Regulation of cold shock-induced RNA helicase gene expression in the Cyanobacterium anabaena sp. strain PCC 7120

Abstract

Expression of the Anabaena sp. strain PCC 7120 RNA helicase gene crhC is induced by cold shock. crhC transcripts are not detectable at 30 degrees C but accumulate at 20 degrees C, and levels remain elevated for the duration of the cold stress. Light-derived metabolic capability, and not light per se, is required for crhC transcript accumulation. Enhanced crhC mRNA stability contributes significantly to the accumulation of crhC transcripts, with the crhC half-life increasing sixfold at 20 degrees C. The accumulation is reversible, with the cells responding more rapidly to temperature downshifts than to upshifts, as a result of the lack of active mRNA destabilization and the continuation of crhC transcription, at least transiently, after a temperature upshift. Translational inhibitors do not induce crhC expression to cold shock levels, indicating that inhibition of translation is only one of the signals required to activate the cold shock response in Anabaena. Limited amounts of protein synthesis are required for the cold shock-induced accumulation of crhC transcripts, as normal levels of accumulation occur in the presence of tetracycline but are abolished by chloramphenicol. Regulation of crhC expression may also extend to the translational level, as CrhC protein levels do not correlate completely with the pattern of mRNA transcript accumulation. Our experiments indicate that the regulation of crhC transcript accumulation is tightly controlled by both temperature and metabolic activity at the levels of transcription, mRNA stabilization, and translation.

FIG. 1

Cold shock-induced increases in crhC transcript abundance is regulated by temperature and metabolic activity. Northern blots of total RNA (15 μg) extracted from Anabaena exposed to different temperatures and/or light-dark conditions were hybridized with either the crhC or the RNase P gene. The autoradiograms are shown. Lane 1, 1 h at 30°C; lane 2, 1 h at 25°C; lane 3, 1 h at 20°C; lane 4, 1 h at 15°C; lane 5, 1 h at 10°C; lane 6, 3 h at 30°C in the dark, followed by 1 h at 20°C in the dark; lane 7, 4 h at 30°C in the dark; lane 8, 3 h at 30°C in the light, followed by 1 h at 20°C in the dark; lane 9, 4 h at 30°C in the light. The blots were probed with crhC, stripped, and reprobed with RNase P, as indicated.

FIG. 2

Time course of cold-induced accumulation and warmth-induced decay of crhC transcripts. (A) Fifteen micrograms of total RNA extracted from each sample was subjected to Northern blot analysis using crhC and RNase P probes, as indicated. RNA was obtained from Anabaena cells grown at 30°C and then cold shocked at 20°C for the following lengths of time: 0 h (lane 1), 0.25 h (lane 2), 0.5 h (lane 3), 1 h (lane 4), 2 h (lane 5), 3 h (lane 6), 6 h (lane 7), 24 h (lane 8), and 48 h (lane 9). Lanes 10 and 11 contain RNAs from control cultures grown at 30°C for 24 and 48 h, respectively. (B) Northern blot analysis of total RNA (15 μg) extracted from Anabaena exposed to 20°C for 1 h and then to 30°C for 0 h (lane 1), 0.25 h (lane 2), 0.5 h (lane 3), 1 h (lane 4), 2 h (lane 5), and 3 h (lane 6). The blot was probed with crhC, stripped, and reprobed with RNase P, as indicated.

FIG. 3

CrhC protein expression mimics transcript accumulation. Total protein was extracted from Anabaena exposed to 20°C for various lengths of time. The Western blot, containing 50 μg of protein per lane and immunodecorated with rabbit anti-CrhC antiserum, is shown. Lane 1, 0 min; lane 2, 15 min; lane 3, 30 min; lane 4, 1 h; lane 5, 2 h; lane 6, 3 h; lane 7, 6 h; lane 8, 24 h; lane 9, 48 h; lane 10, 48 h at 30°C. The position of the 47-kDa CrhC protein is indicated by an arrow.

FIG. 4

crhC transcripts are stabilized at 20°C. Total RNA (15 μg) was extracted from Anabaena exposed to 20°C for 1 h, followed by the addition of rifampin (400 μg/ml) and either continued exposure at 20°C or transferred to 30°C for the indicated times. crhC transcript levels, determined by Northern blot analysis, were quantitated to determine their half-lives at the respective temperatures. Average values from triplicate, independent repetitions of the experiments were subjected to linear regression analysis and are plotted (squares and hatched line, 30°C; diamonds and solid line, 20°C). Shown in the inset are representative autoradiograms of Northern blots used to generate the data points.

FIG. 5

Cold shock-mimicking translational inhibitors partially induce crhC transcript accumulation at 30°C. The autoradiogram of a Northern blot of total RNA (15 μg) extracted from Anabaena exposed to various cold shock-mimicking translational inhibitors for 30 min at 30°C is shown. Lanes 1 and 8, 30 min at 20°C, no inhibitor; lane 2, chloramphenicol (10 μg/ml); lane 3, erythromycin (500 μg/ml); lane 4, fusidic acid (0.5 μg/ml); lane 5, spiramycin (800 μg/ml); lane 6, tetracycline (10 μg/ml); lane 7, ethanol (0.8%) as a control without antibiotics. The blots were probed with crhC, stripped, and reprobed with RNase P, as indicated.

FIG. 6

Translational inhibitors differentially affect crhC induction at 20°C. The autoradiograms of Northern blots of total RNA (15 μg) extracted from Anabaena exposed to either chloramphenicol (40 μg/ml, lanes 1 to 4) or tetracycline (10 μg/ml, lanes 6 to 9) at 20°C for the indicated lengths of time are shown. Lanes 1 and 6, 0 min; lanes 2 and 7, 10 min; lanes 3 and 8, 20 min; lanes 4 and 9, 30 min; lanes 5 and 10, 30 min at 20°C without inhibitor. The blots were probed with crhC, stripped, and reprobed with RNase P, as indicated.