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Project 13. Regulation and specificity of RNase H1 enzymes.

An R-loop is an RNA-DNA hybrid that displaces the second DNA strand. R-loops occur frequently in genomes and have significant physiological importance, regulating gene expression and telomere stability. On the other hand, R-loops are a source of genomic instability e. g. in diseases associated with repetitive sequences including amyotrophic lateral sclerosis, Fragile X syndrome, Friedreich’s ataxia and trinucleotide repeat sequences. Several pathways regulate the stability of R-loops. RNase H1 and RNase H2 are the central endonucleases that process RNA specifically in RNA-DNA hybrids. Loss of either enzyme results in an accumulation of R-loops and genome instability. Conversely, overexpression of RNase H1 is frequently used to counteract the toxic effects of R-loop-associated DNA damage. What regulates the role and activity of the RNase H1 and RNase H2 enzymes at “regulatory” and “toxic” R-loops is largely unknown and mechanistic studies are complicated by the pleiotropic effects of the deletion mutants. Interestingly C. elegans has four RNase H1 enzymes and a single RNase H2 enzyme (Fig. 1a).

 

Figure 1A. C. elegans encodes 4 RNase H1 enzymes that are differentially regulated under stress.

In this project we want to characterize these enzymes to understand:

  1. Is there a natural separation of function for the RNase H1 enzymes?
  2. How are the RNase H enzymes regulated during development, stress, and aging?
  3. What is the binding specificity of the four different RNase H1 enzymes?

We are using C. elegans genetics and the established genomics approaches to compare the role of the five RNase H1/H2 enzymes, in wild-type worms and mutants with increased toxic R-loop levels. This allows us to compare the role of the RNase H enzymes at different sequence contexts, like repetitive sequences, telomeres and protein-coding genes. Taken together this project utilizes a potential natural separation of function of the C. elegans RNase H1 enzymes to study their diverse roles in R-loop processing during stress, aging and development.