Anima Sharma, Katie Doyle, Pat Gillevet, Ancha Baranova
This is a collaborative project between
Molecular and Microbiology Department, College of Science,George Mason University, Fairfax, VA
Dr. Andy Patamawenu, Dr. Mark Connors, HIV-specific immunity section, NIH
In his PhD Dissertation, Dr. Andy Patamawenu discovered very interesting phenomenon - an apparent ADAR-type of RNA editing in human plasma dendritic cells. He studied ony one human gene, though. So, we are planning to PCR fragments of a few other genes on the template of mRNA from these cells and proceed with pyrosequencing of them. Hopefully, we will find a vareity of edited mRNA species.
ADAR FACILITATED RNA EDITING IN HUMAN PLASMACYTOID DENDRITIC CELLS (PDC).
A. Sharma, Lamya Alomair, Katherine Doyle, Patrick Gillevet, Masoumeh Sikaroodi, Aybike Birerdinc, & Ancha Baranova
Adenosine (A) to Inosine (I) RNA editing is facilitated by enzymes known as ADAR (Adenosine Deaminase that Act on RNA). ADARs specifically recognize double stranded RNA structures or RNA duplex structures as their substrates. Inosine is translated as Guanosine, since most enzymes recognize Inosine as Guanosine. Examples of physiological ADAR editing are edits to neuronal Glutamate and Serotonin receptor transcripts. Here we set to find out whether ADAR-editing in human PDCs (Plasmacytoid Dendritic Cell) is limited to TLR7, or whether it covers other known ADAR targets, including other TLR receptors, FLNA, IGFBP7, KCNA1, GABRA3, and CYFIP2. Site specific primers around previously known edited sites were designed using NCBI primer blast and then tested on cDNA derived from universal RNA and adipose tissue. Purified cDNA from PDC cells was used as templates for PCR amplification, tagged, purified, and subjected to Multitagged (MTPS) pyrosequencing on Roche GS-FLX instrument. The pyrosequencing data was assembled using Lasergene’s Seqman Pro to assemble all the contigs together and obtain a SNP report, which will further be subjected to analysis. If successful, these experiments will be the very first demonstration that RNA editing activity is present in PDCs and acts on physiologically important gene targets.