Environmental Factor, January 2011, National Institute of Environmental Health Sciences
Crouch links RNase H to infectious and genetic diseases
By Jeffrey Stumpf
Like most guest speakers in the series, Crouch said he was especially honored to be invited by the fellows. (Photo courtesy of Steve McCaw)
Principal Investigator Thomas Kunkel, Ph.D., was one of several scientists who asked questions following the lecture. Like Kunkel himself, Crouch works at the intersection of molecular genetics and structural biology. (Photo courtesy of Steve McCaw)
The talk appealed to a range of scientists at NIEHS, including Nuclear Magnetic Resonance Group Center Manager Eugene DeRose, Ph.D., left, and Principal Investigator Bob London, Ph.D. (Photo courtesy of Steve McCaw)
Watt watched as several scientists took advantage of the question-and-answer session. She represented LMG trainees in hosting Dr. Crouch's presentation. (Photo courtesy of Steve McCaw)
A presentation by guest lecturer Robert Crouch, Ph.D. provided insight into an RNA cleaving enzyme, whose production he playfully called "the purpose in life." His talk at NIEHS Dec. 10 described cleavage of RNA/DNA hybrids by Ribonuclease H (RNase H), its importance to human health, and its potential applications in the treatment of genetic and environmental disease.
Crouch is the head of the Section on Formation of RNA National Institute of Child Health and Human Development (NICHD). His talk was the latest in the Laboratory of Molecular Genetics (LMG) Fellows Invited Lecture Series and was hosted by postdoctoral fellow Danielle Watt, Ph.D., a member of the DNA Replication Fidelity Group.
Crouch's talk reviewed both RNase H1 and H2 in archeabacteria, E. coli, yeast, mouse, and humans. The two enzymes differ by their substrate preference, determined by the number and location of ribonucleotides in the RNA component of the RNA/DNA hybrid.
An example of a natural RNA/DNA hybrid substrate occurs in the beginning of transcription. RNA is produced but still annealed to the template DNA, forming bubbles called R-loops that lead to genomic instability. Overexpressing RNase H1 can reduce the instability caused by R-loops. However, these R-loops may be a key intermediate in promoting the variety of antibodies needed for the immune system. Therefore, the RNase H expression may be regulated differently depending on the location of the R-loop.
Loss of RNase H1 leads to mitochondrial DNA depletion
Eliminating RNase H1 caused embryonic lethality in mice. Surprisingly, development is aborted because of the lack of mitochondria DNA (mtDNA) replication. Unlike other genes necessary for mtDNA replication, mutations in RNase H1 have not been identified in mitochondrial disease patients. NIEHS mtDNA researcher William Copeland, Ph.D., suggested, "The embryonic lethality and loss of mtDNA in the mouse knockout makes RNase H1 a good candidate for mitochondrial disease-associated mutations, despite its dual role in the nucleus."
Crouch showed two distinct forms of RNase H1 that differed by the presence of a mitochondrial targeting sequence at the beginning. The ratio of mitochondrial to nuclear RNase H1 may fluctuate in different cell types. "It is not yet clear that some cells need both forms," Crouch said. "Cells that are non-dividing may need mtDNA replication but not need the nuclear enzyme."
Linking active site biochemistry to treatments of HIV infection and genetic diseases
Detailed studies by Crouch of the active site of RNase H are extremely relevant to two major health-related topics:
- RNase H of HIV virus is required for production of infectious particles.
- Mutations in RNase H2 associate with Acardi Goutiéres Syndrome (AGS), a rare but fatal disease that mimics in utero viral infection.
HIV RNase H has yet to be successfully targeted for treating HIV/AIDS, but as Crouch pointed out, understanding the active site structure and function may aid this approach.
"So far all of these small molecule inhibitors have failed either because they do not enter cells or because the active site of RNase H is inaccessible to the drug," added Crouch. "The active site is almost always occluded by the presence of the viral RNA or replication intermediates."
The 28 known mutations in human RNase H2 that associate with AGS are located in every region of the gene. The mutations located in the active site likely inhibit cleavage of or accessibility to the RNA/DNA hybrid. Mutations far from the catalytic domain may affect protein stability or interactions with unknown cofactors.
But how does RNase H2 play an essential role in what appears to be an immune disease? The answer, Crouch suggested, may help scientists understand how the immune system responds to different types of DNA.
"RNase H2 may insure that some substrates do not elicit the innate immune response," Crouch explained. "In some cases, RNase H2 may have a role in protection from viruses or other detrimental nucleic acids."
(Jeffrey Stumpf, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group.)