Digoxin and Genistein Treatment by Drosophila Larvae Differentially Modulate the Levels of Arginine Kinase Encoding Transcripts
Arginine kinase (126.96.36.199) catalyzes the addition of a phospho-group of arginine from ATP, which plays an important part in energy homeostasis in high energy-consuming tissues of most invertebrates. Six transcripts can be produced by Drosophila Argk gene (Figure 1) through alternative promoters and splicing. All of the transcripts share 3’ exon7 but differ with unique 5’ exons.
No studies on the function of different Argk isoforms in Drosophila have been reported. Identification of compounds that differentially regulate the expression of specific Argk isoform will be a valuable tool for studying functions of individual Argk isoform. Digoxin, a cardiac glycoside and genistein, an isoflavone from soy, have been demonstrated to be capable of modulating mRNA expression. To investigate the impact of digoxin and genistein on mRNA expression of Argk transcripts, RNA was isolated from 3rd instar larvae fed by digoxin and genistein and subjected to RT-PCR analysis.
Material and methods
Drosophila melanogaster and treatments
Drosophila 3rd instar larvae are used in this project. 3rd instar larvae were kindly provided by Dr. Dubrovsky’s lab, Department of Biological Sciences, Fordham University. Different treatments are DMSO (control), 10 μg/g digoxin, 100 μg/g digoxin, 25 μg/g genistein and 100 μg/g genistein.
Total RNA from different 3rd instar larvae treatment was extracted and purified from the cell lysates using the RNeasy® Plus Mini Kit (QIAGEN) by manufacturer’s instructions. A 10 ng/μl dilute was made from each RNA stock and used in RT-PCR.
RT PCR was performed using QIAGEN® One-Step RT PCR Kit following the instructions. Rp49 was used as the loading control.RT-PCR program is: Reverse transcription of 50°C for 30 min, HotStarTaq DNA Polymerase activation of 95°C for 15 min, 40 cycles of 94°C for 30 sec, 57°C for 30 sec and 72°C for 60 sec, then a final extension of 72°C for 10 min and holding at 4°C.
5 μl products were loaded on 1.0 % agarose gels. The voltage of electrophoresis is 180 V. Gels were visualized in BioRad UV trans-illuminator and pictures were taken.
PCR product purification, gel extraction and sequencing
PCR products were purified using QIAquick® PCR Purification Kit. Some products were cut from gel and purified using QIAquick® Gel Extraction Kit following the manufacturer’s instructions. Purified PCR products were sent out for sequencing.
Information for specific designed primers and location is listed in Figure 1 and 2
Argk-RA, Argk-RC, Argk-RD and Argk-RE are expressed in Drosophila 3rd instar larvae, but Argk-RF is not detected in Drosophila 3rd instar larvae. Argk-RF and Argk-RD are designed to be picked up by same primer pair so that Argk-RF is not expressed in the 3rd instar larvae. Results also indicate that digoxin or genistein treatment does not modulate mRNA expression of Argk-RA/RE or Argk-RD (Figure 3). Argk-RA and Argk-RE are alternative spliced transcripts and Argk-RA has an extra exon4 encoding a proline-rich mitochondria transport signal in the amino terminal of arginine kinase isoform A (Argk-PA). Result reveals that digoxin or genistein has no influence towards the Argk-RA/RE alternative splicing process.
Argk-RC mRNA level is up-regulated by digoxin and genistein treatment and total Argk transcripts also rise up in 3rd instar larvae (Figure 4). High concentration (100 μg/g) digoxin treated larvae has higher Argk-RC mRNA level than low concentration (10 μg/g) digoxin treated larvae. Both 25 μg/g and 100 μg/g genistein treated larvae have same level of total Argk mRNA higher than untreated larvae. Total six Argk isoforms are detected by a primer pair located in exon7 and PCR amplification of this primer pair has no products produced (result no shown) which confirms that Argk band in Figure 4 is produced only by mRNA.
1.Digoxin and genistein treatment up-regulate mRNA expression level of Argk-RC of Drosophila 3rd instar larvae. Total Argk transcripts also increase in response to digoxin and genistein.
2. Digoxin or genistein treatment has no significant impact on mRNA expression levels of Argk-RA, Argk-RE or Argk-RD of Drosophila 3rd instar larvae.
3. Digoxin and genistein may differentially modulate the utilization of Drosophila Argk promoters. The regulatory mechanism remains to be determined.
Figure 1-Six putative transcripts of Drosophila melanogaster arginine kinase gene from Flybase database (Annotation ID: G32031).
Figure 2-RT-PCR primers for Argk-RA/RE, Argk-RB, Argk-RC, Argk-RD/RF, Argk(all isoforms) and Rp49
Figure 3-No significant mRNA expression differences have been observed with regard to Argk-RA, Argk-RE and Argk-RD by digoxin or genistein treatment. RT-PCR was performed on RNA isolated from Drosophila 3rd instar larvae fed with chemicals for 14 hours using primers specific to Argk-RA/RE and Argk-RD/RF. Treatments are DMSO (control), 10 μg/g digoxin, 100 μg/g digoxin, 25 μg/g genistein and 100 μg/g genistein from left to right. Rp49 is a loading control.
Figure 4-Argk-RC mRNA level is up-regulated by digoxin and genistein treatment and total Argk transcripts level is also increased. RT-PCR was performed on RNA isolated from Drosophila 3rd instar larvae fed with chemicals for 14 hours using primers specific to Argk-RC and Argk all isoforms. Treatments are DMSO (control), 10 μg/g digoxin, 100 μg/g digoxin, 25 μg/g genistein and 100 μg/g genistein from left to right. Rp49 is a loading control.
Drosophila melanogaster arginine kinase (Argk) gene encodes 6 different mRNA transcripts: Argk-RA to Argk-RF, due to alternative promoters and splicing. To examine the impact of digoxin and genistein on Drosophila, total mRNA was extracted from 3rd instar larvae after chemical treatment and the level of different transcripts were tested by RT-PCR. Result demonstrates that both digoxin and genistein can up-regulate the level of Argk transcripts in general. Argk-RC is up-regulated by both chemicals whereas Argk-RA, Argk-RD and Argk-RE have no significant changes. This finding indicates that digoxin and genistein can differentially influence the utilization of arginine kinase gene promoters.
I would like to thank Dr. Berish Rubin for his help and guidance during this project. I would like to thank Dr. Edward Dubrovsky for generously providing Drosophila materials. I am deeply grateful to Bo Liu and Xie Xie for continuously support and discussions which have brought me infinite help and new knowledge.