Introns reduce transitivity proportionally to their length, suggesting that silencing spreads along the pre-mRNA.

Endogenes rarely support transitive silencing, whereas most transgenes generally allow the spread of silencing to occur along the primary target. To determine whether the presence of introns might explain the difference, we investigated the influence of introns in the primary target on 3'–5' silencing transitivity. When present in a transgene, an intron-containing endogene fragment does not prohibit the spread of silencing across this fragment, indicating that introns do not preclude silencing transitivity along endogenes. Also, a multiple intron-containing genomic gene fragment that had previously been shown not to support transitivity in an endogenous context could support transitivity when present in a transgene. Nevertheless, genomic intron-containing fragments delayed the onset and diminished the efficiency of transitive silencing of a secondary target compared with the corresponding cDNA fragments. Remarkably, transitivity was impaired proportionally with the length of the pre-mRNA, and not of the mRNA. The latter result suggests that the RNA dependent RNA polymerase-based spreading of silencing progresses along the non-spliced rather than the fully processed mature mRNA.