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Global analysis of posttranscriptional gene expression in response to sodium arsenite.

Qiu LQ, Abey S, Harris S, Shah R, Gerrish KE, Blackshear PJ - Environ. Health Perspect. (2014)

Bottom Line: Most studies have assumed that arsenic-induced changes in mRNA levels result from effects on gene transcription.In arsenite-exposed cells, 186 probe set-identified transcripts were significantly increased and 167 were significantly decreased.We conclude that arsenite modification of mRNA stability is relatively uncommon, but in some instances can result in significant changes in gene expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Research Triangle Park, North Carolina, USA.

ABSTRACT

Background: Inorganic arsenic species are potent environmental toxins and causes of numerous health problems. Most studies have assumed that arsenic-induced changes in mRNA levels result from effects on gene transcription.

Objectives: We evaluated the prevalence of changes in mRNA stability in response to sodium arsenite in human fibroblasts.

Methods: We used microarray analyses to determine changes in steady-state mRNA levels and mRNA decay rates following 24-hr exposure to noncytotoxic concentrations of sodium arsenite, and we confirmed some of these changes using real-time reverse-transcription polymerase chain reaction (RT-PCR).

Results: In arsenite-exposed cells, 186 probe set-identified transcripts were significantly increased and 167 were significantly decreased. When decay rates were analyzed after actinomycin D treatment, only 4,992 (9.1%) of probe set-identified transcripts decayed by > 25% after 4 hr. Of these, 70 were among the 353 whose steady-state levels were altered by arsenite, and of these, only 4 exhibited significantly different decay rates between arsenite and control treatment. Real-time RT-PCR confirmed a major, significant arsenite-induced stabilization of the mRNA encoding δ aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. This change presumably accounted for at least part of the 2.7-fold increase in steady-state ALAS1 mRNA levels seen after arsenite treatment. This could reflect decreases in cellular heme caused by the massive induction by arsenite of heme oxygenase mRNA (HMOX1; 68-fold increase), the rate-limiting enzyme in heme catabolism.

Conclusions: We conclude that arsenite modification of mRNA stability is relatively uncommon, but in some instances can result in significant changes in gene expression.

No MeSH data available.


Related in: MedlinePlus

Decay rates of selected probe set–identified transcripts measured by microarray in cells treated with vehicle or arsenite for 24 hr and then treated with actinomycin D. Probe set–identified transcript levels were determined before and at 1 hr intervals after actinomycin D treatment. The starting levels of each probe set–identified transcript after 24 hr of treatment but before actinomycin D were set at 100%, and data are expressed as mean percentages (± SD) of the average starting value (n = 4 biological replicates per group). Decay curves for vehicle- and arsenite- treated cells are shown for the four most rapidly decaying probe set–identified transcripts (A–D); two expected to be stable under these conditions, encoding GAPDH and ACTB (E,F); and three encoding the TTP family members expressed in human cells, ZFP36 (TTP; G), ZFP36L1 (H), and ZFP36L2 (I). In these examples, there were no differences between the decay rates between arsenite- and vehicle-treated cells. The Affymetrix probe set identifiers for the transcripts shown in this figure are as follows: DUSP1, 201041_s_at; CYR61, 210764_s_at; SGK1, 201739_at; DUSP6, 208892_s_at; ACTB, 224594_x_at; GAPDH, 213453_x_at; ZFP36, 201531_at; ZFP36L1, 211962_s_at; and ZFP36L2, 201368_at.
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f1: Decay rates of selected probe set–identified transcripts measured by microarray in cells treated with vehicle or arsenite for 24 hr and then treated with actinomycin D. Probe set–identified transcript levels were determined before and at 1 hr intervals after actinomycin D treatment. The starting levels of each probe set–identified transcript after 24 hr of treatment but before actinomycin D were set at 100%, and data are expressed as mean percentages (± SD) of the average starting value (n = 4 biological replicates per group). Decay curves for vehicle- and arsenite- treated cells are shown for the four most rapidly decaying probe set–identified transcripts (A–D); two expected to be stable under these conditions, encoding GAPDH and ACTB (E,F); and three encoding the TTP family members expressed in human cells, ZFP36 (TTP; G), ZFP36L1 (H), and ZFP36L2 (I). In these examples, there were no differences between the decay rates between arsenite- and vehicle-treated cells. The Affymetrix probe set identifiers for the transcripts shown in this figure are as follows: DUSP1, 201041_s_at; CYR61, 210764_s_at; SGK1, 201739_at; DUSP6, 208892_s_at; ACTB, 224594_x_at; GAPDH, 213453_x_at; ZFP36, 201531_at; ZFP36L1, 211962_s_at; and ZFP36L2, 201368_at.

Mentions: To confirm the effectiveness of the actinomycin D treatment under these experimental conditions, we examined comparative decay curves for several of the most rapidly decaying probe set–identified transcripts, as well as those expected to be stable in most cell types, such as those encoding ACTB and GAPDH. Figure 1A–D shows the four probe set–identified transcripts that decayed most rapidly under these conditions. Figure 1 also includes data for two probe sets representing transcripts expected to be stable, ACTB and GAPDH mRNAs (Figure 1E,F), as well as transcripts encoding the three members of the tristetraprolin (TTP) mRNA-destabilizing protein family that are expressed in humans and are known to participate in mRNA decay mediated by adenylate-uridylate-rich elements (Figure 1G–I). In these examples, we observed no apparent differences between decay rates in the arsenite and control samples, with decay rates being essentially superimposable in all cases. The observed very rapid decay of some probe set–identified transcripts, the lack of decay of known stable transcripts, the relatively narrow confidence limits, and the essentially identical results in the arsenite and control samples all support the effectiveness of the microarray screen and of actinomycin D as a rapidly acting and effective transcription inhibitor under these conditions.


Global analysis of posttranscriptional gene expression in response to sodium arsenite.

Qiu LQ, Abey S, Harris S, Shah R, Gerrish KE, Blackshear PJ - Environ. Health Perspect. (2014)

Decay rates of selected probe set–identified transcripts measured by microarray in cells treated with vehicle or arsenite for 24 hr and then treated with actinomycin D. Probe set–identified transcript levels were determined before and at 1 hr intervals after actinomycin D treatment. The starting levels of each probe set–identified transcript after 24 hr of treatment but before actinomycin D were set at 100%, and data are expressed as mean percentages (± SD) of the average starting value (n = 4 biological replicates per group). Decay curves for vehicle- and arsenite- treated cells are shown for the four most rapidly decaying probe set–identified transcripts (A–D); two expected to be stable under these conditions, encoding GAPDH and ACTB (E,F); and three encoding the TTP family members expressed in human cells, ZFP36 (TTP; G), ZFP36L1 (H), and ZFP36L2 (I). In these examples, there were no differences between the decay rates between arsenite- and vehicle-treated cells. The Affymetrix probe set identifiers for the transcripts shown in this figure are as follows: DUSP1, 201041_s_at; CYR61, 210764_s_at; SGK1, 201739_at; DUSP6, 208892_s_at; ACTB, 224594_x_at; GAPDH, 213453_x_at; ZFP36, 201531_at; ZFP36L1, 211962_s_at; and ZFP36L2, 201368_at.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383576&req=5

f1: Decay rates of selected probe set–identified transcripts measured by microarray in cells treated with vehicle or arsenite for 24 hr and then treated with actinomycin D. Probe set–identified transcript levels were determined before and at 1 hr intervals after actinomycin D treatment. The starting levels of each probe set–identified transcript after 24 hr of treatment but before actinomycin D were set at 100%, and data are expressed as mean percentages (± SD) of the average starting value (n = 4 biological replicates per group). Decay curves for vehicle- and arsenite- treated cells are shown for the four most rapidly decaying probe set–identified transcripts (A–D); two expected to be stable under these conditions, encoding GAPDH and ACTB (E,F); and three encoding the TTP family members expressed in human cells, ZFP36 (TTP; G), ZFP36L1 (H), and ZFP36L2 (I). In these examples, there were no differences between the decay rates between arsenite- and vehicle-treated cells. The Affymetrix probe set identifiers for the transcripts shown in this figure are as follows: DUSP1, 201041_s_at; CYR61, 210764_s_at; SGK1, 201739_at; DUSP6, 208892_s_at; ACTB, 224594_x_at; GAPDH, 213453_x_at; ZFP36, 201531_at; ZFP36L1, 211962_s_at; and ZFP36L2, 201368_at.
Mentions: To confirm the effectiveness of the actinomycin D treatment under these experimental conditions, we examined comparative decay curves for several of the most rapidly decaying probe set–identified transcripts, as well as those expected to be stable in most cell types, such as those encoding ACTB and GAPDH. Figure 1A–D shows the four probe set–identified transcripts that decayed most rapidly under these conditions. Figure 1 also includes data for two probe sets representing transcripts expected to be stable, ACTB and GAPDH mRNAs (Figure 1E,F), as well as transcripts encoding the three members of the tristetraprolin (TTP) mRNA-destabilizing protein family that are expressed in humans and are known to participate in mRNA decay mediated by adenylate-uridylate-rich elements (Figure 1G–I). In these examples, we observed no apparent differences between decay rates in the arsenite and control samples, with decay rates being essentially superimposable in all cases. The observed very rapid decay of some probe set–identified transcripts, the lack of decay of known stable transcripts, the relatively narrow confidence limits, and the essentially identical results in the arsenite and control samples all support the effectiveness of the microarray screen and of actinomycin D as a rapidly acting and effective transcription inhibitor under these conditions.

Bottom Line: Most studies have assumed that arsenic-induced changes in mRNA levels result from effects on gene transcription.In arsenite-exposed cells, 186 probe set-identified transcripts were significantly increased and 167 were significantly decreased.We conclude that arsenite modification of mRNA stability is relatively uncommon, but in some instances can result in significant changes in gene expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Signal Transduction, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Research Triangle Park, North Carolina, USA.

ABSTRACT

Background: Inorganic arsenic species are potent environmental toxins and causes of numerous health problems. Most studies have assumed that arsenic-induced changes in mRNA levels result from effects on gene transcription.

Objectives: We evaluated the prevalence of changes in mRNA stability in response to sodium arsenite in human fibroblasts.

Methods: We used microarray analyses to determine changes in steady-state mRNA levels and mRNA decay rates following 24-hr exposure to noncytotoxic concentrations of sodium arsenite, and we confirmed some of these changes using real-time reverse-transcription polymerase chain reaction (RT-PCR).

Results: In arsenite-exposed cells, 186 probe set-identified transcripts were significantly increased and 167 were significantly decreased. When decay rates were analyzed after actinomycin D treatment, only 4,992 (9.1%) of probe set-identified transcripts decayed by > 25% after 4 hr. Of these, 70 were among the 353 whose steady-state levels were altered by arsenite, and of these, only 4 exhibited significantly different decay rates between arsenite and control treatment. Real-time RT-PCR confirmed a major, significant arsenite-induced stabilization of the mRNA encoding δ aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. This change presumably accounted for at least part of the 2.7-fold increase in steady-state ALAS1 mRNA levels seen after arsenite treatment. This could reflect decreases in cellular heme caused by the massive induction by arsenite of heme oxygenase mRNA (HMOX1; 68-fold increase), the rate-limiting enzyme in heme catabolism.

Conclusions: We conclude that arsenite modification of mRNA stability is relatively uncommon, but in some instances can result in significant changes in gene expression.

No MeSH data available.


Related in: MedlinePlus