Stalled Spliceosomes Are a Signal for RNAi-Mediated Genome Defense

Using the yeast Cryptococcus neoformans, we describe a mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We show that intron-containing mRNA precursors template siRNA synthesis. We identify a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrate that it physically associates with the spliceosome. We find that RNAi target transcripts are distinguished by suboptimal introns and abnormally high occupancy on spliceosomes. Functional investigations demonstrate that the stalling of mRNA precursors on spliceosomes is required for siRNA accumulation. Lariat debranching enzyme is also necessary for siRNA production, suggesting a requirement for processing of stalled splicing intermediates. We propose that recognition of mRNA precursors by the SCANR complex is in kinetic competition with splicing, thereby promoting siRNA production from transposon transcripts stalled on spliceosomes. Disparity in the strength of expression signals encoded by transposons versus host genes offers an avenue for the evolution of genome defense. [Display omitted] ► Endogenous siRNA is templated by mRNA precursors in Cryptococcus neoformans ► siRNA production requires the spliceosome-associated SCANR complex ► RNAi targets show suboptimal splicing features and accumulate on spliceosomes ► Spliceosomal stalling and lariat debranchase are required for siRNA accumulation In the yeast Cryptococcus neoformans, siRNAs are produced from mRNA precursors that contain suboptimal splicing signals, causing accumulation of these RNAs at spliceosomes. The stalled spliceosomes are a signal recognized by the RNA-silencing machinery, revealing a function for the splicing apparatus in genome defense.