Small interfering RNAs (siRNAs) can target the RNA interference (RNAi) machinery to homologous chromosomal regions where it induces chromatin modifications and transcriptional silencing. Two groups now report that, in fission yeast, RNA polymerase II (pol II) is required for RNAi-mediated chromatin modifications and gene silencing.

In fission yeast, transcription at the centromeric regions generates siRNAs that are loaded into the RNAi targeting machinery. To determine whether RNAi-mediated chromatin modifications require homologous transcripts, Robin Allshire and colleagues constructed a yeast strain (Ter+) that contained a modified ura4 gene. This construct contained a transcription terminator immediately upstream of a gene region that is homologous to a synthetic hairpin RNA (which is processed into siRNAs by the RNAi machinery). A second strain (Ter-M5) contained a mutation in this terminator, which allowed read-through, resulting in 75% full-length transcripts.

In Ter+ cells, truncated transcripts of the ura4 gene were detected, both in the presence and absence of hairpin RNA. In Ter-M5 cells, however, the presence of hairpin RNA resulted in the loss of full-length ura4 transcripts. In addition, the authors detected dimethylation on Lys9 of histone 3 (H3K9me2) on the Ter-M5 ura4 gene, but only in the presence of hairpin RNA. So, transcription is required for gene repression and chromatin modification that is triggered by the presence of siRNAs.

Why is transcription essential for RNAi-mediated chromatin modification and gene silencing? Could it be that an RNA polymerase is required to allow access of siRNAs to the template DNA/homologous region? Possibly — however, Allshire and co-workers found that transcription mediated by bacteriophage T7 polymerase did not allow RNAi-mediated chromatin modification and gene silencing, which implies that a specific RNA polymerase is required.

Next, the authors constructed a strain with a truncated C-terminal domain (CTD) in the largest subunit of RNA pol II (Rpb1). This strain, which did not have a substantial general defect in transcription, was unable to silence the expression of two centromeric marker genes and showed decreased levels of H3K9me2 associated with centromeric repeats. In addition, the Allshire team showed that pol II immunoprecipitated with the RNAi component Argonaute 1 (Ago1), and that this association required an siRNA-loaded RNAi complex. Also, Ago1 associated with centromeric chromatin, and this depended on pol II transcription.

In a complementary report, Yota Murakami and colleagues also showed the requirement for pol II in RNAi-mediated heterochromatin formation. They identified a fission yeast mutation, which they mapped to the second largest subunit of pol II (Rpb2). The mutant showed an accumulation of centromeric transcripts, and a loss of heterochromatic histone modifications and siRNAs. This indicates that pol II might be required for the production of siRNAs. However, the Allshire group did not observe an effect on siRNA generation, and instead proposes that the role of pol II transcription lies downstream — in mediating the RNAi-dependent chromatin modifications.

These findings provide yet another example of the core role of pol II in coordinating transcription with other RNA-processing events. Future studies will no doubt try to establish the precise links between pol II transcription in RNAi-dependent chromatin modification and transcriptional silencing.