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

Homology-dependent gene silencing is achieved in Paramecium by introduction of gene coding regions into the somatic nucleus at high copy number, resul ting in reduced expression of all homologous genes. Although a powerful too l for functional analysis, the relationship of this phenomenon to gene sile ncing mechanisms in other organisms has remained obscure. We report here ex periments using the T4a gene, a member of the trichoeyst matrix protein (TM P) multigene family encoding secretory proteins, and the ND7 gene, a single copy gene required for exocytotic membrane fusion. Silencing of either gen e leads to an exocytosis-deficient phenotype easily scored on individual ce lls. For each gene we have tested the ability of different combinations of promoter, coding and 3' non-coding regions to provoke silencing, and analyz ed transcription and steady-state RNA in the transformed cells. We provide evidence that homology-dependent gene silencing in Paramecium is post-trans criptional and that both sense and antisense RNA are transcribed from the t ransgenes, consistent with a role for dsRNA in triggering silencing. Constr ucts with and without promoters induce gene silencing. However, transgenes that contain 3' non-coding regions do not induce gene silencing, despite an tisense RNA production. We present a model according to which different pat hways of RNA metabolism compete for transcripts and propose that the relati ve efficiencies of dsRNA formation and of 3' RNA processing of sense transg ene transcripts determine the outcome of transformation experiments.