RNAi Functions in Adaptive Reprogramming of the Genome
The regulation of transcribing DNA into RNA, including the production, processing, and degradation of RNA transcripts, affects the expression and the regulation of the genome in ways that are just beginning to be unraveled. A surprising discovery in recent years is that the vast majority of the genome is transcribed to yield an abundance of RNA transcripts. Many transcripts are regulated by the exosome, a multi-protein complex that degrades RNAs, and may also be targeted, under certain conditions, by the RNA interference (RNAi) pathway. These RNA degrading activities can recruit factors to silence certain regions of the genome by condensing the DNA into tightly-packed heterochromatin. For some chromosomal regions, such as centromeres and telomeres, which lie at the center and ends of chromosomes, respectively, silencing must be stably enforced through each cell generation. For other regions, silencing mechanisms must be easily reversible to activate gene expression in response to changing environmental or developmental conditions. Thus, the regulation of gene silencing is key to maintaining the integrity of the genome and proper cellular expression patterns, which, when disrupted can underlie many diseases, including cancer.
Discovering the mechanisms by which cells integrate various signals to regulate expression of the genome remains an important area of research. A recent study showing that RNAi and the exosome can each act to silence centromeres suggested that they could have additional overlapping targets. This idea prompted Soichiro Yamanaka, Ph.D., a postdoctoral fellow working with Shiv Grewal, Ph.D., in CCR’s Laboratory of Biochemistry and Molecular Biology, and their colleagues to embark on a genome-wide search for novel targets of RNAi in the fission yeast Schizosaccharomyces pombe.
The researchers used high-throughput sequencing of small RNAs isolated from yeast lacking Rrp6, the main RNA degrading subunit of the exosome. In these cells, clusters of small RNAs were identified, which were between 20 and 24 nucleotides in length (the approximate size of siRNAs produced by RNAi) and which targeted protein coding genes, including genes for transmembrane domain-containing proteins and genes activated during sexual differentiation, as well as mobile DNA elements called retrotransposons. The generation of these small RNAs was dependent on Ago1, a protein essential for RNAi, supporting their identification as bona fide siRNAs.
Importantly, the siRNA clusters aligned with regions containing the histone 3 lysine 9 methylation (H3K9me) mark, a modification required for heterochromatin formation. This modification also depended on Ago1. The researchers further concluded that heterochromatin and RNAi proteins cooperate to generate the siRNA clusters, because they found that Clr4, a protein essential for generating the H3K9me modification, is also required for siRNA production in exosome deficient cells.
With the identification of these novel RNAi targets, located within domains they termed HOODs (heterochromatin domains), the research team next wondered whether the exosome and RNAi pathways could function together to silence these target regions. They found that, while defects in either pathway alone had little effect on gene silencing, double mutants lacking Rrp6 and an RNAi or a heterochromatin protein significantly increased transcript levels at target regions, indicating that both the exosome and RNAi pathways are necessary for complete silencing of these target genes.
The researchers noted that several gene regions produced transcripts that were known targets of the RNA surveillance factor Red1, which interacts with the poly(A) polymerase Pla1 and the poly(A) binding protein Pab2. Intriguingly, they found that Pla1 and Pab2 are required for siRNA cluster formation and H3K9me at most genes. However, the requirement for Red1 was not universal, indicating that Pla1 and Pab2 may form a common core that recruits various surveillance associated proteins, such as Red1 or other factors, to feed transcripts into the exosome or RNAi pathway.
A novel finding from this work is that the formation of HOODs at particular gene locations can be dynamic and initiated or reversed in response to environmental or developmental conditions. For instance, in wild type yeast, the researchers observed the formation of HOODs when cells were stressed by low temperature or a limited carbon or glucose supply. These results indicate that HOODs, and in particular their regulation, are likely to be important for normal cell growth and survival. Loss of Ago1 impaired both H3K9me and siRNA cluster formation, reinforcing the essential role of RNAi in these processes. In addition, the researchers found that certain established HOODs could be disassembled. In this case, they found that when sexual differentiation was induced, the levels of H3K9me and siRNAs at developmental genes decreased. This effect was specific to developmental genes, since there was little effect at Tf2 retrotransposon regions in response to the differentiation signal. This result revealed the ability of RNAi to not only function in the formation of HOODs, but to selectively regulate them to allow expression of genes required for sexual differentiation while protecting genome integrity by ensuring that mobile elements remain silenced.
The mechanisms underlying HOOD formation in fission yeast also appear to play a role in gene silencing in other organisms, as the researchers observed the accumulation of small RNAs, called piRNAs, in Drosophila fly larvae that lack Rrp6. Thus, this work has revealed a potentially conserved mechanism by which cells can regulate heterochromatin at specific regions throughout the genome. Importantly, this adaptive mechanism alters gene expression in ways that allow cells to respond to cellular and environmental signals. These findings have important implications for understanding genome reprogramming and its role in maintaining the integrity and function of mammalian cells.Summary Posted: 12/2012
RNAi triggered by specialized machinery silences developmental genes and retrotransposons. Yamanaka S, Mehta S, Reyes-Turcu FE, Zhuang F, Fuchs RT, Rong Y, Robb GB, Grewal SI.Nature. 2012 Nov 14. PubMed Link