The 3' untranslated region of a rice alpha-amylase gene functions as a sugar-dependent mRNA stability determinant

Abstract

In plants, sugar feedback regulation provides a mechanism for control of carbohydrate allocation and utilization among tissues and organs. The sugar repression of alpha-amylase gene expression in rice provides an ideal model for studying the mechanism of sugar feedback regulation. We have shown previously that sugar repression of alpha-amylase gene expression in rice suspension cells involves control of both transcription rate and mRNA stability. The alpha-amylase mRNA is significantly more stable in sucrose-starved cells than in sucrose-provided cells. To elucidate the mechanism of sugar-dependent mRNA turnover, we have examined the effect of alphaAmy3 3' untranslated region (UTR) on mRNA stability by functional analyses in transformed rice suspension cells. We found that the entire alphaAmy3 3' UTR and two of its subdomains can independently mediate sugar-dependent repression of reporter mRNA accumulation. Analysis of reporter mRNA half-lives demonstrated that the entire alphaAmy3 3' UTR and the two subdomains each functioned as a sugar-dependent destabilizing determinant in the turnover of mRNA. Nuclear run-on transcription analysis further confirmed that the alphaAmy3 3' UTR and the two subdomains did not affect the transcription rate of promoter. The identification of sequence elements in the alpha-amylase mRNA that dictate the differential stability has very important implications for the study of sugar-dependent mRNA decay mechanisms.

Figure 1

Structures of chimeric genes used for rice transformation. (A) Construction of pLAm and pLAN were described in Chan and Yu (17). All the constructs contain Ubi promoter, Ubi first intron (Ubi-I), Luc coding region, and αAmy3 or Nos 3′ UTR. Domains I, II, and III derived from αAmy3 3′ UTR were inserted between the Luc coding region and the Nos 3′ UTR in pLN. The nucleotide sequences are numbered from the first nucleotide after translation stop codon TGA. (B) Nucleotide sequences of domains I, II, and III. Underline indicates the conserved sequence between domains I and III.

Figure 2

The αAmy3 3′ UTR and its subdomains independently mediate sugar-dependent repression of heterologous mRNA accumulation in transformed rice suspension cells. Rice suspension cells were transfected with plasmids shown in Fig. 1 and cultured as described in Materials and Methods. Four weeks after transfection, the transformed suspension cells were cultured in sucrose-containing or sucrose-free medium for 2 days and total RNA was purified. (A) Levels of Luc mRNA were analyzed with RNA dot blot analysis by using the Luc coding region as a probe. Radioactive signal representing the amount of Luc mRNA in each dot was quantified by using a PhosphorImager (Molecular Dynamics). For each construct, the ratio of mRNA level was determined by dividing the level of Luc mRNA in cells starved for sucrose (−S) by that in cells provided with sucrose (+S). Logarithm value of the ratio (open circle) was then used to plot the graph. Bars represent the average value for each construct. (B) Gel blot analysis of mRNA from one representative transformed cell line of each construct. NT indicates nontransformed control. Five micrograms of total RNA was loaded in each lane. The same blot was stripped and rehybridized with indicated probes as described previously (8). The probes were Luc, Act1 (22), and rRNA (23) cDNAs and αAmy3-specific DNA (8). The Luc and αAmy3 probes hybridized to a same blot, and the Act and rRNA probes hybridized to another parallel-prepared blot.

Figure 3

The αAmy3 3′ UTR functions as a sugar-dependent mRNA stability determinant. (A) Transformed cell line LAm-3 was suspension cultured in sucrose-containing (+S) medium for 96 h (RNA in lane 1) and transferred to sucrose-free (−S) medium for 24 h (RNA in lane 2). ActD was added to the medium to a final concentration of 10 μg/ml. Cells were incubated in the −S medium containing ActD for another 12 h and then divided in half. Half the cells were transferred to a +S medium containing ActD (RNAs in lanes 5–12). The other half were transferred to a −S medium containing ActD (RNAs in lanes 13–20). Cells were collected after 0.5–9 h, and RNAs were purified. RNA gel blot analysis was performed by using various probes used in the experiment described in Fig. 2. The Luc and αAmy3 probes hybridized to the same blot, and the rRNA probe hybridized to another parallel-prepared blot. Five micrograms of total RNA was loaded in each lane. Lanes 3 and 4, cells incubated in −S medium lacking ActD for 36 and 45 h, respectively. (B) Levels of mRNA shown in lanes 5–20 of A were quantified by using the PhosphorImager. Each of the α-amylase mRNA levels of lanes 5–12 and 13–20 was first normalized to the rRNA level for each time point. The relative α-amylase mRNA levels then were determined by dividing the mRNA levels by levels of lane 5 or lane 13. The logarithm values of Luc-αAmy3 (Left) and αAmy3 (Right) mRNA levels were subjected to linear regression analysis and plotted as a function of time. The open and solid circles indicate mRNA from cells grown in −S and +S medium, respectively. Error bar indicates the SE of mRNA levels from three independently transformed cell lines. The error bars for mRNA levels from cells grown in −S medium are too short to be shown on the graph. The dashed line indicates the time at which 50% of mRNA remained. The half-life of mRNA is shown at the bottom of the graph.

Figure 4

Two subdomains of αAmy3 3′ UTR independently mediate sugar-dependent decay of heterologous mRNA. Cell culture, treatment of cells with ActD, and RNA gel blot analysis all were performed as described in the legend of Fig. 3A. (A) Decay pattern of Luc mRNA from transformed cell lines LN-5 (Upper) and LIIN-2 (Lower). (B) Decay pattern of Luc mRNA from transformed cell lines LIN-2 (Upper) and LIIIN-4 (Lower).

Figure 5

The αAmy3 3′ UTR and its subdomains do not affect transcription. Transformed rice suspension cells (same as Fig. 4) were cultured in sucrose-containing or sucrose-free medium for 12 h. Nuclei were isolated and nuclear run-on transcription analysis was performed as described previously (7). Five micrograms each of rRNA, Act1, and Luc cDNAs and αAmy8-specific DNA (8) was dot-blotted on the membrane. pBS, pBluescript DNA (Stratagene).