The germline employs sophisticated mechanisms that provide genome-wide surveillance of gene expression. One such mechanism involves the piRNA/PIWI Argonaute pathway which scans every germline transcript to detect foreign sequences and initiate synthesis of secondary small RNAs (22G-RNAs). These 22G-RNAs, engage both nuclear and cytoplasmic effector Argonautes (WAGOs) that coordinate and maintain mRNA and transcriptional silencing. We were surprised, however, to find that certain transgenes such as a
cas-9::gfp, were silenced in the germline even when the Piwi pathway and an essential WAGO-pathway factor
rde-3 were disarmed by mutations. Prompted by the finding that inserting introns could circumvent this silencing (Frokjaer-Jensen et al., 2016), we decided to ask if removing introns from an expressed transgene would induce its silencing. Indeed, the introduction of a
cdk-1::gfp transgene lacking introns led to potent germline-specific silencing that did not require Piwi or RDE-3 activity. To probe this phenomenon, we set up a forward genetic screen and found that nuclear RNAi defective (
nrde-1,
nrde-2,
nrde-4, and
wago-9/hrde-1) mutants were able to desilence
cas9::gfp. Interestingly, however, these nrde mutants only caused desilencing within the distal region of the germline. The failure of
rde-3 mutants to desilence
cas9::gfp suggests that NRDE-factors can employ an additional RDE-3 independent mechanism to produce 22G-RNAs. Consistent with this idea, small RNA sequencing revealed that
cas9::gfp, and the gfp portion of an intronless
cdk-1::gfp fusion gene are targeted by 22G-RNAs, and that a large spike in reads occurs in the region of the first
cas9 exon. Taken together these findings suggest that the NRDE-pathway is recruited downstream of an as yet unknown mechanisms that senses abnormalities (or insufficiency) in mRNA splicing. This surveillance is initiated independently of the Piwi pathway but requires the NRDE/WAGO-9 small RNA pathway. For example, without the NRDEs, perhaps silencing must be re-initiated each generation, hence a window of expression is limited to the distal region. These findings suggest that splicing abnormalities are sensed and then silenced epigenetically in the germline providing evidence for new layers of intricacy in the surveillance of germline gene expression.