RNA biogenesis is a complex multi-step process mediated by an array of factors that ensure efficient and accurate gene expression (Perales and Bentley, 2009
). Here we show that an additional function for many of these factors is to limit the formation of RNA-DNA hybrids to prevent them from altering genome structure. We find that in addition to elongation and splicing factors, the prevention of hybrid formation is a function of factors required for transcriptional repression, RNA export, and RNA degradation. Thus deleterious interactions between RNA and its template DNA are possible at multiple stages co- and post-transcriptionally. However, we find that not all factors involved in a particular step of RNA biogenesis or within a single complex are needed to prevent hybrid formation. This functional division of transcription factors infers mechanistic functions within RNA processes and complexes yet to be discovered.
How can defects in such diverse aspects of mRNA biogenesis lead to RNA-DNA hybrids? We propose that all these mutants make nascent transcripts more accessible to DNA, potentially by different underlying mechanisms. Mutants affecting the loading of RNA binding proteins onto RNA, may uncoat the RNA and allow for inappropriate interactions with target duplex DNA. Mutants in this class are the PAF complex (Cdc73p and Leo1p) and Spt2p, which are involved in transcriptional elongation and recruit cleavage/polyadenylation factors to RNA (Luna et al., 2005
; Hershkovits et al., 2006
). A similar model was proposed for the THO complex, which is required for co-transcriptional recruitment of the RNA binding proteins Yra1p and Sub2p (Rondón et al., 2010
). Alternatively, mutants defective in processes such as RNA degradation by Kem1p and the TRAMP complex (Air1p, Rrp6p and Trf4p), or subsequent export (Npl3p), may increase the half-life of RNA in the nucleus, hence increasing the time that hybrids can form.
Using our cytological assay we show that hybrids form at many genomic sites in cells compromised for RNAse H or RNA biogenesis factors. Thus, many sites in the genome must be prone to hybrid formation. In recent years, genome-wide studies have shown that widespread transcription occurs from coding and noncoding regions in yeast and higher eukaryotes, much of it originating from sites of cryptic transcription (Cheung et al., 2008
; Wyers et al., 2005
). Numerous transcription and degradation factors have been identified as required for the repression of cryptic transcription, including Bur2p, Cdc73p, Spt2p, Sin3p, Med13p, Cdk8p, Med12p, Rrp6p and Trf4p. We found these genes also suppress hybrid-mediated GCRs, and propose that expression from intergenic promoters may be regulated by RNA biogenesis factors because of the risk cryptic transcription poses to genomic integrity.
Our genetic and cytological data indicates that sites of hybrid formation vary depending upon cellular defect. This is exemplified by the difference in effect on GCR and direct-repeat recombination observed in PAF mutants versus THO mutants, respectively (this work, Gómez-González et al., 2009
). Similarly, the sin3Δ
mutant but not med13Δ
enhances hybrid formation and instability of rDNA repeats. Evidence in yeast indicates that Sin3p may indirectly control polymerase II transcription within rDNA spacer regions (Ciocl et al., 2003
and Smith et al., 1999
). While the functional consequence of this has been unclear, our work suggests it may be critical to prevent polymerase II transcription in the rDNA locus to maintain repeat homeostasis. It will be interesting to know whether other RNA biogenesis mutants also exhibit such locus-enriched formation of hybrids.
Despite their conservation, little is known about the function of nuclear-encoded RNAses H. Recently, RNase H2 has been shown to be involved in the removal of single ribonucleotides misincorporated in duplex DNA (Kim et al., 2011
). We find that compromising RNase H function leads to the accumulation of RNA-DNA hybrids, indicating that RNase H must act as part of a surveillance system to remove hybrids that form as a result of spontaneous errors in RNA biogenesis in wild-type cells. However, it is likely that other mechanisms must exist to resolve RNA-DNA hybrids and/or any resulting DNA damage into non-deleterious outcomes, since the robust levels of hybrids in almost all rnh1Δrnh201Δ
cells leads to GCRs in only 1% of cells.
Strikingly, our studies of the kinetics of hybrid-induced Rad52 foci indicate that hybrids can stimulate DNA damage outside of S phase. Rad52 foci are generally equated with DSBs, but we cannot rule out that these foci mark other types of damage or unusal DNA structures. A number of previous observations had suggested that all endogenous sources of DSBs were linked obligatorily to S phase progression. In WT cells, spontaneous Rad52-GFP foci appear almost exclusively during S phase (Lisby et al., 2001
). In addition, all replication and DNA damage checkpoint mutants increase foci formation in S and G2/M, but not G1 cells. However, recent genetic evidence suggests that 40% of spontaneous mitotic recombination events reflect repair of a G1-inititated DSB (Lee et al., 2009
). We suggest these G1 induced breaks may occur by RNA-DNA mediated hybrids that occur infrequently enough in WT cells so as not to be detected by foci. Induction of DSBs in G1 is a potentially potent cause of changes in the genome as it eliminates the ability to use the sister chromatid as a repair template.
The effects of perturbing RNA biogenesis and RNase H function on GCRs are comparable to those observed in mutants of DNA damage and repair processes, demonstrating the importance of regulating transcription and RNA processing for maintaining genome structure. This finding presents an alternative model for the molecular mechanisms by which genome rearrangements associated with cancer may occur. First, because the number of RNA biogenesis genes that can be mutated to promote hybrids is so large, perturbations of RNA biogenesis are a potentially robust source for genome instability. Second, previous models have suggested that genome instability in cancer cells leads to the changes in transcription profiles necessary for cancer progression (Hanahan and Weinberg, 2000
). However, our work suggests that changes in the transcriptional profile of cells, and ensuing hybrid formation, may precede and in fact be a causative factor in the generation of chromosomal rearrangements. Intriguingly, a recent study suggests that transcription is causative agent for recurrent translocations in prostate cancer cells (Lin et al., 2009
). It will be interesting to see whether these translocations are associated with the formation of an RNA-DNA hybrid in the region. Analysis of rearrangements and transcriptional profiles in other cancer types will further elucidate potential contributions of the transcriptional machinery and RNA-DNA hybrids in promoting DNA rearrangements.