RNA-binding proteins and their role in translational regulation in plants

The chances of a plant, as for any organism, to survive and succeed in an ever-changing environment greatly rely on its capacity to respond appropriately to internal and environmental cues and adjust its growth to the new conditions. Proper adaptation depends on the plant’s ability to produce specific sets of proteins in a precise and timely manner. For this, and because translation is the most energetically demanding process in the cell [1], its regulation is an important and clever way to control gene expression. It allows the generation of very fast and reversible responses to a stimulus because no transcriptional reprograming is needed and the target transcripts are not destroyed. Although the central role of translation in modulating the expression levels of a gene has long been recognized, our understanding of the molecular mechanisms behind this type of regulation has been hampered due to technical limitations. Recent technological developments have fired a renewed enthusiasm towards the study of selective translation, which is becoming a more and more active research field.

The fate of a mRNA is determined by a set of cis- and trans-elements [2]. Cis-elements (Figure 1), usually present in the untranslated regions (UTRs), are involved in plant growth and development [2,3] by being recognized by a set of RNA-binding proteins (RBPs), the trans-acting elements, via their RNA-binding domains. RBPs constitute a diverse class of proteins [4] that have the capacity to bind single or double-stranded RNA and influence its metabolism, orchestrating the translational control of gene expression. mRNA stability, subcellular localization, transport, and translation are all processes controlled by RBPs. Excellent reviews have been recently written about many of the functions of plant RBPs and RBP families regarding post-transcriptional control of gene expression [4–10]. Here, we have focused specifically on the exciting new discoveries made on one of these important roles, the selective mRNA translation, which is a key regulatory step in response to numerous developmental and environmental cues (reviewed in [11–13]). For that, our attention was centered on RBPs that directly regulate the translation of their client RNAs. We have not included the events of translation regulation mediated by the translational machinery per se (thoroughly reviewed in [14]), nor those alterations on translation efficiency due to changes in mRNA stability or decay (reviewed in [15]). To organize the information, we have separated the processes by which RBPs control the translation of several mRNAs that share a specific cis-element from those involved in the translation of a specific mRNA under a certain physiological condition. We are aware that each event of global regulation means the specific binding of the RBP to a specific transcript, and that those conditions in which we relate a specific RBP to a specific transcript may still reflect a lack of knowledge, but we believe it helps to give a wider perspective to the broad range of ways by which RBPs regulate translation. Due to a lack of space, we have only included RBPs involved in the regulation of cytoplasmic translation, although chloroplast translation is strongly influenced by RBPs. For this topic, we refer the reader to excellent recent work [16–19]. Unless specifically stated, in none of the examples below did the steady-state levels of mRNA change.

Certain cellular situations involve massive translational reprogramming. Severe stresses usually turn down the energetically demanding translation machinery [20–25], but also developmental stages such as seed or pollen maturation or dark–light transitions, require global translational switch off and on [26–30]. Here we highlight different mechanisms by which RPBs control global translation.

Hypoxic stress affects cellular translation heavily as it turns down the translation of approximately 90% of cellular mRNAs [23]. However, this response is dynamic and soon after reoxygenation most of the translationally silent transcripts become actively translated again [23,31]. OLIGOURIDYLATE-BINDING PROTEIN 1, UBP1, a hnRNP1 polyuridylate tract-binding protein known to be involved in intron recognition, splicing and mRNA accumulation [32], plays a role in this regulation [33] (Figure 2A). There are three UBP1 members in Arabidopsis (A to C), of which, UBP1C´s expression increases significantly upon unanticipated darkness and submergence and its down-regulation causes hypersensitivity to hypoxic stress in seedlings. UBP1C constitutively binds mRNAs that contain U-rich 3′UTRs, a feature enriched in transcripts encoding regulatory proteins and, thus, whose expression is finely tuned. Under hypoxia, however, UBP1C association with individual cellular mRNAs increased, binding transcripts that are not involved in the stress response and bypassing the need of a U-rich sequence. This binding was largely reversed by reaeration. Also, UBP1C rapidly aggregates into stress granules that disassembled quickly upon reaeration [33]. These UBP1-granules were also observed under heat stress [32]. Ribosome run-off was required for granule formation, which is consistent with the down-regulation of global translation under hypoxic stress. Not surprisingly, mRNAs that escaped global translational repression and UBP1C-sequestration under hypoxia encode for proteins related to anaerobic respiration and stress [33]. It is unclear whether the binding specificities of UBP1C change upon hypoxia or if the detected reduction in U-rich mRNAs is the consequence of UBP1C association withing stress granules. In addition, it is also unknown whether mRNA assembly into UBP1C granules is the cause or the consequence of the global translational inhibition upon hypoxia, but it provides a means for the dynamic regulation of translation in response to a transient energy stress. Interestingly, the triggering signal for UBP1C assembly could be a rise in the oxidative intracellular state [33], an aspect that should be further explored.

Other RBPs work positively regulating translation under stress. COLD SHOCK PROTEIN 1, CSP1, is a cold shock domain-containing RBP that in Arabidopsis associates to polysomes by directly binding to mRNA [34]. Upon cold treatment, CSP1 increased its association to polysomes and that of mRNAs involved in energy-consuming processes, like ribosome synthesis, biogenesis of RNP complexes, and components of the mitochondrial envelope. These mRNAs share a high tendency to form double-stranded RNA regions in their 5′UTR which typically disfavors translation under conditions of low cell energy [35,36]. As Arabidopsis CSPs denature RNA secondary structures in vitro [37] CSP1-binding could melt these structures and facilitate the scanning by the 43S pre-initiation complex, thus enhancing translation. The fact that Arabidopsis plants overexpressing CSP1 performed better than WT upon drought stress supports this hypothesis [34].

La and La-related proteins (LARPs) are RBPs present in almost all eukaryotes and characterized by the presence of the extremely well-conserved La motif (LAM) followed by an RNA recognition motif [38]. Arabidopsis presents two LARP families, LARP1 and LARP6, with three members each. Both play important roles in the translational state of their target mRNAs, although are involved in very different biological processes. While LARP1A is involved in energy state-dependent translational regulation, LARP6C controls the translational state of mRNAs during pollen development [30,39]. LARP1A promotes translation through the of target of rapamycin (TOR) pathway. Upon activation when cellular conditions are favorable [40] TOR phosphorylates LARP1, which in turn, promotes the translation of a specific set of mRNAs that begin with a 5′TOP motif [39]. The promotion of translation of mRNAs encoding ribosomal proteins is the main function assigned to the TOR-LARP1-5′TOP in humans due to the 5′TOP motifs in these transcripts [41]. However, only a few plants cytosolic ribosomal proteins possess 5′TOP while other mRNAs involved in chromatin remodeling, auxin signaling, and ribosome biogenesis possess them. This means that although the function is conserved in eukaryotes, the mechanisms by which the TOR-LARP1-5′TOP enhances ribosome biogenesis are different between humans and plants [39].

Pollen maturation and germination are two developmental stages that require extensive post-transcriptional regulation. All mRNAs required for pollen germination and pollen tube growth are already present in mature pollen grains, but maintained translationally inactive until pollen enters the progamic phase, when these mRNAs engage in active translation and sustain pollen tube growth [42]. However, only very recently, has some light been shed on how this translation regulation takes place [30] (Figure 2B). LARP6C is exclusively expressed in pollen and plays important roles in pollen tube growth and guidance that require its RNA-binding activity through an intact LAM [30]. LARP6C accumulates both in the cytoplasm and nucleus and under hypoxic stress aggregates in stress granules [43]. These granules also form as pollen matures and colocalize with microtubules along the pollen tube [30]. LARP6C directly binds mRNAs involved in pollen gametogenesis, germination, tube growth and guidance, and tip-polarized growth. Most of these transcripts share a U-rich box in their 5′UTR that LARP6C binds directly. Studying mRNA and protein levels of MGD2, a LARP6C client mRNA, it was found that LARP6C played a dual role, both as translational repressor and as activator of the same set of target genes depending on the pollen developmental stage, as it kept MGD2 protein levels low in dry pollen and drastically increased them upon germination [30]. Remarkably, MGD2 protein distributes along a gradient in the pollen tube, and this gradient attenuated in the larp6c mutant. This raises the hypothesis that the movement of LARP6C granules along microtubules is responsible for the site-specific translation of MGD2 [30]. These roles are exclusive to LARP6C as LARP6A that has a wider expression pattern and is expressed at a higher level in pollen, does not act redundantly in these functions. How LARP6C shifts from being a translational repressor to an activator when the pollen enters the progamic phase; whether LARP6C plays a role in controlling protein gradients in polarized cells; and if so, which molecular mechanisms are involved in this site-specific translation are important questions that are still open. Local protein synthesis has been extensively studied in neurons [44], but not much is known about this process in plants besides the role of the peptide RALF1 and its receptor FER sustaining local translation at the tip of the root hair [45]. Whether a similar mechanism could regulate local translation along the pollen tube needs to be determined.

miRNAs play a critical role in many developmental and physiological processes in both plants and animals. After being generated, miRNAs bind AGO1 and are incorporated into the RNA-induced silencing complex (RISC), where they repress the expression of their target mRNAs by inducing either transcript cleavage or translational inhibition [46]. miRNA-directed translational inhibition takes place in the cytoplasm in membrane-bound polysomes through the interaction of AGO1 with AMP1 on the endoplasmic reticulum [47]. Excellent reviews describe the proteins known to be involved in plant miRNA-mediated translational inhibition [48–50]. Recently an important component of the miRNA biogenesis pathway in the nucleus has been found to play a crucial role in the cytoplasmic miRNA translational inhibition pathway [51] (Figure 2C). HYPONASTIC LEAVES 1, HYL1, is a dual-localized nuclear and cytoplasmic double-stranded RNA-binding protein that controls miRNA biogenesis and, thus, is involved in many aspects of plant development [52,53]. In a series of elegant experiments using exclusively nuclear or cytoplasmic-localized forms of HYL1, Yang and collaborators have discovered two independent functions for HYL1 in the control of gene expression [51]). While nuclear HYL1 is involved in miRNA biogenesis, cytoplasmic HYL1 is involved in translational inhibition and its absence from the cytoplasm caused an increase in the protein levels of its target mRNA. Both locations are required to complement the defects of hyl1 mutants. Cytoplasmic HYL1 colocalizes with AGO1 and AMP1 on the ER membrane. It copurifies with polysomes and its presence is required for the proper distribution of AGO1 in them. Interestingly, HYL1 had no effect on the accumulation of these mRNAs in polysomes, meaning that the translational inhibition did not occur by preventing initiation [51]. How HYL1 recognizes its target mRNAs, whether it requires AGO1 loaded with the miRNA or depends on the specific recognition by HYL1 of yet-unknown cis-elements is still unknown.

Despite these examples, most of the players controlling the global translational reprogramming events still need to be found.

Some RBPs play a crucial role in determining the translation efficiency of a specific mRNAs under certain circumstances and these constitute beautiful examples of how gene expression is finely tuned at single-mRNA level. This regulation is normally of key importance for the plant, as is evidenced by the severe phenotypes its disruption causes.

Phytochromes, the red and far-red light receptors, can interconvert between the Pr and Pfr forms in response to red and far-red light, respectively [54]. The Pfr form moves to the nucleus to promote changes in gene expression leading to photomorphogenesis [55]. Seedlings moved from far-red to white light show photobleaching, partly because phyA represses the expression of the protochlorophyllide reductase (PORA) mRNA. In addition, phytochromes also repress translation of PORA in the cytosol, leading to a concerted transcriptional and translational inhibition of PORA during the dark–light transition [56]. This translational inhibition is mediated by PENTA 1, PNT1, a cytosol-localized quintuple ZnF domain RBP. PNT1 interacts with phyA and phyB and represses the translation of PORA by binding its 5′UTR in a light-independent, but phyA and phyB-dependent, manner. Light is in the equation because the Pfr form of the phytochromes binds PNT1, and thus PORA, with higher affinity than the Pf form [56]. What the motif or secondary structure in the PORA 5′UTR that PNT1 recognizes is, and the mechanism by which the phytochromes-PNT1 interaction inhibits PORA´s translation are still open questions.

The gaseous hormone ethylene plays numerous roles in the development and environmental responses of the plant [57]. The ethylene signaling cascade is represented in Figure 3A. ETHYLENE INSENSITIVE 2, EIN2 is a key player the pathway, and the ein2 mutant is defective in all examined ethylene responses [58]. EIN3/EIL1 are the major transcriptional regulators in response to ethylene and although transcriptional regulation constitutes a major fraction of the ethylene response, there are some rapid ethylene growth responses that are EIN2-dependent but don’t require EIN3/EIL1, which led to speculation that the pathway branched after EIN2 [59]. Two independent works provided the molecular basis for this second pathway in Arabidopsis. Upon ethylene perception, the C-terminal end of EIN2 (C-EIN2) is cleaved form the membrane and moves to the nucleus to activate EIN3/EIL1 [60]. In addition to this nuclear function, C-EIN2 plays a role in cytoplasmic translational control [61,62] (Figure 3A). It was found that although EBF2 is transcriptionally induced by ethylene, its translation, as well as that of EBF1, is inhibited by this hormone. This translational regulation is mediated by C-EIN2, that directly binds the long 3′UTRs of EBF1/2 in the presence of ethylene and relocates them to p-bodies [61,62]. Interestingly, in the upf2 mutant, deficient in the non-sense mediated decay (NMD) machinery [15], this regulation is lost, so most likely, the NMD proteins collaborate to translocate the EIN2-3′EBFs complex to p-bodies in the presence of the hormone [61]. There, EBF1/2 are stored and ready to return to translation when the ethylene signal is withdrawn. Newly synthetized EBF1/2 will then be able to bind EIN3/EIL1 in the nucleus and shut the signaling pathway down [61]. The fact that the 3′UTR of EBF1/2 is sufficient to confer ethylene-sensitivity to the translation of a reporter gene, and that the 3′UTRs of EBF1/2 are required for the proper response to the hormone highlights the biological importance of this regulation. A conserved motif was found multiple times within the 3′UTRs of EBF1/2 and significantly enriched in the mRNAs translationally regulated by ethylene [61,62]. Interestingly, EIN2 also binds RNA during seed germination [63]. EIN2 mutants show a strong dormancy phenotype due to high ABA levels in the dry seeds [64]. It is possible that EIN2 is involved in repressing the translation of mRNAs that promote dormancy via their sequestration in p-bodies.

More recently, a regulatory loop involving translation regulation has been reported within the ABA signaling pathway [65] (Figure 3B). SOAR1, for SUPPRESSOR OF THE ABAR-OVEREXPRESSOR 1, is a nuclear and cytoplasmic-localized pentatricopeptide repeat protein (PPR) that negatively regulates ABA signaling [66]. It prevents the formation of the cap-binding complex by binding to eIFiso4Gs, thus inhibiting global translation [65]. Interestingly, SOAR1 reduces polysome binding of positive regulators of ABA signaling, like ABI5 and PYR1, while enhancing translation of negative regulators, like ABI1, ABI2, and HAB1 [65,67]. Curiously, Bi and colleagues showed that SOAR1 also binds ABI5 mRNA, which inhibits the interaction of SOAR1 to eIFiso4Gs, allowing them to participate in eIFiso4F complexes and promoting translation of the positive regulators of the ABA pathway. The molecular mechanisms involved in this gene-specific modulation of translation mediated by SOAR1 are still unknown.

Phloem transports photosynthates, organic compounds and signaling molecules throughout the plant, playing a fundamental role in plant growth and development, and serving as communication network. Phloem differentiation involves a series of non-reversible steps that end up in enucleated cells that form a sieve tube [68–70] meaning that phloem cells lose their transcriptional ability and need to rely on post-transcriptional regulation to stablish phloem networks in the plant. The interaction between JULGI (JUL)—Korean word for “plant shoot” or “stream”—and the G-quadruplex of SMXL mRNAs constitutes a beautiful example of translation regulation controlling phloem development in vascular plants [71] (Figure 3C). JUL is a sucrose-induced, phloem and cambium-specific RanBP2-type ZnF RBP present in all vascular plants. It negatively regulates phloem differentiation, and its RNA-binding activity is crucial for this role. JUL binds to G-rich sequences with potential to form G-quadruplexes, which are extremely stable secondary structures that are assembled as G-quartet stacks through Hoogsteen bonds [72]. SMXL4/5, key positive regulators of phloem development, also exclusively present in vascular plants, have G-quadruplex-forming regions in their 5′UTRs that JUL binds directly inducing their folding. Binding of JUL to SMLX5 down-regulates its translation restricting the amount of phloem cells [71]. Giving its strong dependance on post-transcriptional regulation, phloem development constitutes an excellent model to search for new translation regulation events. In addition, there are more phloem-specific genes with the capacity to form G-quadruplexes [71]. If they are also subject to translational regulation, whether new regulatory mechanisms are controlling phloem development, and which RBPs are involved should be further investigated. Interestingly, the seeds of JUL-down-regulated plants were bigger and heavier than WTs, and this effect is not due to the actual genotype of the seed, which means there is a close relationship between the number of phloem cells and the sink capacity of the seeds.

Panicle development greatly influences grain number in rice and was known to be tightly controlled at the transcriptional level. Now, a role for translational control in this process has just been uncovered [73]. FRIZZY PANICLE, FZP, is an AP2 transcription factor that negatively regulates panicle size and grain number per panicle, and is a QTL that increased rice yield during domestication [74]. Interestingly, the length of the 3′UTR of FZP correlated directly with the number of secondary branches and grains in the rice panicle [75]. Chen and colleagues [73] observed that the 3′UTR of FZP contains three CU-rich elements (CUREs). CUREs are common cis-elements in the 3′UTR and in mammal cells are involved in splicing, polyadenylation, mRNA location, stability, and translation [76]. In plants, CUREs were known to facilitate mRNA long-distance transport [77,78], but this is the first report of CUREs mediating translational repression in plants. Deletion or overexpression of the CUREs of FZP produced rice plants with smaller panicles, fewer primary and secondary branches, lower grain number per panicle, and increased FZP translation. These results strongly suggested the existence of a trans-acting factor binding to CUREs that negatively regulates FZP translation levels. Two rice PTBs/hnRNPI (PTB1/2) were found to bind to CUREs in FZP. PTB1/2 are RBPs with four RNA-recognition motifs that bind RNA molecules that contain UCUU or CUCUCU sequences [79]. They are involved in alternative splicing and were also suggested to play a role in translational control in Arabidopsis [80]. PTB1/2 bound the 3′UTR of FZP at the CUREs region and repressed its translation. The biological importance of the CUREs in the regulation of FZP translation was demonstrated with the dramatical decrease of both the rice plant and panicle size, and in yield of plants lacking or overexpressing the CUREs [73]. How PTB1/2 mediate the translational repression of FZP is still unknown. Interestingly, in Drosophila, a similar translation inhibition mechanism was described by which another PTB/hnRNP1 is necessary for the translational repression of Oskar (Osk), a determinant for primordial germ cells and posterior patterning [81]. Cytoplasmic PTB binds directly to multiple sites along the Osk 3' UTR and mediates the assembly of RNP granules that entail Osk translational silencing [82]. It will be worth exploring if this is also the molecular mechanism behind the PTB1/2-mediated translational repression of rice FZP and, if so, whether it is a conserved mode of action of cytoplasmic PTB in eukaryotic regulation of translation.

The research included herein demonstrates the key role that regulation at the translational level plays in the control of gene expression in plants. However, this is just a very small bite of what is yet to be unveiled. There are still many open questions, molecular mechanisms to be discovered and numerous aspects that should be further studied.

Many techniques that allow the study of translation have been successfully implemented in plants (thoroughly reviewed in [83]). TRAP-seq and Ribo-seq have specially contributed to the identification of genome-wide targets of translational regulation under many different environmental and developmental conditions, and, in most cases, the molecular mechanisms and RBPs responsible of the regulation have not yet been uncovered. Remarkably, the last years have seen a surge of studies characterizing the plant mRNA interactome using different tissues and developmental stages [63,84–88] greatly expanding the number of potential RBPs in the Arabidopsis genome to more than 1400 [3] and whose role in translation is yet to be discovered. Research combining these experiments under different conditions would allow the identification of many new translation regulation modules modulating gene expression. These studies are, at present, laborious, expensive and greatly rely on the expertise, but hopefully, with standardized protocols they will become easier to perform. Years of exciting research on the field of translation regulation are ahead.

• Translation regulation plays important roles in fine-tuning gene expression.

• RBPs govern the post-transcriptional life of mRNA. By recognizing specific cis-elements in their target transcripts, RBPs adjust translation efficiency in response to developmental or environmental cues.

• RBPs can both repress or enhance translation of one or several transcripts at a time, employing a wide variety of molecular mechanisms.

• The number of RBPs found to be involved in important biological processes is increasing, so the discovery of many more exciting events of RBP-governed translational regulation in the future years is expected.

The authors declare that there are no competing interests associated with the manuscript.

This work was supported by grants RYC2017-22323 (Ministerio de Economía, Industria y Competitividad, cofinanced by the European Regional Development Funds; Spain), BIO2017-82720-P (Ministerio de Economía, Industria y Competitividad, Spain), P18-RT-1218 (Junta de Andalucía, Spain), and UMA20-FEDERJA-100 (Junta de Andalucía and University of Málaga, Spain) to C.M; and a PRE2018-083348 (Ministerio de Ciencia, Innovación y Universidades, cofinanced by the European Union Social Funds; Spain) to J.A.D.C..

Open access for this article was enabled by the participation of Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC) in an all-inclusive Read & Publish agreement with Portland Press and the Biochemical Society.

NMD

non-sense mediated decay

RBP

RNA-binding protein

RISC

RNA-induced silencing complex

TOR

target of rapamycin