Characterization of adapt33, a stress-inducible riboregulator

Abstract

We have identified adapt33 as a multiple stress-responsive gene that is induced under conditions of a cytoprotective "adaptive response." adapt33 RNA does not contain any appreciable open reading frame nor produce a protein product and is therefore classified as a stress-inducible riboregulator. Although a number of oxidant stress-modulated, protein-encoding genes have been reported and characterized, very few stress-inducible riboregulator RNAs are known. Here we extend previous studies toward understanding the underlying regulation of expression and function of this rare mammalian riboregulator. mRNA stability and transcription studies determined that adapt33 induction by hydrogen peroxide is at the mRNA stability level, and that adapt33 has a very short half-life. Surprisingly, adapt33 mRNA also exhibits altered electrophoretic migration in response to both hydrogen peroxide and cis-platinum treatment. Although no transcriptional modulation in response to hydrogen peroxide was observed, fusion promoter constructs revealed that adapt33 has an unusually strong promoter that is active in both hamster and human cells. Analysis of expression following the stimulation of apoptosis with hydrogen peroxide and staurosporine revealed a strong correlation with apoptosis, suggesting a possible novel, noncoding RNA component of the apoptotic mechanism. We conclude that adapt33 is a stress-inducible, apoptosis-associated RNA with unique structural and gene promoter characteristics.

Figure 1

The effect of hydrogen peroxide on adapt33 RNA levels. (A) Northern blot containing RNA from HA-1 cells treated with 160 μM of hydrogen peroxide for 90 min and 5 h was probed with radiolabeled adapt33 cDNA. C, control. P, peroxide treated. The Northern blot was also treated with GAPDH cDNA as a loading control. (B) adapt33 stability. HA-1 cells were treated with 0, 4, or 10 μmol of hydrogen peroxide per 10⁷ cells. Three hours later, all cells were treated with 40 μg/ml of DRB. Total RNA was extracted at indicated times after DRB treatment and subjected to Northern blot analysis. (C) adapt33 transcription. HA-1 cells were treated with or without (control) 4 μmol of hydrogen peroxide per 10⁷ cells. At 1.5 and 4.5 h time points after treatment, cell nuclei were extracted from both control and treated cells and run-on reactions were performed on the nuclei and quantified as described in Materials and Methods.

Figure 2

The altered migration of adapt33 mRNA. HA-1 cells were exposed to 4 μmol of hydrogen peroxide per 10⁷ cells. RNA was extracted 5 h later and subjected to Northern blot analysis as described above. Horizontal lines were drawn through the middle of adjacent lanes to more easily assess the accelerated migration observed for adapt33 in the peroxide (P) lanes compared with control (C). (A) Single blot probed successively with radiolabeled cDNA probes for adapt33, adapt15, and GAPDH. (B) Single blot probed successively with radiolabeled cDNA probes for adapt33 and GAPDH. (C) The effect of the DNA-damaging agent cis-platinum (CisPt; 25 mg/ml) on adapt33 expression analyzed as described above.

Figure 3

adapt33 promoter studies. (A) 5′ RACE was used to identify the transcription start site of the adapt33 gene using gene-specific primers. The major PCR product was cloned and sequenced. The sequence shown represents the 5′ end of the cDNA. (B) adapt33 genomic clone and subcloning into the pBluescript vector. The shaded box represents the genomic sequence that is overlapping with the 5′ end of adapt33 cDNA. N, Not1 site; R, EcoR1 site. (C) adapt33 promoter–CAT fusion constructs. The top figure shows the pBluescript/adapt33 genomic subclone. A 2.5-kb fragment containing the 5′ end of adapt33 cDNA and the upstream region was generated from this genomic subclone by PCR and ligated upstream of a reporter CAT gene in a promoter-less pCAT vector. A subsequent 5′ deletion at an internal HindIII site generated a construct containing the 1.4-kb adapt33 insert. The 5′ end of adapt33 cDNA is shaded. R, EcoR1 site; H, HindIII site; N, Not1 site; S, Sac1 site. (D) After 6-h transfection, cells were allowed to recover for 24 h and then treated with 4 μmol of hydrogen peroxide per 10⁷ cells. Cell extracts from control and treated cells were prepared as described in Materials and Methods at indicated time points after treatment.

Figure 4

adapt33 RNA expression and apoptosis. (A) HA-1 cells were exposed to 0, 4, 10, or 25 μmol of hydrogen peroxide per 10⁷ cells and incubated for the indicated times prior to RNA extraction. A Northern blot containing total RNA from these sample was then probed with a cDNA to adapt33. This blot was also probed with GAPDH cDNA as a loading control. (B) HA-1 cells treated with 0, 0.8, 4, 10, 25, or 50 μmol of hydrogen peroxide per 10⁷ cells and staurosporine for 24 h were collected, lysed, centrifuged, and treated. After precipitation, the DNA was run out on a 1.5% agarose gel and stained with SYBR green.

Figure 5

The effect of staurosporine on adapt33 mRNA levels. HA-1 cells were exposed to 200 μM staurosporine and incubated for the indicated times prior to RNA extraction. (A, B) Northern blots containing total RNA from two independent experiments. The blots were probed with radiolabeled cDNAs to (A) adapt33, bax, and GAPDH, and (B) adapt33, actin, and GAPDH, respectively. (C) Densitometric quantification of induction of adapt33 by staurosporine. Relative mRNA levels of adapt33 were normalized to that of GAPDH.