Regulation of plant translation by upstream open reading frames

ARTICLE IN PRESS

G Model PSL 8856 1–12

Plant Science xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Plant Science journal homepage: www.elsevier.com/locate/plantsci

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Review

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Regulation of plant translation by upstream open reading frames

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Q1

Albrecht G. von Arnim a,b,∗ , Qidong Jia b , Justin N. Vaughn a,1

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Q2

a

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b

Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, United States Graduate School of Genome Science and Technology, The University of Tennessee, Knoxville, TN 37996-0840, United States

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a r t i c l e

i n f o

a b s t r a c t

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Article history: Received 17 July 2013 Received in revised form 8 September 2013 Accepted 10 September 2013 Available online xxx

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Keywords: Protein synthesis Translation initiation factor Upstream open reading frame Sensor Development Arabidopsis thaliana

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Contents

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We review the evidence that upstream open reading frames (uORFs) function as RNA sequence elements for post-transcriptional control of gene expression, specifically translation. uORFs are highly abundant in the genomes of angiosperms. Their negative effect on translation is often attenuated by ribosomal translation reinitiation, a process whose molecular biochemistry is still being investigated. Certain uORFs render translation responsive to small molecules, thus offering a path for metabolic control of gene expression in evolution and synthetic biology. In some cases, uORFs form modular logic gates in signal transduction. uORFs thus provide eukaryotes with a functionality analogous to, or comparable to, riboswitches and attenuators in prokaryotes. uORFs exist in many genes regulating development and point toward translational control of development. While many uORFs appear to be poorly conserved, and the number of genes with conserved-peptide uORFs is modest, many mRNAs have a conserved pattern of uORFs. Evolutionarily, the gain and loss of uORFs may be a widespread mechanism that diversifies gene expression patterns. Last but not least, this review includes a dedicated uORF database for Arabidopsis. © 2013 Published by Elsevier Ireland Ltd.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Definitions and early cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Types of uORFs and their distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. How does the ribosome get past uORFs: Leaky scanning, shunting, and reinitiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How does the ribosome engage with uORFs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Elongation on CPuORFs with inhibitory peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Termination and reinitiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . uORFs as regulators of metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Regulation of polyamine metabolism by uORFs and polyamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Regulation of carbohydrate metabolism by uORFs and sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . uORFs mediate developmental gene regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. uORFs and translation reinitiation modulate the auxin response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The development of leaf dorsoventral polarity is sensitive to defects in translation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and hypotheses to guide future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AGI#, Arabidopsis gene identifier; CaMV, cauliflower mosaic virus; eIF, eukaryotic translation initiation factor; mORF, major open reading frame; NMD, nonsense mediated decay; RPL, ribosomal protein of the large subunit; RPS, ribosomal protein of the small subunit; uORF, upstream open reading frame; CPuORF, conserved peptide uORF; UTR, untranslated region. ∗ Corresponding author at: Department of Biochemistry, Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, United States. Q3 Tel.: +1 865 974 6206. E-mail addresses: [email protected] (A.G. von Arnim), [email protected] (Q. Jia), [email protected] (J.N. Vaughn). 1 Current address: Department of Genetics, University of Georgia, Athens, GA 30602-7223, United States. 0168-9452/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.plantsci.2013.09.006

Please cite this article in press as: A.G. von Arnim, et al., Regulation of plant translation by upstream open reading frames, Plant Sci. (2013), http://dx.doi.org/10.1016/j.plantsci.2013.09.006

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1. Introduction

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1.1. Definitions and early cases

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Upstream open reading frames (uORFs) are protein coding regions in mRNAs that lie upstream of the main protein coding region, i.e. in the 5 untranslated region of the mRNA (Fig. 1). Although counterintuitive, the so-called 5 untranslated regions (5 UTR) of mRNAs are often partially translated. According to Kozak’s scanning model of translation initiation, the ribosome scans the mRNA from the 5 cap and engages at the first AUG triplet that it encounters. If the first AUG is the start codon of a uORF, it typically reduces the efficiency of translation of the main coding region of the mRNA (major open reading frame or mORF) [1,2]. However, uORFs usually do not eliminate translation altogether. This was shown in planta with the maize transcription factors, R/Lc and Opaque-2 [3,4]. One of the earliest and most unconventional uORF-based translational control systems was discovered in the pararetrovirus, cauliflower mosaic virus (CaMV)[5,6]. Briefly, the long CaMV 5 leader contains six uORFs (Supplemental Fig. 1) and a long hairpinloop structure downstream from the first uORF. A ribosome that has translated the first short uORF is competent to scan past the hairpin without unfolding it, an event called shunting. The shunting mechanism ensures that a specific region of the 5 UTR remains free of ribosomes. This region contains an RNA encapsidation signal that binds to the CaMV coat protein [6,7]. It is notable that these viruses do not use an internal ribosome entry mechanism that maintains their 5 UTR free of ribosomes. Internal ribosome entry sites are RNA sequence elements commonly used by metazoan viruses that direct ribosomes to specific sites adjacent to their translation start codons and circumvent cap-dependent translation initiation. Until recently, shunting was thought to be a peculiarity of the pararetroviruses such as CaMV and the related pararetrovirus, rice tungro bacilliform virus. However, uORF-stimulated shunting was recently discovered in a picorna-like RNA virus (rice tungro spherical virus). Because the two rice viruses coexist together in the same host, it seems very likely that the RNA virus may have acquired the shunting mechanism by cohabitation with rice tungro bacilliform virus [8].

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1.2. Types of uORFs and their distribution

41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74

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The preponderance of uORFs is clearly biased with respect to gene function. Highly expressed mRNAs such as those of many housekeeping genes tend to have short 5 UTRs that are devoid of uORFs. Poorly expressed mRNAs such as mRNAs for transcription factors and kinases often have longer 5 UTRs and are rich in uORFs [9]. This feature is pan-eukaryotic and was first observed in 1987 [10]. These results suggest that the presence or absence of uORFs is most likely adaptive. However, not many uORFs have been directly examined for their functional significance at the whole plant level. Only in a handful of cases has the mutation of the uORF revealed significant growth defects [11–13]. In addition, very few uORFs were discovered by classical forward genetic analysis, that is, because a mutation in the uORF altered the phenotype of the plant [12,14]. Some uORFs overlap the major open reading frame of the mRNA (major ORF). A recent study in yeast concluded that, among all the possible mutations in a gene’s 5 upstream region, mutations that cause uORFs to overlap with the major ORF have the most dramatic inhibitory effect on gene expression [15]. uORFs are classified along evolutionary lines. For a fairly small fraction of uORFs the peptide sequence is noticeably conserved in evolution (Conserved peptide uORFs or CPuORFs). In these cases, the peptide sequence is key for translational repression. Several surveys on CPuORFs have been published [16–19]. CPuORFs have rightfully been assigned their own gene identifiers (AGI numbers)

in Arabidopsis. In some cases, and in keeping with similar CPuORF peptides in fungi [20], the conserved peptide is hypothesized to stall the ribosome as a nascent peptide while located in the ribosome exit tunnel, thus blocking the progression of upstream ribosomes or suppressing reinitiation [11,21]. In other cases, the uORF peptide may exert its function after it has been released from the ribosome. Only two such cases are known. In one case, the peptide binds to the mRNA and destabilizes it [22]. In another case, the synthesized uORF peptide can inhibit translation when added to an in vitro translation system [23]. CPuORFs are currently sorted into more than 30 homology groups that are spread over more than 79 Arabidopsis genes [17,19,24], while up to 150 cereal genes are now estimated to have CPuORFs [18]. Most CPuORFs are conserved between monocots and dicots [16–18,25]. Lineage specific gain or loss of CPuORFs is uncommon [19], but it does occur. Arabidopsis often has uORF features different from those of other dicots [25]. For example, the highly conserved CPuORF in the mRNA for ribosomal protein S6 kinase has lost its AUG in the Brassicaceae lineage, and has been replaced by a different uORF in a different frame. About 35% of Arabidopsis genes give rise to a uORF-containing mRNA, and about half of these have multiple uORFs [9]. Other plant species have similar fractions of transcripts with uORFs (Fig. 2A). The Arabidopsis genome encodes more than 20,000 uORFs, almost as many as major ORFs (Supplemental Data File S1). Because the AUG triplet is only slightly underrepresented in Arabidopsis 5 UTRs [9], the number of uORFs is only slightly lower than predicted by chance alone. However, longer uORFs are more underrepresented than shorter ones (Fig. 2B). The CPuORFs represent only a small fraction of all uORFs. However, the uORFs that do not qualify as CPuORFs (nonCPuORFs) commonly influence the level of gene expression of the major ORF (Table 1). Therefore, a large number of nonCPuORFs are functional. Then, what fraction of the nonCPuORFs was, or still is, subject to selection? What fraction of them has adaptive significance? There is evidence that uORFs other than the known CPuORFs are subject to selection. First, the presence of the nonCPuORF is sometimes conserved, even if their amino acid sequence is not [25]. Similar to the situation in mouse and human [26], the AUG triplet is the most frequently conserved triplet when 5 UTRs from two plant families are aligned (Fig. 2C), consistent with the notion that many uAUGs are under stabilizing selection. Furthermore, certain gene networks that possess CPuORFs also include other genes with nonCPuORFs, for example the polyamine gene network (Fig. 3) and the auxin response transcription factors [27]. Finally, it should be recognized that the definition of a uORF as a CPuORF is constrained by our statistical power. All of these nonrandom patterns suggest that many nonCPuORFs are biologically significant. Such observations notwithstanding, it is also evident that many uORFs are unconserved. It was speculated that uORFs might counterbalance evolutionary changes in transcriptional control [4]; this interesting idea of coevolution between transcriptional and posttranscriptional regulation has not been scrutinized. 1.3. How does the ribosome get past uORFs: Leaky scanning, shunting, and reinitiation In vitro, uORFs suppress translation in a length dependent manner [2,28]. One of the more detailed in vivo surveys was performed in human cells [26] with >25 uORFs that inhibited protein expression between a little and 100%. Although this study did not detect a correlation between uORF length and protein expression, evidence from plants (Fig. 2D) shows a moderate correlation between inhibition of gene expression and uORF length. uORFs of more than 16 codons can be expected to inhibit translation. In contrast, uORFs shorter than 16 codons often inhibit expression by less than three fold and sometimes not at all, although there are exceptions.

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5’ UTR Alternative Splice Sites

3

3’ UTR

Major ORF (mORF)

Alternative Transcription Initiation Site overlap-uORF

5' Cap

AAAAA uAUG

mAUG

Stop

Stop

Alternative Translation Initiation Site Upstream ORFs (uORFs) Fig. 1. Schematic of an mRNA with uORFs. Not all possibilities illustrated in this figure are likely to apply simultaneously to a single mRNA. uORFs (red) can overlap with each other. In this review, overlap-uORFs are the subset of uORFs that overlap the major ORF (mORF, green). uORFs can be affected by alternative transcription initiation sites or alternative splicing (blue arrows) [100,101]. Therefore, not all mRNA molecules transcribed from a given gene will necessarily contain all the annotated uORFs. An alternative translation initiation site may extend the coding sequence of the mORF (light green). Such N-terminal extensions of major ORFs do not qualify as uORFs. The sequence between a uORF stop and the next start codon is referred to as the intercistronic spacer (not marked). In the literature, uORFs are occasionally referred to as short ORFs, upstream ORFs, and micro ORFs. 165 166 167 168 169

How does the ribosome overcome the inhibition by the uORF? First, the ribosome may ignore the uAUG by leaky scanning. The likelihood of leaky scanning is difficult to predict with precision. The extent of leaky scanning is estimated by fusing a reporter gene a few codons downstream of the uAUG. These experiments

show that most AUGs are detected at least some of the time [29,30]. A weak sequence context such as uugAUGa or uuuAUGu may allow between 3% and 15% initiation as compared to a strong context, such as aaaAUGg [2,29,31]. However, depending on the broader sequence context, even weak AUGs such as uugAUGc can

Fig. 2. Characteristics of uORFs in the 5 UTRs of plants. (A) The fraction of known transcripts that contain at least one uAUG in the 5 UTR is given for several genera of angiosperms. (B) The number of genes with a given uORF length in Arabidopsis. The line marked ‘Real’ indicates the authentic data. The two other lines are simulations of uORF lengths obtained from shuffling each 5 UTR 1000 times. Shuffling was performed with the 5 UTR split into mononucleotides or dinucleotides. Arabidopsis uORFs are slightly biased toward shorter lengths compared to the simulation. The inset shows the full distribution. (C) Comparison of orthologous pairs of genes. Alignable regions of 5 UTRs were scored for the fraction at which each triplet is identical in both species (see [26] for a similar analysis). The datapoint for the AUG triplet is marked. Boxes and whiskers indicate the distribution of conservation frequencies for all other 63 triplets. The median number of aligned triplets scored for each taxon was 3281 (Brassicaceae), 14,136 (Solanaceae), and 3780 (mammals). The whiskers represent the minimum and maximum values in the distribution. Boxes represent 25% below the median (thick horizontal line) and 25% above the median. uAUGs are conserved in a higher percentage of alignment columns than any other triplet. Whole-transcriptome pairwise comparisons: Brassicaceae - Arabidopsis thaliana and Brassica napus, Solanaceae - tomato and potato, Mammals – human and mouse. (D) The relationship between uORF length and repression of gene expression. Each datapoint represents the fold repression of expression from the major ORF when a uORF-containing 5 UTR is compared with a uORF-less mutant version. R2 denotes the correlation coefficient. Original data are in Supplemental Table 1.

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4 Table 1 Experimental case studies of uORFs in plants. Gene AGI # Functional class A. Transcription factors AtbZip11 At4g34590 S-type bZip Opaque-2 Maize bZip myb7 Rice Myb ATR1 At5g60890 Myb R/Lc Maize basic helix loop helix SAC51 At5g64350 Basic helix loop helix HsfB1/HSF4/TBF1 At4g36990 Heat shock factor ARF3/ETTIN At2g33860 Auxin response factor ARF5/MONOPTEROS At1g19850 Auxin response factor ATH1 At4g32980 BELL-type homeodomain ABI3 At3g24650 MtHAP2-1 Medicago C/EBP B. Polyamine network SAMDC/AdoMetDC At3g02470 S-adenosylmethionine decarboxylase Arginine decarboxylase Dianthus (carnation) ODC Ornithine decarboxylase (tomato) C. Other genes CGS1 At3g01120 Cystathione gamma synthase pma1, pma3 Nicotiana plumbaginifolia H+-ATP-ases XIPOTL1 At3g18000 Phosphoethanolamine N-methyltransferase AtMHX1 At2g47600 Vacuolar magnesium/zinc – proton antiporter CaMV CaMV a b

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Number of uORFs CPuORF classa

uORF affectsb

uORF lengths (codons)

Other characteristics

References

4 uORFs CPuORF HG1

Translation

18, 42, 5, 19

42 amino acid (aa) CPuORF peptide detected in vitro; sucrose exacerbates repression;

[21,27,31,51]

3 uORFs

Translation

3, 21, 20

uORFs in cluster repress translation redundantly

[3]

1 uORF

Translation

40

[103]

3 uORFs

mRNA level

33, 3, 4

uORF repression depends on downstream spacer sequence; peptide detected in vitro 33 amino acid uORF reduces mRNA level; uORF mutant allele from suppressor screen (atr1-d)

1 uORF

Translation

38

Peptide involved in repression; poor reinitiation

[4,38]

5 uORFs CPuORF HG15 2 uORFs CPuORF HG18 2 uORFs

mRNA level (NMD)

20, 16, 48, 53, 6

[12,81,87]

Translation

15, 36

CPuORF truncation from 53 to 3 amino acids rescues expression and thermospermine deficiency; uORF mutant allele from suppressor screen (sac51-d) CPuORF has 36 amino acids

mRNA level (minor) Translation

92, 5

Large uORF1 peptide detected in vitro; ARF3 translation stimulated by RPL24 and eIF3h

[27,69]

6 uORFs

Translation

3–23

ARF5 translation stimulated by RPL24 and eIF3h

[27,69]

4 uORFs

Translation

9, 12, 13, 1

uORF cluster with 7 uAUGs

[106]

26, 11, 12

Cluster of 3 uORFs inhibits ABI3 expression

[107]

3 uORFs

[14]

[104,105]

3 uORFs

mRNA level

62, 50, 34

uORF cluster in alternatively spliced intron; uORF1 peptide binds and represses its own mRNA

[22]

2 uORFs CPuORF HG3

Translation

3–4 48–54

Dual overlapping uORFs; 4 paralogous genes; polyamine suppresses uORF1 translation and triggers uORF2 translation and SAMDC repression

[11,49]

1 uORF

Translation

7

Synthesized uORF peptide inhibits in vitro translation

[23]

1 uORF

Translation

5

Two to three fold repression of ODC in vitro, independent of uORF peptide sequence

[111]

mainORF

Translation elongation

does not apply

S-adenosylmethionine blocks translation elongation

[52–54]

1 uORF

Translation

9 or 5

[108–110]

CPuORF HG13

Translation

25

uORFs mildly repress translation; uORF counteracts activation of expression by the 5 leader; uORFs also in tomato and Arabidopsis homologs Translation repressed by phosphocholine; peptide is arginine-serine rich

1 uORF

Translation, mRNA level (NMD)

13

uORF recognized despite poor AUG context; secondary structure in 5 UTR; peptide-independent; disallows efficient reinitiation

[32,33,80]

6 uORFs 6 uORFs

Shunting Translation

<15 Various

The most 5 uORFs, A and B, trigger shunting uORFs C to F trap ribosomes and disallow reinitiation

[6,74,112] [6]

[48,50]

The homology group (HG) of the CPuORF is indicated [17]. If nonsense mediated decay was also shown, it is indicated by NMD.

sometimes intercept a majority of scanning ribosomes [32,33]. For example, a computational model from an allelic series of the uORF-containing AtbZIP11 mRNA that included uAUG context as a variable yielded the best match with our experimental data when the parameter for uAUG recognition in a weak context was set as high as 0.7 [31]. There are only two clear scenarios in which the ribosome will definitely ignore the uAUG. In the first case, the

ribosome bypasses an AUG while it is actively translating another uORF in a different frame. Second, ribosomes may potentially bypass the uORF via an internal ribosome entry site downstream from the uAUG [34], a mechanism that has yet to be seen in plants. Third, ribosomes can scan past a uAUG by shunting [6]. During shunting, the 40S subunit bypasses a hairpin loop in the mRNA without unfolding it, which gives the impression that the ribosome

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has jumped. Documented cases of shunting are very rare, especially outside the virus kingdom [35], although shunting has been demonstrated in a system reconstituted from purified mammalian components [36]. In the best-characterized example, the CaMV 5 leader, the hairpin loop contains multiple uORFs [6]. Overall, when an mRNA contains a uORF followed by the requisite hairpin loop, the possibility of shunting should not be neglected. Ribosomes overcome uORFs by reinitiation [1]. For reinitiation to occur, the regular translation termination process [37] is interrupted before the 40S ribosomal subunit releases the mRNA [2]. Next, the ribosome reacquires a fresh methionyl-tRNAi , and other necessary translation factors, and resumes scanning downstream from the uORF until it reaches the next start codon. The extent of reinitiation after a uORF can be estimated by moving the uORF’s stop codon into the major ORF, which eliminates the possibility of reinitiation, yet leaves leaky scanning unaffected. The reduction in expression represents the degree of reinitiation [31,32,38].

Tryptic peptides are from Mars, uORFs are from Venus Proteomic surveys have made major inroads toward the goal of characterizing the entire coding potential of Arabidopsis and other plant genomes. Yet these surveys have not discovered any uORF peptides, the best direct evidence that the peptides are produced. One possible explanation would be that uORF peptides do not accumulate because short peptides may typically be turned over rapidly in the cell. A more plausible reason is that few if any uORF peptides are included in the standard predicted-protein databases used in proteomic analyses. It seems as if uORFs and tryptic peptides are on different orbits; uORF peptides have been ‘dark matter’ for proteomics. In addition, the mass spectrometry pipelines are focused on larger proteins, while short peptides are lost during sample preparation. Mass spectrometry is also typically set to analyze proteolytic peptides derived from these larger proteins, and uORF peptides may lack protease cleavage sites. However, when present in an mRNA, uORFs are the first coding sequence that the scanning ribosome encounters. Therefore, uORF peptides should be included in the proteomic databases that are used to search mass spectrometry data for expressed gene products.

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2. How does the ribosome engage with uORFs?

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2.1. Elongation on CPuORFs with inhibitory peptides

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During translation elongation, some uORF peptides can cause the ribosome to stall, as will be described in Section 3. A stalling peptide can be a platform to regulate translation by a small molecule [21,48–51]. The mechanism whereby uORFs render translation sensitive to small molecules is not known, and therefore we cannot a priori disregard mechanisms involving elaborate signaling pathways. However, in keeping with Occam’s razor, the simplest hypothesis is that the uORF peptide serves as the receptor for the small molecule. No direct evidence exists for this hypothesis for plant mRNAs, only circumstantial proof of concept. First, in an unusual case of elongation stalling, the nascent peptide of the CGS1 protein most likely serves as a receptor of S-adenosylmethionine [52–54]. Second, in bacteria, nascent peptides can bind to small molecules in the ribosome exit tunnel, and stall ribosome progression [55,56]. A nascent peptide would be expected to assume many possible conformations, an unlikely platform for a receptor. However, within the ribosome exit tunnel, certain peptides may adopt a well-defined structure [57], which may well bind a small molecule, perhaps in conjunction with rRNA or ribosomal protein contacts. Given that certain amino acid sequences in nascent proteins are known to impede the elongation step [52], it is plausible that a small molecule might then stall ribosome elongation. There are a few theoretical concerns with this model. If true, the repression mechanism should operate in vitro or in a heterologous system, as is the case for mammalian S-adenosylmethionine decarboxylase (SAMDC) [58,59]. Moreover, in all known cases of uORF mediated metabolic regulation, the metabolite represses rather than derepresses translation. If the nascent peptide does not have an inherent stalling ability, then the nascent peptide would continue to elongate, and thus spend only a fraction of a second in the correct conformation that can bind the given metabolite. In this situation, the metabolite would have to exist at a sufficiently high concentration, or would have to be pre-bound in the ribosome, to provide a realistic chance of binding at the critical moment and stall elongation. Finally, nature has evolved other mechanisms that allow metabolic control of gene specific translation through a uORF pattern, mechanisms that do not require the uORF peptide to bind a metabolite [60]. 2.2. Termination and reinitiation

The evidence for uORF translation in other organisms Evidence that uORF peptides actually accumulate in the cell is nearly as lacking in other organisms as it is in plants. However, a number of recent studies have tackled this problem. Briefly, a few surveys have started to yield the first proteomic glimpses of uORF peptides in the cell. Most recently, a search for human peptides yielded evidence for more than 15 uORF peptides [39]. More comprehensive experimental evidence for uORF translation has emerged from ribosome footprinting. Footprinting has demonstrated that many uORFs are indeed translated in yeast as well as in mammalian cells [40–42]. Surprisingly, ribosomes frequently seem to initiate at non-AUG codons in the 5 UTR, especially at CUG. The mapping of translation initiation sites was performed with various ribosome binding inhibitors, including cycloheximide [40], lactimidomycin [43], harringtonine [41], and puromycin [44]. While each inhibitor may suffer from its own source of false-positive signals, it is remarkable that the recent peptidomics search for uORF peptides also uncovered non-AUG initiation [39]. Alternative initiation at non-AUG codons does occur with some regularity at major ORFs of plants [45–47], and may well apply to uORFs as well (spermine synthase, SPDS3, At5g53120 [25]).

Reinitiation is an exceptional phenomenon. Reinitiation does not occur spontaneously after long major ORFs, i.e. in 3 UTRs. If it did, one would expect that the ∼107 bp of plant 3 UTRs would contain at least a few conserved ORFs, yet none were found in an open-ended search for conserved RNA sequence elements [25]. Thus plant 3 UTRs appear to be truly non-coding, while 5 UTRs code for thousands of peptides, as we have seen. Second, the exceptional reinitiation after long mORFs on the CaMV mRNA requires a specialized viral cofactor (transactivator), as well as a novel host protein (reinitiation supporting protein) [6,61]. Because short uORFs are often not highly inhibitory to major ORF translation, while long uORFs are, reinitiation must be dependent on uORF length. Most likely, the translating ribosome measures the time since the initiation event rather than the number of codons translated [28], which implies that the translating ribosome harbors a form of molecular short-term memory. But before we can understand the complexity of reinitiation, we first need to understand two other processes; regular translation initiation, and regular termination. For reinitiation to occur, the regular termination process must be interrupted and lead into a new round of initiation.

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Fig. 3. uORF distribution across the polyamine metabolic network and its regulatory genes (after [102]). (A) The polyamines spermidine, spermine, and thermospermine are produced from one of two precursors, arginine or ornithine. Although the ornithine pathway does not appear to operate in Arabidopsis, ODC genes are present in other plant species. SAMDC/AdoMetDC is a commitment step in polyamine synthesis. (B) Many of the genes harbor uORFs (data from TAIR10), including CPuORFs (black rectangles). Other colors are merely meant to provide contrast. For some but not all genes several gene models are shown to illustrate the diversity of uORF patterns due to alternative splicing and alternative transcription initiation. SAMDC, S-adenosylmethionine decarboxylase; ADC, arginine decarboxylase; AIH, agmatine iminohydrolase; CPA, N-carbamoylputrescine amidohydrolase; SPDS, spermidine synthase; SPMS, spermine synthase (SPDS3); ACL5, thermospermine synthase ACAULIS5; CuAO, copper amine oxidase; PAO, polyamine oxidase. PAO4 uORF2 is a weakly conserved CPuORF in dicots; PUT, polyamine uptake transporters. (C) A model depicting the regulation of SAMDC translation in response to polyamine by its uORFs. For details see text. The tiny uORF1 (yellow) is not drawn to scale in order to illustrate its translation by ribosomes (blue ovals). The uORF peptide products are shown as yellow and black triangles (after [49]).

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During regular translation initiation, the 40S ribosomal subunit binds the initiation factors, eIF3, eIF1, eIF1A, and eIF5, and is charged with a methionyl-initiator tRNAi in the context of the trimeric GTP-binding protein, eIF2, forming the 43S preinitiation complex. The 43S preinitiation complex binds to the 5 cap of the mRNA via the eIF4F cap binding complex. As part of eIF4F, the eIF4G protein serves as a scaffold for protein-protein interactions. Meanwhile, the eIF4A helicase melts secondary structure in the 5 end of the mRNA and facilitates the subsequent scanning of the 43S, aided by the eIF4B protein. Scanning ends when the Met-tRNAi recognizes an AUG start codon. Next, the 60S ribosome joins, and translation elongation begins [62]. Translation termination is less well understood than initiation [37]. Briefly, the synthesized polypeptide is released by eukaryotic release factors, eRF1 and eRF3, and the 60S subunit is released by ABCE1. The tRNA in the peptidyl (P-) site of the ribosome and the mRNA are released by eIF3, eIF1, eIF1A, and eIF3j [63], a surprising role for these initiation factors, although we note that the eIF functions in termination were identified in the context of a very

short ORF of only four codons [63]. How does the ribosome segue from termination into reinitiation, specifically after translation of a uORF? Two requirements are certain: The termination process needs to preserve the memory of the uORF length; and the 40S ribosome must capture a fresh methionyl-tRNA at its P-site. Theoretically, the first requirement would favor an early transition, before the memory of uORF length is lost. The second requirement would favor a late transition, after the peptide, 60S, eRFs, and Psite tRNA have been removed, because it is easier to envisage an unobstructed 40S binding fresh eIF2-tRNA-GTP and other initiation factors. The exact stage of the transition is still unknown. We suspect that a special nonribosomal factor is required for reinitiation after uORFs and functions as a molecular reinitiation licensing factor. But which? It is not known whether Reinitiation Supporting Protein, which stimulates reinitiation on the multicistronic mRNA of CaMV, affects reinitiation after uORFs [6,61]. The protein with the most credentials as a reinitiation factor is eIF3. eIF3 is a highly conserved protein complex. It has thirteen subunits (a to m) that co-purify as a complex [64] with the exception

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of eIF3j, which is loosely associated in all eukaryotes. First, eIF3 interacts with Transactivator and Reinitiation Supporting Protein, which support reinitiation after long ORFs in CaMV [61,65,66], and eIF3 is also involved in a similar case of reinitiation in mammalian viruses [67]. Second, mutants with defects in the eIF3h subunit are compromised in reinitiation of numerous mRNAs, especially after long uORFs and on 5 UTRs with multiple uORFs [9,31,68]. A computational simulation is consistent with the notion that eIF3h contributes to the molecular memory that coaxes the ribosomes to reinitiate after short but not long uORFs [31]. Third, genetic evidence suggests that eIF3h works in conjunction with a ribosomal protein of the large subunit, Arabidopsis RPL24 (rpl24e)[27]. RPL24 has been implicated in reinitiation in its own right on multiple occasions [66,69]. Finally, eIF3 assists with translation termination in vitro by releasing deacylated tRNA from the P-site [63,70]. Taken together, substantial evidence indicates that eIF3 supports translation reinitiation. How eIF3 performs this function remains the subject of speculation. In yeast, eIF3a recognizes an RNA sequence element that triggers reinitiation in that species [71]. In vertebrates, it has been suggested that eIF3 can remain associated with a translating 80S ribosome for a short period of time [67,72] and thereby serve as the postulated reinitiation licensing factor. In plants, strong biochemical evidence for an association of eIF3 with translating 80S ribosomes mostly comes from the CaMV system, where Transactivator enhances the association of eIF3 with polysomes [73]. In general, the molecular machinery that performs reinitiation is a topic of exciting ongoing research. The shunt model system of CaMV revealed unexpected insights into the biochemistry of the reinitiating ribosome. First, it appears that the ribosome will only shunt efficiently when it is located downstream from a uORF. Thus, a post-termination ribosome may be more prone to shunt than a regular ribosome. Second, immediately following the shunt, the 40S ribosome is prone to recognize non-AUG triplets as start codons, but only near the shunt landing site, not further downstream on the mRNA [74]. It is conceivable that the relaxed start codon recognition by the 40S is due to an imbalance in initiation factors (e.g. eIF1 and eIF5; [75,76]). The question whether reinitiating ribosomes have relaxed start codon recognition is important and has gathered urgency with the recent discovery of widespread translation initiation at non-AUG triplets in 5 UTRs [39,41,43,44]. Finally, recent in vitro experiments have thrown the door wide open regarding possible noncanonical ribosome behaviors [70]. For example, the post-termination 80S ribosome can, under some circumstances, lose its P-site tRNA, bind a fresh methionyl-tRNA, slide up and down the RNA and be trapped on an AUG triplet, the essential hallmarks of reinitiation. Before moving on, we shall address the question whether uORFs trigger nonsense mediated decay of the mRNA. mRNAs harboring premature termination codons are substrates for the nonsense mediated decay pathway, an RNA quality-control mechanism. The stop codon of a uORF can be considered a premature termination codon. Indeed, mRNAs with uORFs are enriched among the mRNAs that are targeted by nonsense mediated decay, especially if the uORF overlaps the major ORF, or if the uORF is exceptionally long [77–80]. Strikingly, about half of all mRNAs that harbor CPuORFs are upregulated in Arabidopsis mutants with defects in the nonsense mediated decay machinery [24,81]. However, the vast majority of uORFs, including some fairly long ones, are not conserved at the peptide level, and do not seem to trigger obvious nonsense mediated decay. Why do CPuORFs preferentially predispose an mRNA to nonsense mediated decay? It is tempting to invoke a threshold effect. Quite possibly nonsense mediated decay only occurs when the translation of the major ORF is suppressed below a low threshold. The majority of uORFs may allow a residual degree of leaky scanning past the uORF, or reinitiation downstream of the

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uORF, and thus populate the major ORF with enough ribosomes and muffle the nonsense mediated decay response. CPuORFs, however, may have evolved to stall ribosomes in such a fashion that the nonsense mediated decay response is triggered. The threshold hypothesis has the advantage of being simple. However, it does not account for the fact that more than three dozen CPuORF peptides have no apparent sequence features in common. How each of these different peptide sequences triggers enough ribosome stalling to block ribosome traffic on the major ORF remains unknown. In summary, certain uORFs clearly predispose the mRNA harboring them for mRNA turnover by nonsense mediated decay, but the precise sequence features causing this are only beginning to come into focus. 3. uORFs as regulators of metabolism Much is known about mechanisms that activate gene expression, but less about mechanisms that stop gene expression. Off switches for protein synthesis would be particularly important in metabolism, where dramatic changes in concentrations can occur within minutes in response to environmental changes. Transcriptional repression only works well as an Off switch if the mRNA is highly unstable. mRNAs can be destabilized by a variety of mechanisms [79,82–84], yet most mRNAs are comparatively stable [85]. Once translation initiation is repressed, protein synthesis stops within seconds or minutes, as determined by the speed of translation elongation (∼5 amino acids per second [41]), even on stable mRNAs. When uORFs overlap with each other, ribosomes traversing the 5 UTR adopt various discrete patterns of scanning and translation. If we agree to refer to scanning as ‘0’ and active translation as ‘1’, overlapping uORFs become a system of modular biobricks that perform a calculation using a binary number system. In the three cases discussed below, ribosome behavior is additionally regulated by a small molecule as an external factor. Such a system of uORFs then constitutes a regulatory switch. uORFs are common among metabolic genes. While many mRNAs for enzymes in primary and secondary metabolism do not have uORFs, uORFs are enriched in certain enzymatic pathways, especially polyamine metabolism (see below), in certain transporters, and certain transcription factors that regulate metabolism. An exciting recently emerging trend of thought is that certain uORFs confer regulation of translation by a metabolite from the given pathway. To date, three cases have been published [21,48–51]. In the first case the translation efficiency of the phosphocholine biosynthesis enzyme, phosphoethanolamineaminomethyltransferase, is dramatically inhibited by phosphocholine or choline via a CPuORF in the 5 UTR [48,50]. The level of the mRNA remains essentially unaffected, i.e. no nonsense mediated decay can be detected [48,79]. Considering that the physiological inhibitor is most likely phosphocholine rather than choline [48], the CPuORF mediates endproduct repression. The molecular mechanism of translational inhibition remains unknown. Phosphocholine may stall the ribosome on the uORF and thus impede reinitiation; however, it may also stimulate start codon recognition at the uORF. 3.1. Regulation of polyamine metabolism by uORFs and polyamine Polyamines are ubiquitous, positively charged, aliphatic metabolites that bind RNA, stimulate translation, and affect start codon recognition. In addition, the translation factor eIF5A requires polyamine for its posttranslational modification [86]. Many mammalian genes in the polyamine synthesis pathway are regulated by

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uORFs; the polyamine stimulated translational repression on the MAGDIS hexapeptide uORF of S-adenosylmethionine decarboxylase (SAMDC or AdoMetDC) is a classical example of regulated uORF mediated translation, where the MAGDIS peptide stalls the ribosome in a polyamine dependent manner [86]. Likewise, many plant polyamine metabolism genes harbor uORFs (Fig. 3). Polyamine metabolism would surely lend itself to uORF mediated feedback control if indeed start codon recognition and thus uORF selection were sensitive to polyamine levels, as has been suggested in the case of SAMDC [49]. Translation of SAMDC mRNA in plants is also regulated by uORFs and polyamine, although the mechanism differs from that of the MAGDIS uORF in vertebrates. Plants harbor two conserved uORFs, a CPuORF of 48–55 codons and a tiny uORF of 2–3 codons that overlaps the AUG of the CPuORF (Fig. 3C). Surprisingly, overexpression of the wild-type SAMDC mRNA in transgenic plants did not lead to any change in SAMDC activity or polyamine levels, unless the uORFs were removed, leading to the development of a negative feedback model [49]: When polyamine levels are low, mandating new synthesis, the scanning ribosome initiates at the uAUG of the tiny uORF, and therefore bypasses the uAUG of the short uORF. Because reinitiation after a very short uORF is efficient, the ribosome resumes scanning and reinitiates translation efficiently at the SAMDC major ORF. In contrast, when polyamine levels are high, the ribosome scans past the uAUG of the tiny uORF, which is in a weak sequence context, and therefore recognizes the second uORF, which is in a strong context. The second uORF precludes efficient reinitiation by virtue of its peptide sequence, which dissociates the ribosome from the mRNA, prevents new synthesis of SAMDC, and maintains polyamine homeostasis (Fig. 3C) [49]. A mutation that disrupts synthesis of the polyamine thermospermine revealed yet another CPuORF control system. Here, it is unknown whether the uORFs sense polyamine levels. The bHLH transcription factor SAC51 (At5g64340), which supports stem elongation in Arabidopsis, harbors five uORFs, one of which is a CPuORF (Supplemental Fig. 1). Expression of SAC51 is inhibited in the thermospermine-deficient dwarf mutant, acaulis5. It appears that the thermospermine deficiency exacerbates the CPuORF, while a wild-type thermospermine level keeps the inhibition by the CPuORF in check [12]. The best evidence for this notion is that a premature stop codon in the uORF improves SAC51 translation and rescues the acl5 phenotype. Shortening the uORF stabilizes the mRNA [12], perhaps because it allows for more efficient reinitiation at the main SAC51 start codon [28]. A second acl5 suppressor mutation works through a ribosomal protein. This mutation also appears to ameliorate the post-transcriptional repression of SAC51, yet exactly how it suppresses the acl5 phenotype is less clear [87]. 3.2. Regulation of carbohydrate metabolism by uORFs and sucrose Two classes of genes in carbohydrate regulation are controlled by uORFs: bZIP transcription factors, and trehalose-6-phosphate phosphatase [16,18]. The bZIP11 transcription factor and several of its paralogs harbor a cluster of four uORFs, the second of which encodes a sucrose-control peptide, which renders translation repressible by sucrose [51] (Supplemental Fig. 1). bZIP11 has been implicated in metabolic reprogramming of carbon and amino acid pathways [88,89]. At least in tobacco, uORF2 is part of a negative feedback loop that keeps sucrose levels stable [13]. The C-terminus of uORF2 is extremely highly conserved at the peptide level. The peptide of uORF1 is less conserved, while those of uORF3 and 4 are not [21,51]. Detailed structure function analyses of the sucrose-control-peptide uORF indicate that sucrose repression occurs during translation termination of the uORF and suggest that the sucrose-control peptide must occupy a specific location in the

ribosome exit tunnel [21]. Translation of the major ORF of AtbZIP11 relies on reinitiation after the uORF cluster [27,31] and is stimulated by auxin through the Target of Rapamycin (TOR) kinase pathway [90]. Exactly how sucrose triggers translational repression is not clear [21]. It is tempting to speculate that sucrose may be present in the ribosome exit tunnel and may bind to the nascent sucrosecontrol peptide in a way that causes ribosome stalling. Stalling may then prevent subsequent reinitiation, physically block other scanning ribosomes from traversing the uORF cluster and reaching the major AUG of bZIP11, or may potentially stimulate new ribosomes in initiating at the uAUG codons, which are in a poor sequence context. Since the uORF2 alone can mediate the sucrose repression, the role of the remaining conserved uORFs remains to be clarified. While uORF1 is clearly anti-inhibitory [31] and might be expected to cause the sucrose repression to be leaky, this does not seem to be the case [51]. uORFs3 and 4 may suppress residual leakiness of sucrose repression [21]. Taken together, a variety of small metabolites can regulate the translation of specific mRNAs through a uORF or uORF cluster. The biochemical mechanism of metabolite recognition, which may be indirect or direct, remains to be elucidated. It has been proposed that the uORF peptide may bind the small molecule directly [17], an elegant concept. Evidence for or against this proposition will not be easy to obtain. Biochemical experiments would have to contend with the strong possibility that the peptide functions while still located in the ribosome exit tunnel. Computational models of the ribosome and its exit tunnel will most likely play a part in this investigation. Genetic evidence may possibly emerge from modifier mutations. If the model were correct, then the regulatory mechanism would possibly be sensitive to specific alleles of ribosomal protein genes. The uORF-mediated regulation of translation by small molecules is conceptually similar yet chemically distinct from riboswitches and tRNA-sensing attenuator peptides (Table 2; [17]), which have not been detected in plant 5 UTRs. An elaborate pair of alternative secondary structures lies at the core of a riboswitch. The structures regulate ribosome binding, transcription termination, or RNA cleavage. Eukaryotic 5 UTRs do not lend themselves to riboswitches, because the scanning ribosome regularly disrupts any secondary structure. The subset of uORFs that respond to small metabolites affords an analogous form of translational regulation, as well as a rapid, gene-specific block in gene expression. 4. uORFs mediate developmental gene regulation 4.1. uORFs and translation reinitiation modulate the auxin response Compared to the multitude of uORFs in genes for metabolic enzymes and their regulators, uORF control of plant developmental mechanisms is less well established, even though over 40% of mRNAs for protein kinases and transcription factors harbor uORFs [9]. The transcription factors of the Auxin Response Factor class are particularly rich in uORFs [27], which can be conserved in their position, if not their sequence, as exemplified by ARF2 [25]. ARF3/ETTIN, which regulates leaf polarity and reproductive development, and ARF5/MONOPTEROS, which regulates embryo and shoot apex development, harbor two and six uORFs, respectively, that dramatically attenuate expression of the Auxin Response Factor proteins. Plants expressing a uORF-less version of ARF3/ETT had undisclosed ‘morphological differences’ indicating that attenuation by the uORFs is functionally significant [69]. The residual expression of ARF3 and ARF5 most likely occurs by translation reinitiation, given that two different mutations that have been implicated previously in translation reinitiation compromised the expression of ARF3 and ARF5. Specifically, a mutation that deletes one of two copies of RPL24 (rpl24b/shortvalve1) suppresses ARF3 expression, resulting in

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Table 2 Comparison between three mRNA-specific regulatory principles that operate at the translation level.

Kingdom Small molecule detected Small molecule binds uORF present uORF peptide features mRNA folding Ribosome affects Actuator

5 riboswitch

5 attenuator

uORF (subset)

Prokaryotes Various mRNA No Not applicable Alternate forms RNA folding Transcription termination or 30S binding

Prokaryotesa Amino acid tRNA Yes Repeated codons Alternate forms RNA folding Transcription termination

Eukaryotes Various ? Yes Conserved peptideb Unknown Translation Translation reinitiation inhibited (RNA degradation)

a Attenuators exist in amino acid biosynthesis genes and are characterized by a series of codons requiring the given amino acid. We have not detected any uORFs with the hallmarks of attenuator peptides in Arabidopsis (Jia and von Arnim unpublished). b Regulatory CPuORFs have been referred to as peptoswitches [17].

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apical-basal patterning defects in the gynoecium, which resemble those in arf3 mutants [69]. Likewise, a mutation in the h subunit of eIF3 has similar effects [27]. It was possible to partially rescue the gynoecium defects of the rpl24b mutant using ARF3 transgenes, and the uORF-less transgene was slightly more effective than the wild-type version [79]. Rescue of the severe rpl24b phenotype with a single ‘client’ gene is a remarkable result, because the rpl24b/stv1 mutant misexpresses and mistranslates more than a hundred mRNAs (Kim, Tiruneh and von Arnim, unpublished data). Moreover, the arf3/ettin phenotype was dramatically enhanced by the rpl24b and eif3h mutants [27,69], which indicates that the ARF3-regulated developmental pathways require additional client mRNAs of RPL24 and eIF3h. The enhanced phenotype included pinformed shoots, a characteristic phenotype that is a hallmark of auxin transport defects, and that can also be seen in certain alleles of ARF5/MP [91]. These results are in keeping with the notion that ARF5/MP is also a client of RPL24 and eIF3h. The mutant phenotypes seen in rpl24b and eif3h may point to the role of translation reinitiation in developmental gene regulation. eIF3h supports reinitiation rather than leaky scanning [31] and would not be expected to affect ribosome assembly or translation elongation. One cannot rule out that RPL24B affects elongation or ribosome assembly, but the rpl24b mutant has translation defects that partially overlap those in eif3h [27]. These findings raise the possibility that the defects observed in the rpl24b/stv1 ribosomal mutant are due in part to flaws in translation reinitiation [69]. Moreover, the RPL24B paralog, RPL24A, has already been implicated in reinitiation in cauliflower mosaic virus [66]. Mutations in RPL4 also cause mistranslation of ARF mRNAs [92]. Interestingly, the auxin pathway is not simply a target of uORF-mediated translational attenuation. In fact, auxin actively stimulates gene expression of Auxin Response Factors and other uORF containing mRNAs [90]. The auxin-stimulated polysome loading and translation of uORF containing mRNAs requires the TARGET OF RAPAMYCIN (TOR) kinase, as was evident from experiments using TOR kinase inhibitors and TOR RNA silencing. eIF3h has an active role in this pathway, since it is phosphorylated by the TOR client, ribosomal protein S6 kinase (S6K), and a phosphomimic allele of eIF3h stimulated translation of the uORF-containing ARF5 mRNA [90]. How auxin activates TOR kinase in the first place is still open to speculation [93]. Overall, the Auxin Response Factor mRNAs have been used as a productive experimental system to explore how the basal translation machinery and its peripheral signaling pathways control the translational attenuation and developmental regulation by uORFs. In fact, among the various gene families in the auxin network, the Auxin Response Factors are specifically enriched in uORFs, whereas others, such as AUX/IAA transcriptional repressors, YUCCA auxin biosynthesis enzymes, PIN auxin co-transporters, and TIR auxin receptor mRNAs, have few or no uORFs [27]. Other families of developmental regulator genes, such as homeodomain-basic leucine

zippers and receptor-like kinases also have proportionally more uORFs. While circumstantial, these findings suggest that development may be as much under the control of uORFs as metabolism. The case of auxin also demonstrates that small molecules can regulate development via uORFs, although in this case the regulation operates at least in part via a conserved signaling pathway, the TOR pathway. Whether or not small molecules such as hormones might regulate translation by more direct interaction with specific uORFs, analogous to the paradigm established by polyamines and sucrose in metabolism (Section 3), remains to be seen. 4.2. The development of leaf dorsoventral polarity is sensitive to defects in translation Mutations among the approximately 80 ribosomal proteins cause a variety of growth and developmental abnormalities. Some of these have been attributed to quantitative ribosome insufficiency, others to ribosome aberrancy (mutants defective in translating a subset of mRNAs), and a few may be due to ribosome heterogeneity (the wild-type allele provides a specialized function to a subset of ribosomes, which is lacking in the mutant) [94]. The classical ribosomal protein mutant phenotype in Arabidopsis is a pointed first leaf, which has been attributed to reduced translational activity, i.e. ribosome insufficiency [94]. A more complex, yet also very common, phenotype is that rpl mutations enhance the mild leaf polarity defects of mutations in the transcription factor genes ASYMMETRIC LEAVES1 (AS1) and AS2. Mutations in RPS6, RPS21, RPL4, RPL5, RPL10a, RPL27a, RPL28, and RPL9 fall into this category [94–98]. Plants that are double heterozygous for mutations of the two paralogs, RPS6A and 6B, also have leaf polarity defects, even in the absence of the as1 or as2 mutation [99]. We would like to speculate that the common leaf development phenotypes of ribosomal protein mutants may be a sign of ribosome aberrancy in a class of mRNAs having special needs during translation, specifically uORF-containing mRNAs. What might be the mechanistic explanation? A uORF-containing mRNA simply requires a series of at least two successful initiation events to generate one protein from the major ORF. Such an mRNA may therefore be more sensitive to general mutations in the translation machinery. This hypothesis is particularly intriguing given that the reinitiation event is performed by the same individual 40S subunit that performed the first, cap-dependent, initiation. The first piece of evidence in favor of this idea is as follows. The mutant phenotypes of rpl24b and eif3h and rpl4 attributed to translation reinitiation defects are not all too different from the defects seen in other ribosomal protein mutants. For example, the as1 and as2 mutations are not only enhanced by the previously listed rps and rpl mutations, but also by eif3h and rpl24b (Zhou and von Arnim, unpublished data). Next, we should review some of the findings in more detail. In essence, mutations in the adaxial leaf determinants, AS1, AS2, and

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Homeodomain-zipper class III genes, are enhanced by rpl mutations, whereas mutations in abaxial determinants, KANADI1 and 2, are suppressed by these mutations [97]. This observation is of interest for several reasons. First, it pinpoints that genes in the same pathway with AS1, AS2, and Homeodomain-zipper class III are extremely dependent on effective ribosome function. Second, the mutations in genes that specify adaxial fate, AS1, AS2 and Homeodomain-zipper class III mRNAs, which are subject to enhancement by ribosomal protein mutations, are characterized by prominent clusters of uORFs in their 5 UTR ([25]; see Supplemental Fig. 1 for AS1 and Supplemental Data File S1 for other genes). In contrast, many genes that specify abaxial fate in the leaf, such as YABBYs and KANADIs, have few or no uORFs. We note that ARF3 and ARF4 are exceptions among the determinants of abaxial fate, in that they carry uORFs. Third, most target mRNAs that are known to be affected by the translation factor mutations harbor uORFs [27,69,92]. Finally, a mutation in the A paralog of RPL10, which suppresses an unrelated developmental defect caused by thermospermine misregulation, also works through a uORF-containing 5 UTR [12,87]. In summary, it is plausible - and worthy of further examination - that the striking phenotypes of ribosomal protein mutations in development really point to a pervasive role of uORFs in adjusting expression levels of individual regulatory genes. However, alternative explanations that do not attribute a role to uORFs also remain possible. 5. Conclusions and hypotheses to guide future work 1. uORFs are common control elements that posttranscriptionally affect gene expression. 2. Certain uORFs function as sensors of metabolites. While the mechanism is still not understood, this elegant regulatory principle may be more common than is currently appreciated. 3. uORFs that are regulated by external signals function as stopgo signals, analogous to traffic lights. Most likely the majority of uORFs throttle translation in a more quantitative manner and thus resemble a speed bump. 4. Repression of translation initiation, which can be accomplished with uORF based sensors, adds an important regulatory capability in gene regulation. 5. Most uORFs are not conserved, yet are predicted to inhibit translation. They may function as rheostats by adjusting gene expression up or down during evolution, or they may counterbalance mutations in the promoter that affect transcription of the gene. 6. Translation reinitiation downstream from a uORF must be coordinated very carefully with regular translation termination. The molecular biochemistry of both of these processes is still not completely understood. 7. To the best of our knowledge, ribosomal initiation on a uORF start codon is no different from initiation on the major AUG; most likely, the ribosome does not recognize when its activity is being initiated at an upstream AUG rather than the major AUG. 8. Several mutants with defects in leaf polarity are enhanced by mutations affecting ribosomal proteins. This phenotype may indicate that critical mRNAs contain uORFs and thus require that a single ribosome (40S) performs not just one but two or more translation initiation events on the same mRNA. Acknowledgments Research in the von Arnim lab was supported by funding from the National Science Foundation (DBI-0820047) and the US Department of Energy (DE-FG02-96ER20223). We thank Tyler White for collecting data for Fig. 3.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci. 2013.09.006.

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