Trans-splicing in C. elegans is known as a mechanism by which short, spliced leader sequences are spliced to the 5 ends of mRNAs. Trans-splicing between two mRNAs has not been observed in worms, though in Drosphila, the (mod)
mdg4 locus undergoes trans-splicing to form a functional protein. In mammals, rare chimeric transcripts have been found that are thought to be the consequence of aberrant splicing. Here we describe apparent trans-splicing between the mRNAs of two genes in C. elegans that each, when mutated, produce an Enhanced RNAi (Eri) response:
eri-6 and
eri-7. We identified multiple alleles of two adjacent eri genes residing on opposite DNA strands. cDNA analysis revealed that
eri-6 and
eri-7 exist as two separate pre-mRNAs and as a single mature mRNA encoding a helicase. The pre-mRNAs are likely to be trans-spliced in order to form a functional mRNA, orthologous to the mRNA encoded by a single gene in several, other natural isolates of C. elegans and in C. briggsae. Trans-splicing may be facilitated by base pairing of the two pre-mRNAs over a repeated sequence that flanks
eri-6.
eri-6 and -7 mutants were isolated in a forward genetic screen that sought to identify negative regulators of post-transcriptional silencing and/or transcriptional gene silencing. Looking for mutants that show an enhanced silencing of an
unc-47::gfp transgene upon gfp feeding RNAi, we identified mutations that generally enhance RNAi responses to exogenous dsRNA. We isolated a class of eri mutants, including
eri-6 and -7, that are distinct from previously characterized eri mutants. This class of mutants does not show the ts-sterility phenotype seen in mutants of
eri-1-5 and
rrf-3. Also, unlike another class of eri mutants that includes
lin-35 and
lin-9, these mutants are not SynMuvB. While the ERI-6/7 helicase suppresses exogenous RNAi, it is required for endogenous siRNAs. Like
eri-1 mutants,
eri-6/7 mutants are hyper-responsive to injected siRNAs. Epistasis analysis shows that
rde-1,
rde-4 and
rrf-1 are epistatic to
eri-6/7, suggesting that
eri-6/7 acts upstream or in parallel to these rde genes; analysis of
eri-1;
eri-7 doubles indicates that
eri-1 and
eri-7 may act in the same pathway. Together, these data and published data on ERI-1 complexes suggest that ERI-6/7 and ERI-1 are part of distinct complexes but may function in the same pathway negatively regulating exogenous RNAi.