Fed-1 mRNA Research Articles

The Fed-1 (CAUU)4 element is a 5' UTR dark-responsive mRNA instability element that functions independently of dark-induced polyribosome dissociation.

Darkness rapidly induces a decline in the stability and translation of the pea Ferredoxin-1 (Fed-1) mRNA in transgenic tobacco. Direct half-life measurement showed that mutation of the (CAUU)4 stabilizes Fed-1 mRNA in the dark. (CAUU)1, a feature more common in plant 5' UTRs than (CAUU)4, confers slight light-responsive mRNA accumulation. At least three but less than 11 CAUU repeats near the 5' end of the 5' UTR are required for full light-responsive accumulation. Furthermore, 26 nt of the 5' UTR, including the (CAUU)4 repeat, is sufficient to confer a significant approximately 2.5-fold increase in light-regulated mRNA accumulation when fused to the 5' end of a heterologous plant mRNA. A mutation of the (CAUU)4 repeat that compromises light-regulated mRNA stability changes in vitro the accessibility of the region to ribonuclease V1 and ribonuclease A suggesting the geometry formed by the repeat may be important for instability. Finally, dark-induced Fed-1 mRNA instability occurs even when most of the mRNA is retained on polyribosomes, and thus is likely an independent event regulated by darkness.

Light control of nuclear gene mRNA abundance and translation in tobacco.

Photosynthetic signals modulate expression of nuclear genes at the levels of mRNA transcription, mRNA stability, and translation. In transgenic tobacco (Nicotiana tabacum), the pea (Pisum sativum) Ferredoxin 1 (Fed-1) mRNA dissociates from polyribosomes and becomes destabilized when photosynthesis is inhibited by photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We used polymerase chain reaction suppressive-subtractive hybridization to identify similarly regulated endogenous tobacco genes. This screen identified 14 nuclear-encoded tobacco mRNAs whose light-induced increase in abundance is suppressed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Sequence analysis of the cognate cDNAs revealed that nine of the mRNAs encode putative chloroplast-targeted proteins. We asked whether the abundance of these mRNAs was regulated transcriptionally or posttranscriptionally. Of the five mRNAs with sufficient abundance to detect using nuclear run-on assays, we observed transcriptional regulation of alpha-tubulin, thiazole biosynthetic enzyme, and pSKA10 (an unknown gene). Photosystem A subunit L and, to a lesser extent, alpha-tubulin and pSKA10 mRNAs, may also be stabilized in the light. In contrast, Rubisco small subunit mRNA abundance appears to be transcriptionally up-regulated but posttranscriptionally down-regulated in the light. To determine whether, like Fed-1 mRNA, the mRNAs identified in this screen were translationally responsive to light, we characterized the polyribosome association of these mRNAs in the light and after a 15-min dark treatment. A subset of the mRNAs showed dramatic dark-induced polyribosome dissociation, similar to Fed-1 mRNA, and all of the mRNAs showed at least slight polyribosome dissociation. Thus, both posttranscriptional and translational regulation appear to be important mechanisms regulating the expression of many nuclear-encoded mRNAs encoding proteins involved in photosynthesis.

Dark-Induced Destabilization

Expression of the Ferredoxin-1 (Fed-1) gene product is regulated by photosynthetic activity at multiple levels: transcription, mRNA stability, and translation. Hansen et al. studied how the association of Fed-1mRNA with polyribosomes was regulated by photosynthesis and how this interaction influenced Fed-1 mRNA stability. Kinetic experiments with a Fed-1 internal light regulatory element (iLRE) fused to the chloramphenicol acetyltransferase gene showed that the reporter mRNA is released from the polyribosomes within minutes in the dark and that the mRNA can reassociate with the ribosomes upon reillumination. The degradation of the mRNA occurs after the ribosome disassociation, suggesting that Fed-1 mRNA is more stable when complexed with the polyribosomes. The regulation of translation by illumination and by its effects on the association of mRNA with the ribosome was not unique to Fed-1, but was also seen with several, but not all, nuclear-encoded genes involved in photosynthesis.E. R. Hansen, M. E. Petracek, L.F. Dickey, W. F. Thompson, The 5' end of the pea Ferredoxin-1 mRNA mediates rapid and reversible light-directed changes in translation in tobacco. Plant Physiol. 125, 770-778 (2001). [Abstract] [Full Text]

The 5' end of the pea ferredoxin-1 mRNA mediates rapid and reversible light-directed changes in translation in tobacco.

Ferredoxin-1 (Fed-1) mRNA contains an internal light response element (iLRE) that destabilizes mRNA when light-grown plants are placed in darkness. mRNAs containing this element dissociate from polyribosomes in the leaves of transgenic tobacco (Nicotiana tabacum) plants transferred to the dark for 2 d. Here, we report in vivo labeling experiments with a chloramphenicol acetyl transferase mRNA fused to the Fed-1 iLRE. Our data indicate that the Fed-1 iLRE mediates a rapid decline in translational efficiency and that iLRE-containing mRNAs dissociate from polyribosomes within 20 min after plants are transferred to darkness. Both events occur before the decline in mRNA abundance, and polyribosome association is rapidly reversible if plants are re-illuminated. These observations support a model in which Fed-1 mRNA in illuminated leaves is stabilized by its association with polyribosomes, and/or by translation. In darkness a large portion of the mRNA dissociates from polyribosomes and is subsequently degraded. We also show that a significant portion of total tobacco leaf mRNA is shifted from polyribosomal to non-polyribosomal fractions after 20 min in the dark, indicating that translation of other mRNAs is also rapidly down-regulated in response to darkness. This class includes some, but not all, cytoplasmic mRNAs encoding proteins involved in photosynthesis.

Heat shock protein HSP101 binds to the Fed-1 internal light regulator y element and mediates its high translational activity.

The internal light-regulatory element (iLRE) of ferredoxin (Fed-1) mRNA, comprising the 5' leader and at least the first 13 codons of the open reading frame, controls transcript abundance after illumination of the plant in a translation-dependent manner. We have characterized the RNA binding activities associated with the Fed-1 iLRE and have identified one activity as the heat shock protein HSP101, a protein shown to bind the 5' leader of tobacco mosaic virus. HSP101 was sufficient and necessary to mediate a high level of translational activity from a Fed-1 iLRE-containing mRNA in yeast. Moreover, the Fed-1 iLRE substantially enhanced translation of reporter mRNAs in plant protoplasts expressing HSP101. Expression of HSP101 was subject to developmental regulation in leaves in that expression was highest in young leaves. These data suggest that Fed-1 mRNA may use the HSP101 regulatory mechanism as a means of ensuring a high level of translation required for the light-mediated regulation of Fed-1 mRNA stability.

Premature termination codons destabilize ferredoxin-1 mRNA when ferredoxin-1 is translated.

Ferredoxin-1 (Fed-1) mRNA is poorly translated in dark-treated tobacco (Nicotiana tabacum) leaves, resulting in destabilization of Fed-1 mRNA and a differential light/dark accumulation of the mRNA. Insertion of nonsense codons within the Fed-1 coding sequence disrupts the light regulation of Fed-1 mRNA abundance. Here we show that the nonsense codon effect results primarily from lowering the Fed-1 mRNA stability in light-treated leaf tissue and in rapidly growing tobacco cell cultures, but not in dark-treated leaf tissue. These results suggest that nonsense codons trigger a decay pathway distinct from that seen for Fed-1 mRNA in the dark. We propose that nonsense-mediated decay of nonsense-containing Fed-1 mRNA occurs in light-treated leaves and in non-photosynthetic tobacco culture cells where Fed-1 mRNA is being actively translated.

Ferredoxin-1 mRNA is destabilized by changes in photosynthetic electron transport.

In transgenic tobacco, pea Ferredoxin-1 (Fed-1) mRNA accumulates rapidly in response to photosynthesis even when the transgene is driven by a constitutive promoter. To investigate the role of photosynthesis on Fed-1 mRNA stability, we used the tetracycline repressible Top10 promoter system to specifically shut off transcription of the Fed-1 transgene. The Fed-1 mRNA has a half-life of approximately 2.4 hr in the light and a half-life of only 1.2 hr in the dark or in the presence of the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These data indicate that cessation of photosynthesis, either by darkness or DCMU results in a destabilization of the Fed-1 mRNA. Furthermore, the Fed-1 mRNA half-life is reduced immediately upon transfer to darkness, suggesting that Fed-1 mRNA destabilization is a primary response to photosynthesis rather than a secondary response to long-term dark adaptation. Finally, the two different methods for efficient tetracycline delivery reported here generally should be useful for half-life measurements of other mRNAs in whole plants.

Light regulation of Fed-1 mRNA requires an element in the 5′ untranslated region and correlates with differential polyribosome association : Dickey LF, Petracek ME, Nguyen TT, Hansen ER, Thompson WF: Plant Cell 1998, 10:475–484

Light regulation of Fed-1 mRNA requires an element in the 5′ untranslated region and correlates with differential polyribosome association : Dickey LF, Petracek ME, Nguyen TT, Hansen ER, Thompson WF: Plant Cell 1998, 10:475–484

Light regulation of Fed-1 mRNA requires an element in the 5' untranslated region and correlates with differential polyribosome association.

Light regulation of Fed-1 mRNA abundance in the leaves of green plants is primarily a post-transcriptional process. Previously, we have shown that the Fed-1 mRNA light response requires an open reading frame, indicating that the light regulation of the mRNA depends on its concurrent translation. We now show that light-induced increases in Fed-1 mRNA abundance are associated with increases in polyribosome association that require both a functional AUG and a normal Fed-1 translational start context. We also present evidence that light regulation of Fed-1 mRNA levels requires more than efficient translation per se. Substitution of the efficiently translated tobacco mosaic virus Omega 5' untranslated region resulted in a loss of Fed-1 light regulation. In addition, we identified a CAT T repeat element located near the 5' terminus of the Fed-1 5' untranslated region that is essential for light regulation. We introduced two different mutations in the CAT T repeat element, but only one of these substitutions blocked the normal light effect on polyribosome association, whereas both altered dark-induced Fed-1 mRNA disappearance. The element may thus be important for Fed-1 mRNA stability rather than polyribosome loading. We propose a model in which Fed-1 mRNA is relatively stable when it is associated with polyribosomes in illuminated plants but in darkness is not polyribosome associated and is thus rapidly degraded by a process involving the CAT T repeat element.

Light Regulation of Fed-1 mRNA Requires an Element in the 5' Untranslated Region and Correlates with Differential Polyribosome Association

Light Regulation of Fed-1 mRNA Requires an Element in the 5' Untranslated Region and Correlates with Differential Polyribosome Association

Light-regulated changes in abundance and polyribosome association of ferredoxin mRNA are dependent on photosynthesis.

In transgenic tobacco plants containing a pea ferredoxin transcribed region (Fed-1) driven by the cauliflower mosaic virus 35S promoter (P35S), light acts at a post-transcriptional level to control the abundance of Fed-1 mRNA in green leaves. To determine whether the light signal for this response involves photosynthesis, we treated transgenic seedlings with or without 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport. DCMU prevented the normal light response by blocking reaccumulation of Fed-1 transcripts when dark-adapted green plants were returned to the light. In contrast, reaccumulation of light-harvesting complex B (Lhcb) transcripts was unaffected by DCMU treatment. Because Fed-1 light regulation requires translation, we also examined polyribosome profiles. We found that Fed-1 transcripts accumulated on polyribosomes in the light but were found primarily in non-polyribosomal fractions in dark-adapted plants or in illuminated plants exposed to lower than normal light intensity or treated with DCMU. Surprisingly, although Lhcb mRNA abundance was not affected by DCMU, its polyribosomal loading pattern was altered in much the same way as was that of Fed-1 mRNA. In contrast, DCMU had no effect on either the abundance or the polyribosome profiles of endogenous histone H1 or transgenic P35S::CAT transcripts. Thus, our results are consistent with the hypothesis that a process coupled to photosynthesis affects the polyribosome loading of a subset of cytoplasmic mRNAs.

Light modulation of ferredoxin mRNA abundance requires an open reading frame.

Ferredoxin I (Fed-1) mRNA abundance is modulated by an internal light regulatory element that includes sequences both 5' and 3' of the translational initiation site. To test the hypothesis that the light response mediated by this element might be coupled to translation, we transformed tobacco plants with gene constructs blocked in translational initiation or elongation. Here, we report that such mutations abolish the light response in vivo. A nonsense mutation could be rescued by restoring the open reading frame with a different sequence, even when the new codon caused an amino acid substitution. Our data establish that the light response requires a translatable reading frame and thus provide strong circumstantial evidence for post-transcriptional modulation of Fed-1 mRNA levels. The Fed-1 system is presently the only higher plant example of a developmentally regulated change in mRNA abundance that requires translation of the affected mRNA.

Light regulatory sequences are located within the 5' portion of the Fed-1 message sequence.

We have previously shown that element(s) mediating a light-induced increase in the abundance of Fed-1 mRNA in the leaves of transgenic tobacco plants are located within the transcribed portion of the gene. As part of an effort to define the mechanism of this effect, we report here that cis-acting elements capable of mediating a 5-fold light-induced increase in the abundance of this mRNA are located within a region comprising the 5' leader and first third of the Fed-1 coding sequence. No activity was detected in the 3' untranslated region of the gene. In a gain-of-function assay, the 5' region was found to be capable of conferring light responsiveness on three different reporter sequences, although experiments with the gusA reporter were complicated by an apparent negative light effect on the stability of this mRNA. Deletion experiments show that at least one essential light regulatory element is located in the 5' untranslated region of Fed-1 between nucleotides +19 and +57. Additional Fed-1 sequences, including a portion of the protein coding region, are required to confer positive responsiveness on the gusA reporter. These additional sequences may include specific light regulatory elements or simply provide an environment in which the leader element can function normally.

The pea ferredoxin I gene exhibits different light responses in pea and tobacco.

We monitored Fed-1 (encoding ferredoxin I) mRNA levels in etiolated transgenic tobacco seedlings containing the intact pea Fed-1 gene to determine if the characteristic light responses of this gene in pea seedlings are also observed in transgenic tobacco. Fed-1 transcript levels in transgenic tobacco seedlings closely paralleled those of the native gene in pea buds when etiolated seedlings were transferred to white light. However, the response to red light was much smaller in tobacco than in pea and was not efficiently reversed by far-red light. The red light response of endogenous tobacco ferredoxin transcripts is closely comparable to that of the Fed-1 transgene, with a similar lack of photoreversibility. Thus, the pea Fed-1 transgene responds normally to tobacco gene-regulatory factors, but these factors are less influenced by phytochrome in tobacco cotyledons than in pea buds.

Cis-Acting Elements for Light Regulation of Pea Ferredoxin I Gene Expression Are Located within Transcribed Sequences.

An intact pea gene encoding ferredoxin I (Fed-1) and several chimeric constructs containing portions of Fed-1 were introduced into tobacco plants by Agrobacterium-mediated transformation. The intact gene was correctly transcribed and translated to produce a protein that was imported into the chloroplast and processed to its mature size. Fed-1 mRNA accumulation in these plants was strongly light-dependent, as it is in pea leaves. In chimeric constructs, the Fed-1 promoter was active but no light responses were seen, even when as much as 2 kilobases of 5[prime] -flanking sequence were included. We also failed to observe clear light responses with a construct containing 3[prime] -flanking sequences from Fed-1 attached to a [beta]-glucuronidase gene driven by the cauliflower mosaic virus 35S promoter. However, the transcribed portion of Fed-1 conveyed normal light responsiveness when driven by the 35S promoter. The results are discussed in terms of the hypothesis that light determines Fed-1 mRNA abundance by affecting RNA stability rather than by affecting transcription.