NEW YORK – A team led by researchers at Columbia University has uncovered a bacterial defense-associated reverse transcriptase (DRT) system that involves the production of novel, unexpected genes using noncoding RNAs as the template.
"Our discovery of de novo genes synthesized by [reverse transcriptases] adds a new dimension to the conventional view of the genome," first author Stephen Tang, a researcher at Columbia University, said in an email, adding that the team is "excited about opportunities to harness this system for biotechnological applications."
In a paper published in Science on Thursday, Tang and colleagues at Columbia University and its Taub Institute for Research on Alzheimer's and the Aging Brain used a combination of complementary DNA (cDNA) immunoprecipitation sequencing (cDIP-seq) and RNA immunoprecipitation sequencing (RIP-seq) to dig into prokaryotic reverse transcriptase and DRT activity in Klebsiella pneumoniae.
They identified a "DRT2" system that uses rolling circle reverse transcription of noncoding RNA and programmed template jumping to produce immune-related genes de novo that are not predicted from genome sequence data.
"We were excited to go after this puzzling question of how DNA synthesis activity could lead to an antiviral immune response," Tang said, "and this investigation led us to a highly unexpected answer: DRT2 systems synthesize de novo genes that encode immune effector proteins."
"Whereas the paradigm of genetic information flow is that RNA serves as an intermediate between DNA and protein, we show that RNA can be used to template the creation of new genes that do not exist on the one-dimensional axis of the chromosome," he explained.
In their follow-up experiments, the investigators found that the presence of foreign phage material prompted concatemeric cDNA to switch from single- to double-stranded, leading to cell growth arrest in response to the expression of "neo" genes produced through the DRT2 system to make so-called Neo proteins.
"The exact mechanism by which these effector proteins work is still unclear, but from what we know so far, it has parallels with some other recently discovered phage defense systems that work by activating toxin proteins to arrest cell growth," Tang said.
He noted that the team is "in the process of searching for such mechanisms in other organisms."
"The identification of Neo proteins disrupts conventionally held notions of features that define protein-coding genes, as well as our broader understanding of genome composition," he and his coauthors wrote in their study. The findings "seem especially important when considering the large proportion of noncoding DNA in higher eukaryotes," they added, noting that "additional examples of Neo-like, non-canonical protein-coding genes likely await future discovery in our own genomes."
More broadly, Tang explained, the unique properties and capabilities of the reverse transcriptase enzymes involved in the DRT2 system are expected to provide "a valuable new capability to the genome engineering and synthetic biology toolbox."
Columbia University has filed a patent application related to the work, according to the paper.