The effect of novel proteins on RNA editing

Project Summary

RNA editing is the conversion of particular cytidines (Cs) to uridines (Us) in organelle mRNAs.  It is regulated by the editosome, a complex of several ­trans-factors, which bind to cis-elements upstream of the editing site (Barkan and Small, 2014).  The identification of proteins involved in the editosome and characterization of their functions is a current topic of investigation.

The objective of this project is to examine the effect of several candidate genes on chloroplast RNA editing in vitro using single nucleotide polymorphism (SNP) analysis.  T-DNA insert seed lines of the candidate genes were ordered from the Arabidopsis Resource Center (ABRC).  Each line was genotyped and sequenced.  RNA was extracted from homozygous mutant lines and will be reverse transcribed to generate cDNA.  The cDNA will undergo SNP analysis, in which mass spectrometry will quantify the ratio of As (edited) to Gs (unedited).

Thirty one out of 48 T-DNA insert lines were found to contain at least one homozygous mutant plant.  RNA was extracted from these plants and stored for further use (RT-PCR).  The 31 homozygous lines, along with 5 lines found to contain heterozygous plants, were transplanted in soil to set seed to be stored for future use.

While many elements of the editosome have been identified, several key factors are still unknown.  Identification and characterization of these unknown components will further elucidate how RNA editing works.  This will contribute to overall knowledge of chloroplast gene expression, which has the potential to contribute to efforts to increase photosynthetic efficiency in plants.

My Experience

Prior to this internship, I was unfamiliar with the subject of RNA editing.  I enjoyed studying this process and working within this system.  I was able to refine my skills in genotyping and learned new techniques of RNA extraction.  Additionally, I had the great opportunity to attend two thesis defense seminars, which gave me insight into the graduate school experience from the perspective of a graduate student.

My experience working in the Hanson lab was wonderful.  The atmosphere was inviting and productive.  While the lab has a diverse array of projects, each lab member takes an active interest in each other’s work, which fosters a supportive and close-knit group.  In particular, my mentor, Stéphane Bentolila, was very supportive and encouraging, and provided excellent instruction in lab techniques.  I would like to thank him, Dr. Hanson, and the entire lab for an amazing summer experience.