Insufficient Radiofrequency Ablation Promotes Hepatocellular Carcinoma Metastasis Through N6-Methyladenosine mRNA Methylation-Dependent Mechanism.

BACKGROUND AND AIMS: The dynamic N6-methyladenosine (m6 A) mRNA modification is essential for acute stress response and cancer progression. Sublethal heat stress from insufficient radiofrequency ablation (IRFA) has been confirmed to promote HCC progression; however, whether m6 A machinery is involved in IRFA-induced HCC recurrence remains open for study. APPROACH AND RESULTS: Using an IRFA HCC orthotopic mouse model, we detected a higher level of m6 A reader YTH N6-methyladenosine RNA binding protein 1-3 (YTHDF1) in the sublethal-heat-exposed transitional zone close to the ablation center than that in the farther area. In addition, we validated the increased m6 A modification and elevated YTHDF1 protein level in sublethal-heat-treated HCC cell lines, HCC patient-derived xenograft (PDX) mouse model, and patients' HCC tissues. Functionally, gain-of-function/loss-of-function assays showed that YTHDF1 promotes HCC cell viability and metastasis. Knockdown of YTHDF1 drastically restrains the tumor metastasis evoked by sublethal heat treatment in tail vein injection lung metastasis and orthotopic HCC mouse models. Mechanistically, we found that sublethal heat treatment increases epidermal factor growth receptor (EGFR) m6 A modification in the vicinity of the 5' untranslated region and promotes its binding with YTHDF1, which enhances the translation of EGFR mRNA. The sublethal-heat-induced up-regulation of EGFR level was further confirmed in the IRFA HCC PDX mouse model and patients' tissues. Combination of YTHDF1 silencing and EGFR inhibition suppressed the malignancies of HCC cells synergically. CONCLUSIONS: The m6 A-YTHDF1-EGFR axis promotes HCC progression after IRFA, supporting the rationale for targeting m6 A machinery combined with EGFR inhibitors to suppress HCC metastasis after RFA.

RNA Modification Regulatory Genes in DNA Damage.

Expression of genetic information is a multistep process which needs to be tightly regulated. One of the regulatory mechanisms is posttranscriptional modification of RNA, which can alter the stability, expression, or protein composition. Therefore, misregulation of this important cellular process can lead to pathological consequences, such as cancer development. It has been shown that alteration in the expression of certain RNA-modifying genes can promote tumorigenesis. Here, we present a mRNA expression analysis-based approach to comprehensively determine the expression of RNA readers/writers/erasers using DNA damage as an example, and then to validate the effect of altered RNA reader/writer/erasers in regulating the DNA damage response.

Design, synthesis and activity of light deactivatable microRNA inhibitor.

miRNAs are key cellular regulators and their dysregulation is associated with many human diseases. They are usually produced locally in a spatiotemporally controlled manner to target mRNAs and regulate gene expression. Thus, developing chemical tools for manipulating miRNA with spatiotemporal precise is critical for studying miRNA. Herein, we designed a strategy to control miRNA biogenesis with light controllable inhibitor targeting the pre-miRNA processing by Dicer. By conjugating two non-inhibiting units, a low affinity Dicer inhibitor and a pre-miRNA binder, through a photocleavable linker, the bifunctional molecule obtained could inhibit miRNA production. Taking advantage of the photocleavable property of the linker, the bifunctional inhibitor can be fragmented into separate non-inhibiting units and therefore be deactivated by light. We expect that this strategy could be applied to generate chemical biological tools that allow light-mediated spatiotemporal control of miRNA maturation and contribute to the study of miRNA function.

Radiation induced transcriptional and post-transcriptional regulation of the hsa-miR-23a~27a~24-2 cluster suppresses apoptosis by stabilizing XIAP.

The non-coding transcriptome, in particular microRNAs (miRNA), influences cellular survival after irradiation. However, the underlying mechanisms of radiation-induced miRNA expression changes and consequently target expression changes are poorly understood. In this study we show that a single dose of 5Gy ɣ-radiation decreases expression of the miR-23a~27a~24-2 cluster in the human endothelial cell-line EA.hy926 and the mammary epithelial cell-line MCF10A. In the endothelial cells this was facilitated through transcriptional regulation by promoter methylation and also at the post-transcriptional level by reduced miRNA processing through phosphorylation of Argonaute (AGO). Furthermore, we demonstrate that all three mature cluster miRNAs reduce apoptosis by increasing expression of the common target protein XIAP. These findings link a temporal succession of transcriptional and post-transcriptional regulatory mechanisms of the miR~23a~24-2~27a cluster, enabling a dynamic stress response and assuring cellular survival after radiation exposure.

NB-LRR signaling induces translational repression of viral transcripts and the formation of RNA processing bodies through mechanisms differing from those activated by UV stress and RNAi.

Plant NB-LRR proteins confer resistance to multiple pathogens, including viruses. Although the recognition of viruses by NB-LRR proteins is highly specific, previous studies have suggested that NB-LRR activation results in a response that targets all viruses in the infected cell. Using an inducible system to activate NB-LRR defenses, we find that NB-LRR signaling does not result in the degradation of viral transcripts, but rather prevents them from associating with ribosomes and translating their genetic material. This indicates that defense against viruses involves the repression of viral RNA translation. This repression is specific to viral transcripts and does not involve a global shutdown of host cell translation. As a consequence of the repression of viral RNA translation, NB-LRR responses induce a dramatic increase in the biogenesis of RNA processing bodies (PBs). We demonstrate that other pathways that induce translational repression, such as UV irradiation and RNAi, also induce PBs. However, by investigating the phosphorylation status of eIF2α and by using suppressors of RNAi we show that the mechanisms leading to PB induction by NB-LRR signaling are different from these stimuli, thus defining a distinct type of translational control and anti-viral mechanism in plants.

MicroRNA-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous cell carcinoma cells.

BACKGROUND AND PURPOSE: Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation. MATERIALS AND METHODS: A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p. RESULTS: A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo. CONCLUSION: miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells.

Proteasome activity influences UV-mediated subnuclear localization changes of NPM.

UV damage activates cellular stress signaling pathways, causes DNA helix distortions and inhibits transcription by RNA polymerases I and II. In particular, the nucleolus, which is the site of RNA polymerase I transcription and ribosome biogenesis, disintegrates following UV damage. The disintegration is characterized by reorganization of the subnucleolar structures and change of localization of many nucleolar proteins. Here we have queried the basis of localization change of nucleophosmin (NPM), a nucleolar granular component protein, which is increasingly detected in the nucleoplasm following UV radiation. Using photobleaching experiments of NPM-fluorescent fusion protein in live human cells we show that NPM mobility increases after UV damage. However, we show that the increase in NPM nucleoplasmic abundance after UV is independent of UV-activated cellular stress and DNA damage signaling pathways. Unexpectedly, we find that proteasome activity affects NPM redistribution. NPM nucleolar expression was maintained when the UV-treated cells were exposed to proteasome inhibitors or when the expression of proteasome subunits was inhibited using RNAi. However, there was no evidence of increased NPM turnover in the UV damaged cells, or that ubiquitin or ubiquitin recycling affected NPM localization. These findings suggest that proteasome activity couples to nucleolar protein localizations in UV damage stress.

DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization.

Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the posttranscriptional regulation of gene expression as part of the DNA damage response in cancer cells.

Processing of 3'-phosphoglycolate-terminated DNA double strand breaks by Artemis nuclease.

The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double strand breaks. To assess the possibility that Artemis acts on oxidatively modified double strand break termini, its activity toward model DNA substrates, bearing either 3'-hydroxyl or 3'-phosphoglycolate moieties, was examined. A 3'-phosphoglycolate had little effect on Artemis-mediated trimming of long 3' overhangs (> or =9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3'-phosphoglycolates on overhangs of 4-5 bases promoted Artemis-mediated removal of a single 3'-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3' overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was completely dependent on DNA-dependent protein kinase and ATP and was largely dependent on Ku, which markedly stimulated Artemis activity toward all 3' overhangs. Together, these data suggest that efficient Artemis-mediated cleavage of 3' overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3' to the cleavage site, as well as 2 unpaired nucleotides 5' to the cleavage site. Shorter 3'-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis but much more slowly. Consistent with a role for Artemis in repair of terminally blocked double strand breaks in vivo, human cells lacking Artemis exhibited hypersensitivity to x-rays, bleomycin, and neocarzinostatin, which all induce 3'-phosphoglycolate-terminated double strand breaks.

Role of the C-terminal domain of RNA polymerase II in U2 snRNA transcription and 3' processing.

U small nuclear RNAs (snRNAs) and mRNAs are both transcribed by RNA polymerase II (Pol II), but the snRNAs have unusual TATA-less promoters and are neither spliced nor polyadenylated; instead, 3' processing is directed by a highly conserved 3' end formation signal that requires initiation from an snRNA promoter. Here we show that the C-terminal domain (CTD) of Pol II is required for efficient U2 snRNA transcription, as it is for mRNA transcription. However, CTD kinase inhibitors, such as 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), that block mRNA elongation do not affect U2 transcription, although 3' processing of the U2 primary transcript is impaired. We show further that U2 transcription is preferentially inhibited by low doses of UV irradiation or actinomycin D, which induce CTD kinase activity, and that UV inhibition can be rescued by treatment with DRB or H7. We propose that Pol II complexes transcribing snRNAs and mRNAs have distinct CTD phosphorylation patterns. mRNA promoters recruit factors including kinases that hyperphosphorylate the CTD, and the CTD in turn recruits proteins needed for mRNA splicing and polyadenylation. We predict that snRNA promoters recruit factors including a CTD kinase(s) whose snRNA-specific phosphorylation pattern recruits factors required for promoter-coupled 3' end formation.

Elliptically polarized magnetic fields do not alter immediate early response genes expression levels in human glioblastoma cells.

Expression of immediate early response genes such as c-fos, c-jun, and c-myc in response to 1-500 microT resultant (r) 60 Hz elliptically polarized (EP) magnetic fields (MFs), typical of environmental MFs polarization under overhead power lines, was analyzed in both at transcriptional and translational levels using human glioblastoma (T98G) cells. Pseudo synchronized T98G cells at G1 phase were exposed to EP-MFs (1, 20, 100, and 500 microTr) for up to 3 h, but produced no statistical difference (P>0.05) in the levels of expression ratio at both the transcriptional and translational levels at 30 min for c-fos and c-jun and at 180 min for c-myc after serum stimulation. In addition, exposure of T98G cells to linearly (vertical and horizontal) and/or circularly polarized MFs (500 microTr) produced no significant change (P>0.05) in the expression ratio at both transcriptional and post-transcriptional levels. Thus, there was no evidence that linearly or rotating polarized MFs enhanced early response gene expression in these studies. These results suggest that environmental MFs at 1-500 microT flux density are unlikely to induce carcinogenesis through a mechanism involving altered expression of the immediate early response genes.

Transcriptional and posttranscriptional regulation of the glycolate oxidase gene in tobacco seedlings.

The roles of light and of the putative plastid signal in glycolate oxidase (GLO) gene expression were investigated in tobacco (Nicotiana tabacum cv. Samsun NN) seedlings during their shift from skotomorphogenic to photomorphogenic development. GLO transcript and enzyme activities were detected in etiolated seedlings. Their respective levels increased three- and six-fold during 96 h of exposure to light. The GLO transcript was almost undetectable in seedlings in which chloroplast development was impaired by photooxidation with the herbicide norflurazon. In transgenic tobacco seedlings, photooxidation inhibited the light-dependent increase in GUS activity when it was placed under the regulation of the GLO promoter P(GLO). However, even under these photooxidative conditions, a continuous increase in GUS activity was observed as compared to etiolated seedlings. When GUS expression was driven by the CaMV 35S promoter (P35S), no apparent difference was observed between etiolated, deetiolated and photooxidized seedlings. These observations indicate that the effects of the putative plastid development signal and light on GUS expression can be separated. Translational yield analysis indicated that the translation of the GUS transcript in P(GLO)::GUS seedlings was enhanced 30-fold over that of the GUS transcript in P35S::GUS seedlings. The overall picture emerging from these results is that in etiolated seedlings GLO transcript, though present at a substantial level, is translated at a low rate. Increased GLO transcription is enhanced, however, in response to signals originating from the developing plastids. GLO gene expression is further enhanced at the translational level by a yet undefined light-dependent mechanism.

UV-induced modifications in the peptidyl transferase loop of 23S rRNA dependent on binding of the streptogramin B antibiotic, pristinamycin IA.

The naturally occurring streptogramin B antibiotic, pristinamycin IA, which inhibits peptide elongation, can produce two modifications in 23S rRNA when bound to the Escherichia coli 70S ribosome and irradiated at 365 nm. Both drug-induced effects map to highly conserved nucleotides within the functionally important peptidyl transferase loop of 23S rRNA at positions m2A2503/psi2504 and G2061/A2062. The modification yields are influenced strongly, and differentially, by P-site-bound tRNA and strongly by some of the peptidyl transferase antibiotics tested, with chloramphenicol producing a shift in the latter modification to A2062/C2063. Pristinamycin IA can also produce a modification on binding to deproteinized, mature 23S rRNA, at position U2500/C2501. The same modification occurs on an approximately 37-nt fragment, encompassing positions approximately 2496-2532 of the peptidyl transferase loop that was excised from the mature rRNA using RNAse H. In contrast, no antibiotic-induced effects were observed on in vitro T7 transcripts of full-length 23S rRNA, domain V, or on a fragment extending from positions approximately 2496-2566, which indicates that one or more posttranscriptional modifications within the sequence Cm-C-U-C-G-m2A-psi-G2505 are important for pristinamycin IA binding and/or the antibiotic-dependent modification of 23S rRNA.

Generation of a tropoelastin mRNA variant by alternative polyadenylation site selection in sun-damaged human skin and ultraviolet B-irradiated fibroblasts.

The goal of this research was to delineate the post-transcriptional mechanisms responsible for the increased elastin synthesis characteristic of sundamaged skin. In this study, a unique molecular variant of the tropoelastin mRNA transcript was identified in human sundamaged skin that was derived from the usage of an alternate polyadenylation site. Nonsolar exposed human skin expressed one tropoelastin mRNA species whereas sundamaged human skin expressed the primary tropoelastin mRNA and a larger, alternate tropoelastin mRNA formed from the utilization of a second polyadenylation site. Cultured human skin fibroblasts expressed both tropoelastin transcripts and in vitro UV treatment increased the amount of the unique tropoelastin mRNA. Hairless mouse skin (normal and UV treated) expressed the primary tropoelastin transcript although UV irradiation increased the length of its poly (A) tail two-fold. Therefore, UV radiation may stimulate elastin production by affecting polyadenylation site selection and the poly (A) tail length of tropoelastin mRNA.

Altered processing of precursor transcripts and increased levels of the subunit I of mitochondrial cytochrome c oxidase in Syrian hamster fetal cells initiated with ionizing radiation.

Treatment of Syrian hamster fetal cells (SHFC) with ionizing radiation resulted in the establishment of 21 transformed cell lines. Relative to unirradiated controls, cells from early post-irradiation passages (p.3) showed marked morphologic alterations, increased growth rate and extended life span, and they were contact-inhibited and not tumorigenic in nude mice, although they became tumorigenic after extended passaging in culture (p. > 30). Differential mRNA display analyses of normal cells (84-3) and radiation-initiated cell lines at early passage showed that the latter contained increased steady-state levels of the precursor (4-fold) and mature (1.7-fold) transcripts of the mitochondrial (mt) gene encoding the subunit I of cytochrome c oxidase (CO I). These molecular alterations were consistently observed in 57% of the irradiated (HDR) cell lines, and were stably maintained during continuous passaging (p. > 50). Further analyses of one of these cell lines (HDR-3) demonstrated that the accumulation of CO I precursor transcripts was the result of mRNA stabilization and increased replication and/or amplification of the mt DNA. Radiation-initiated cells contained elevated levels of the CO I protein, showed a 75% reduction in cytochrome c oxidase (CO) activity, and a 5-fold increase in the concentration of hydrogen peroxide secreted into their culture medium compared with cells with no alterations in CO I mRNA processing. Our findings suggest that alterations in mt CO I processing may play a role in the neoplastic conversion of mammalian cells by ionizing radiation.

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.

Evidence for post-transcriptional regulation of cyclin B1 mRNA in the cell cycle and following irradiation in HeLa cells.

Cyclin B1 mRNA expression varies markedly through the cell cycle with its peak in G2/M and lowest level in G1. Cyclin B1 mRNA levels are also transiently reduced in HeLa cells after gamma-irradiation, coincident with the radiation-induced G2 block. In order to understand the mechanisms underlying these variations, we have measured cyclin B1 mRNA stability in HeLa cells during different phases of the cell cycle. The half-life of the mRNA measured after actinomycin D administration is 1.1-1.8 h in both early and late G1, 8 h in S and 13 h in G2/M. We therefore conclude that altered RNA stability is important in modulating cyclin B1 mRNA levels through the HeLa cell cycle. Furthermore, 3 h after irradiation of HeLa cells in S phase with 10 Gy, the half-life of cyclin B1 mRNA is reduced to 5 h; it is further reduced to 2-3 h at 14 h after irradiation. Thus, decreased stability contributes to the reduction in cyclin B1 mRNA following irradiation.

Gene-specific modulation of RNA synthesis and degradation by extremely low frequency electromagnetic fields.

Pulse-labeling studies from our laboratory and others have shown that extremely low frequency (ELF) electromagnetic fields can produce a transient increase in gene transcription. In this study, the synthesis, degradation and processing, and steady state levels of specific RNA species during exposure to ELF radiation were determined in human leukemia HL-60 cells. The overall steady state RNA levels, assessed by continuous and equilibrium labeling with 3H-uridine, were not affected by ELF exposure. Northern blot analysis using probes specific for c-myc, beta-actin, and 45S ribosomal RNA gene products revealed that ELF did not alter the steady state levels of these RNAs. Examination of gene-specific transcription by a novel nuclease protection assay revealed that while ELF did not substantially alter the transcription rates for c-myc and beta-actin, transcription of the 45S ribosomal RNA gene was increased by 40-50%. To explain the observed increase in the synthesis of 45S ribosomal RNA without an associated increase in its steady state level, the degradation and processing of the ribosomal gene transcript in the presence and absence of an ELF field were followed by pulse-chase 3H-uridine labeling. This revealed that ELF radiation accelerated both the processing and degradation of the ribosomal RNA transcript. During ELF exposure, the half-life of the 45S ribosomal RNA was decreased from 115 min. to 85 min. These results show that ELF can selectively affect RNA levels by modulating either the transcription rate and/or RNA post-transcriptional processing and turnover.

Transcriptional and posttranscriptional components of psbA response to high light intensity in Synechococcus sp. strain PCC 7942.

The psbA genes, which encode the D1 protein of photosystem II, constitute a multigene family in the cyanobacterium Synechococcus sp. strain PCC 7942. Levels of messages from the three psbA genes change rapidly when cells are shifted from low-light to high-light conditions: the psbAI message level drops, whereas psbAII and psbAIII message levels increase dramatically. We examined the potential contributions of transcriptional and posttranscriptional processes in these high-light responses by subjecting cells that had been grown in a turbidostat at a standard light intensity (130 microeinsteins [microE] m-2 s-1) to either the same or a higher light intensity (500 microE m-2 s-1) in the presence or absence of rifampin. Northern (RNA blot) analysis of RNA isolated from cells subjected to high light showed that the increases in psbAII and psbAIII transcripts were blocked by rifampin. This suggests a transcriptional induction of these genes at high light intensities. Increased mRNA stability does not contribute to their accumulation in high-light conditions, since their half-life values did not increase relative to the half-lives measured at the standard light intensity. The rate of disappearance of the psbAI transcript in cells shifted to high light was diminished when either transcription or translation was blocked by rifampin or chloramphenicol, suggesting that accelerated degradation of the message requires de novo synthesis of a protein factor. When rifampin was added 10 min after the shift to high light intensity rather than before the shift, psbAI and psbAIII messages, but not the psbAII message, decayed at a faster rate. Susceptibility of the psbAIII transcript to the high-light-induced factor was also demonstrated by addition of chloramphenicol prior to the shaft to high light. psbAIII transcript levels went up more than twofold higher in chloramphenicol-treated cells than in untreated cells, whereas psbAII transcript levels were affected by the inhibitor. These experiments provide evidence that either new or increased synthesis of a degradation factor which affects a subset of Synechococcus transcripts occurs in cells subjected to high light intensity.