Stalled Spliceosomes Are a Signal for RNAi-Mediated Genome Defense

Abstract

SUMMARY 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.

Conserved factor Dhp1/Rat1/Xrn2 triggers premature transcription termination and nucleates heterochromatin to promote gene silencing

“…In human cells, transcripts with defective splicing or impaired 3′-end formation are also prematurely terminated and cotranscriptionally degraded by the Dhp1 human homolog, Xrn2 (44). Given that splicing mutants affect the generation of small RNAs at loci containing cryptic introns in S. pombe (11,46) and that splicing is coupled to 3′-end formation in many cases (47)(48)(49)(50)(51), it is possible that the stalled spliceosome engages Dhp1/Xrn2 to trigger transcription termination and RNA degradation (44,52). This Dhp1-mediated early termination of cryptic intron-containing transcripts with its associated cotranscriptional degradation activity perhaps provides an early and effective way to suppress aberrant or untimely gene expression and supports the emerging view that the fate of the transcripts is determined during 3′-end formation (53).…”

The spliceosome as a transposon sensor

“…27 These observations raised the possibility that a kinetic competition determines transcripts from which small RNAs are produced. In this model, rapidly spliced transcripts are not targeted for dsRNA synthesis, whereas transposon transcripts, by virtue of their poor splicing kinetics, accumulate in spliceosomes and are targeted for dsRNA synthesis by SCANR (Fig.…”

“…21,27 Surprisingly, however, siRNAs map not only to the exons but also to the introns of their corresponding transcripts, implicating incompletely spliced mRNA precursors as substrates for siRNA biogenesis. These initial observations suggested that transposon-derived transcripts may be recognized as triggers of RNA silencing while still undergoing splicing in the nucleus.…”

Protecting genes from RNA silencing by destroying aberrant transcripts

“…Recently it was proposed that splicing is likely an important feature by which the cell senses foreign/aberrant transcripts. The study in human pathogenic yeast Cryptococcus neoformans shows that transposons tend to have sub-optimal splicing sites that may be sensed by spliceosomes and enhance siRNA biogenesis [9]. Indeed Arabidopsis intron-less genes have higher exon siRNA density, and introducing an intron into a GFP transgene suppresses spurious transgene silencing in plants [10].…”