transcriptional processes. Leysin, VD, Switzerland, February 25-28, 1999. EMBO J. 18, 5153–5158. Bruick, R.K., and Mayfield, S.P. (1999). Light-activated translation of chloroplast mRNAs. Trends Plant Sci. 4, 190–195.[CrossRef][Web of Science][Medline]
Burks, E.A., Bezerra, P.P., Le, H., Gallie, D.R., and Browning, K.S. (2001). Plant initiation factor 3 subunit composition resembles mammalian initiation factor 3 and has a novel subunit. J. Biol. Chem. 276, 2122–2131.
Dinesh-Kumar, S.P., and Baker, B.J. (2000). Alternatively spliced N resistance gene transcripts: Their possible role in tobacco mosaic virus resistance. Proc. Natl. Acad. Sci. USA 97, 1908–1913. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.[CrossRef][Medline] Franceschetti, M., Hanfrey, C., Scaramagli, S., Torrigiani, P., Bagni, N., Burtin, D., and Michael, A.J. (2001). Characterization of monocot and dicot plant S-adenosyl-L-methionine decarboxylase gene families including identification in the mRNA of a highly conserved pair of upstream overlapping open reading frames. Biochem. J. 353, 403–409.[CrossRef][Web of Science][Medline] Gallie, D.R., and Bailey-Serres, J. (1997). Eyes off transcription! The wonderful world of post-transcriptional regulation. Plant Cell 9, 667–673.[CrossRef][Web of Science][Medline] Guo, L., Allen, E., and Miller, W.A. (2001). Base-pairing between untranslated regions facilitates translation of uncapped, nonpolyadenylated viral RNA. Mol. Cell 7, 1103–1109.[CrossRef][Web of Science][Medline] Hudak, K.A., Wang, P., and Tumer, N.E. (2000). A novel mechanism for inhibition of translation by pokeweed antiviral protein: Depurination of the capped RNA template. RNA 6, 369–380.[Abstract] Ji, L.H., and Ding, S.W. (2001). The suppressor of transgene RNA silencing en-coded by Cucumber mosaic virus interferes with salicylic acid-mediated virus resistance. Mol. Plant-Microbe Interact. 14, 715–724.[Medline]
Johnson, M.A., Perez-Amador, M.A., Lidder, P., and Green, P.J. (2000). Mutants of Arabidopsis defective in a sequence-specific mRNA degradation pathway. Proc. Natl. Acad. Sci. USA 97, 13991–13996. Lambermon, M.H., Simpson, G.G., Wieczorek Kirk, D.A., Hemmings-Mieszczak, M., Klahre, U., and Filipowicz, W. (2000). UBP1, a novel hnRNP-like protein that functions at multiple steps of higher plant nuclear pre-mRNA maturation. EMBO J. 19, 1638–1649.[CrossRef][Web of Science][Medline]
Le, H., Browning, K.S., and Gallie, D.R. (2000). The phosphorylation state of poly(A)-binding protein specifies its binding to poly(A) RNA and its interaction with eukaryotic initiation factor (eIF) 4F, eIFiso4F, and eIF4B. J. Biol. Chem. 275, 17452–17462. Leh, V., Yot, P., and Keller, M. (2000). The cauliflower mosaic virus translational transactivator interacts with the 60S ribosomal subunit protein L18 of Arabidopsis thaliana. Virology 266, 1–7.[CrossRef][Medline] Lindbo, J.A., Silva-Rosales, L., Proebsting, W.M., and Dougherty, W.G. (1993). Induction of a highly specific antiviral state in transgenic plants: Implications for regulation of gene expression and virus resistance. Plant Cell 5, 1749–1759.[Abstract] Lorkovic, Z.J., Wieczorek Kirk, D.A., Lambermon, M.H., and Filipowicz, W. (2000). Pre-mRNA splicing in higher plants. Trends Plant Sci. 5, 160–167.[CrossRef][Web of Science][Medline] Simpson, C.G., Hedley, P.E., Watters, J.A., Clark, G.P., McQuade, C., Machray, G.C., and Brown, J.W. (2000). Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants. RNA 6, 422–433.[Abstract]
Vance, V., and Vaucheret, H. (2001). RNA silencing in plants: Defense and counterdefense. Science 292, 2277–2280. Waterhouse, P.M., Wang, M.-B., and Lough, T. (2001). Gene silencing as an adaptive defence against viruses. Nature 411, 834–842.[CrossRef][Medline]
Xiang, C., and Oliver, D.J. (1998). Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell 10, 1539–1550. This article has been cited by other articles:
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POST-TRANSCRIPTIONAL GENE...
SPLICING: BRINGING THE PIECES...
21-nucleotide RNAs. His group's data seemed to show that the hpRNA-directed methylation and RNA degradation did not spread outside of the target sequences defined by the self-complementary sequences of the hpRNA. This contrasted with the results reported by Fabian Vaistij, of David Baulcombe's laboratory (John Innes Centre, Norwich, UK), in which virus-induced PTGS directed the DNA methylation and RNA degradation of sequences in the transcript outside of those of carried by the virus. All of the transcribed sequence of the target gene, not just the region represented in the virus, was methylated, and all of the target gene mRNA appeared to act as a template for the production of guides to direct further RNA degradation. This allowed a virus-containing sequence identical to the 3' portion of the transgene to induce methylation of the entire gene, which then conferred resistance to a virus containing the 5' portion of the transgene (i.e., to a virus lacking any homology with the virus that induced silencing). This methylation could "spread" in either direction on the transcribed portion of the transgene. After much discussion, the contradictory results from these two reports were thought to be attributable to the different ways in which the PTGS was induced. 
-sarcin loop of the large rRNA, stopping all translation. This was thought to act as a suicide defense mechanism. Nilgun Tumer's (Rutgers University, New Brunswick, NJ) research during the last decade has shown that the multiple functions of the ribosome-inactivating protein pokeweed antiviral protein (PAP) are much more diverse, subtle, and complex. She reported on the ability of PAP to bind to the m7GpppG cap of mRNAs and to catalyze depurination of the mRNA (Hudak et al., 2000
-glutamylcysteine synthase (GSH1) gene. GSH is one of the gene products necessary for the production of glutathione. David Oliver and co-workers (Iowa State University) reported that exposure to cadmium or jasmonic acid increases the transcription of the genes for glutathione biosynthesis. However, despite the increased transcription of GSH1 mRNA, there is no increase in GSH1 protein levels (Xiang and Oliver, 1998
