Extended Data Figure 5. Expression of synthetic siRNAs in paf1-Q264Stop cells is sufficient to trigger stable repression of protein coding genes in trans.

a, Left: The paf1-Q264Stop mutation was introduced into cells that express synthetic ura4-hp siRNAs¹⁵. Right: Wild type (paf1+) and paf1-Q264Stop were grown in the presence or absence of 5-FOA. Red arrow indicates paf1-Q264Stop colonies growing on FOA-containing medium. Note that these colonies could be propagated in non-selective medium without losing the repressed state. b, In S. pombe, artificial tethering of the RITS complex to mRNA expressed from the endogenous ura4+ locus using the phage λN protein results in de novo generation of ura4+ siRNAs. These siRNAs load onto RITS and are necessary to establish heterochromatin at the ura4+ locus in cis. However, like ura4-hp siRNAs, they are incapable of triggering the repression of a second ura4+ locus in trans¹⁸. To test whether ura4+ siRNAs produced as a result of Tas3λN tethering to ura4+::5BoxB mRNA (chromosome III) can act in trans to silence a second ura4+ allele (leu1Δ::ura4+, chromosome II), paf1+ was mutated and ura4+ repression was assessed by FOA silencing assays. Whereas 5-FOA was toxic to both paf1+ and paf1-Q264Stop cells in the absence of ura4+ siRNAs (Tas3 not fused to λN), FOA resistant colonies appeared upon Tas3-λN tethering, demonstrating that siRNAs generated from the ura4+::5BoxB locus can initiate repression of the second ura4+ copy expressed from the leu1+ locus. Notably, siRNA-mediated ura4+ repression in trans was more pronounced in the absence of the RNAse Eri1. We have previously shown that the levels of ura4+::5BoxB-derived siRNA are higher in eri1Δ cells⁴¹. We note that trans-silencing of the second ura4+ allele occasionally occurs in paf1+ cells in the absence of Eri1¹⁸. However, in contrast to paf1-Q264Stop cells, the repressed state of ura4+ is not stably propagated. Hairpin symbols downstream of the ura4+ ORF denote BoxB sequences. They form stem loop structures when transcribed and are bound by the λN protein. c, ade6+ silencing assay demonstrating that also the endogenous ade6+ gene is repressed if ade6-hp siRNAs are expressed from the nmt1+ locus in paf1-Q264Stop cells. Silencing assay was performed with two freshly generated (naïve) paf1-Q264Stop mutant strains. A few white colonies in which heterochromatin has not yet formed are discernable. Such white colonies were picked to determine heterochromatin initiation frequencies shown in figure 2.