Project Summary:
The family of ACA genes encodes Ca2+ ATPases, pumps that transport calcium ions out of the cytosol. A total of fourteen genes in Arabidopsis encode these pumps that reside in the plasma membrane, endoplasmic reticulum, or vascular membrane. The ACA10 gene is found to have a 5’-uORF (upstream open reading frame), positioned in the 5’ UTR. These uORFs could function as “translational control elements,” and can serve as regulators for the function of the gene in question. The goal of my experimentation is to determine if the uORF of ACA10 has a regulatory role in transcriptional or translational regulation. I also explored the growth conditions (such as temperature) that may alter uORF activity, due to a potential change in the transcription start site. Additionally, I sought to investigate the existence of uORF in other ACAs. I began my project with bioinformatic analysis, utilizing an online program that can predict the existence of uORFs in a select number of organisms. I opted to analyze maize, in addition to Arabidopsis, to glean insight on these elements in both monocots and dicots. Ultimately, I concluded that uORFs are not unique to Arabidopsis alone, and ACA homologs among varying species contain a uORF region. To investigate the regulatory capabilities of uORFs, I conducted a series of experiments including a Western blot, two dual-luciferase assays with protoplasts, as well as RNA isolations and qPCRs. My research demonstrates that the 5’uORF can increase translation when compared to transcripts manipulated to eliminate the uORF. As for my experimentation on the uORF temperature response, there is possibly a transcriptional/ translational effect, but further research would lead to more definitive conclusions regarding the extent of this response and at what temperatures ACA10 gene regulation is most susceptible to change.
My Experience:
I began this summer excitedly, expecting just to learn new lab techniques and hone my scientific communication skills. While I have greatly refined these abilities through my experimentation and presentations, the full extent to which I have improved as a learning scientist is immeasurable. Through practicing techniques I have never done before, such as RNA isolation, dual-luciferase assays, and Western blot, as well as analyzing data, I feel more competent as a researcher in a technical aspect. By presenting journals and research updates to my lab as well as participating in the symposium, I have gained newfound confidence in my ability to effectively communicate my own knowledge and research to a large and diverse audience. The help I received from my mentor and the rest of my lab will forever be unforgettable to me; I was able to learn so much due to their unwavering support. Most importantly, my summer here has solidified my desire to pursue a career in not only academia but more specifically my most beloved subject of plant genetics. I am eternally grateful for what I learned over the course of my internship and for the opportunity to be surrounded by such intelligent and dedicated individuals.