By David N. Leff
MIAMI - "This is the first documentation," said the speaker, "of RNA-editing-induced messenger RNA transcript mutations in pathogenesis of systemic lupus erythematosus and in other autoimmune disorders."
Molecular immunologist Dama Laxminarayana was addressing a session Tuesday morning on kinase cascades at the Miami/Nature Biotechnology Winter Symposium. Laxminarayana and Gary Kammer, both on the faculty at Wake Forest University School of Medicine in Winston-Salem, N.C., presented a lecture titled "The mRNA transcript editing in type I protein kinase A (PKA-I) regulatory subunit of systemic lupus erythematosus (SLE) T lymphocytes."
Before reporting his methods and results, Laxminarayana gave his audience a brief SLE-101 rundown on the disease. "It's an idiopathic autoimmune disorder of indeterminate etiology, "he told them, "characterized by protean clinical symptoms and signs. They range from a benign skin disorder to severe, life-threatening multisystem disease."
SLE's U.S. prevalence ranges from 146 to 508 victims per million people, with 18 to 76 new cases diagnosed annually. It strikes women of childbearing years eight to 10 times more frequently than males, and afflicts more African-Americans than Caucasians.
"The disease is characterized by diverse T cell immune effector dysfunctions," Laxminarayana went on, "including impaired proliferation to antigen and diminished antibody and cell-mediated toxicity." These and other T-cell defects in SLE "raised the possibility that a disorder primary to the T cell exists in the disease."
Molecular Mechanism Linked To Rla Gene
He and laboratory colleagues previously had identified the first derangement of signal transduction in the T cells of SLE patients. "This disordered signal transduction," he said, "involves impaired protein phosphorylation, due to deficient protein kinase A-I isozyme activity."
Seeking to nail down the molecular mechanisms leading to this deficiency, he and his colleagues discerned "mRNA transcript editing in the RIa gene in SLE T cells. That gene encodes a protein that is a principal regulatory subunit of PKA-I."
They synthesized the cDNA from RNA they extracted from SLE patients' T cells, and from normal controls.
"Messenger RNA editing," Laxminarayana told his audience, "is one means of producing phenotypic variability. Transcript mutation induced by mRNA editing is a widely occurring phenomenon in all eukaryotypes. RNA editing," he pointed out to BioWorld Today, "is a process of post-transcriptional changes occurring in the RNA."
"When the RNA is transcribed from DNA," he explained, "it will be spliced into exons, and that's how it becomes a functional gene product, which can translate into protein. After the transcription, some structural changes are induced in the RNA by several factors. Really, we don't know in humans, but we know in lower organisms like Trypanosoma, in which I showed a guide RNA, which comes and binds with this RNA and edits it, that is, deletes or inserts some bases.
"Another thing," he continued, "there are enzymes that can convert bases like adenine and cytidine in SLE-damaged T lymphocytes to guanine and thymidine, at rates 50 times higher than normal T cells. That's how it has an impact on its protein product. These base substitutions and deletions will have an impact on the protein structure. The protein will be abnormal, so it cannot do its function in the diseased patient. So in these patients, the PK is defective. And what the precise role of protein kinase is in immune function we cannot define. It's a very complex system, but it basically plays a role in degradation of the immune system.
"In my lab," Laxminarayana recounted, "people showed that protein kinase is a vital protein. If it is knocked out - if you take out its gene, RIa, from the animal - the animal becomes lethal."
He and his colleagues isolated the T cells from SLE patients, isolated the RNA transcripts from these cells, and subjected them to PCR amplification of their gene of interest. They were able to identify mutant transcripts, which have an altered structure compared to normal ones.
"That definitely showed," he reported, "a structural change in that transcript. And then we cloned into the vectors to analyze those structural changes at the base level, and sequenced them. So we have showed the impairment of the functional region of the enzyme.
"But the important thing," Laxminarayana concluded, "is to determine its role in the disease. If we characterize this, others are going to take it a step better. So, if we can eliminate what is causing this, it is going to help get results in curing SLE. That is our future focus."