Transgene-mediated post-transcriptional gene silencing is inhibited by 3′ non-coding sequences in Paramecium

Abstract

Homology-dependent gene silencing is achieved in Paramecium by introduction of gene coding regions into the somatic nucleus at high copy number, resulting in reduced expression of all homologous genes. Although a powerful tool for functional analysis, the relationship of this phenomenon to gene silencing mechanisms in other organisms has remained obscure. We report here experiments using the T4a gene, a member of the trichoeyst matrix protein (TMP) multigene family encoding secretory proteins, and the ND7 gene, a single copy gene required for exocytotic membrane fusion. Silencing of either gene leads to an exocytosis-deficient phenotype easily scored on individual cells. For each gene we have tested the ability of different combinations of promoter, coding and 3′ non-coding regions to provoke silencing, and analyzed transcription and steady-state RNA in the transformed cells. We provide evidence that homology-dependent gene silencing in Paramecium is post-transcriptional and that both sense and antisense RNA are transcribed from the transgenes, consistent with a role for dsRNA in triggering silencing. Constructs with and without promoters induce gene silencing. However, transgenes that contain 3′ non-coding regions do not induce gene silencing, despite antisense RNA production. We present a model according to which different pathways of RNA metabolism compete for transcripts and propose that the relative efficiencies of dsRNA formation and of 3′ RNA processing of sense transgene transcripts determine the outcome of transformation experiments.