Trans-splicing in C. elegans generates the negative RNAi regulator ERI-6/7

Abstract

Mutations that enhance the response to double-stranded RNA (dsRNA) have revealed components of the RNA interference (RNAi) pathway or related small RNA pathways. To explore these small RNA pathways, we screened for Caenorhabditis elegans mutants displaying an enhanced response to exogenous dsRNAs. Here we describe the isolation of mutations in two adjacent, divergently transcribed open reading frames (eri-6 and eri-7) that fail to complement. eri-6 and eri-7 produce separate pre-messenger RNAs (pre-mRNAs) that are trans-spliced to form a functional mRNA, eri-6/7. Trans-splicing of eri-6/7 is mediated by a direct repeat that flanks the eri-6 gene. Adenosine to inosine editing within untranslated regions of eri-6 and eri-7 pre-mRNAs reveals a double-stranded pre-mRNA intermediate, forming in the nucleus before splicing occurs. The ERI-6/7 protein is a superfamily I helicase that both negatively regulates the exogenous RNAi pathway and functions in an endogenous RNAi pathway.

Figure 1. Theeri-6/7 locus in various strains and its gene products in C. elegans N2

a, Structure of eri-6/7 in C. elegans N2 and the syntenic region in C. briggsae AF16, based on RT-PCR, 3″-RACE and WormBase. The eri-7 operon is conserved. C. briggsae eri-6 is encoded on the opposite strand from C. elegans and is one gene with C. briggsae eri-7 (CBG03999). Exons are black or red rectangles, depending on the strand on which they are encoded. Open rectangles: predicted exons, but unconfirmed. Striped exons: discovered experimentally. eri-6 splices to eri-7 24 nucleotidesupstream of the predicted eri-6 stop codon. b, Relative orientation of eri-6 and eri-7 in four C. elegans isolates. Blue arrows: the approximate 930-bp direct repeat flanking eri-6. Orange arrowheads: 25-bp inverted repeats.

Figure 2. The eri-6/7 mRNA is formed by local trans-splicing

a, Schematic of eri-6/7 locus used for rescue: 9.1-kb fragment; and 8.2-kb fragment missing the second repeat. b, Rescue of eri-6/7(mg411)-induced silencing of the mgIs30 transgene-conferred rolling phenotype. Number of broods scored = 28, 28, 18, 19, 38, 29 and 18, respectively. Multiple transgenic lines assayed. Error bars: s.e.m. c, The eri-6 and eri-7 promoters express in overlapping tissues (hypodermis and head neurons). eri-6 and eri-7 promoters were fused to rfp and gfp, respectively. d, 5′-RACE experiments show that eri-6 and eri-7 are expressed as separate pre-mRNAs with sufficient nucleotide homology to base-pair, thus facilitating trans-splicing. eri-7 pre-mRNA starts 775 nucleotides (nt) upstream of exon 1, whereas eri-6 pre-mRNA starts 100 nt upstream of eri-6 exon 1 (black arrows). Edited nucleotides (A to G indicative of A to I) in eri-7 pre-mRNA are in bold capital letters. Blue arrows: direct repeats. Capital letters: exon sequence. Curved line: trans-splicing juncture. e, Model of eri-6/7 trans-splicing. eri-6 and eri-7 pre-mRNAs are locally co-transcribed in the same cells. The pre-mRNAs may form a dsRNA intermediate that facilitates co-transcriptional trans-splicing.

Figure 3. Chimeric ERI-6/7 protein is expressed through trans-splicing

a, RT-PCR on gfp-expressing worms using primers in gfp (G) and eri-6 (E1 and E2). Sequencing of the RT-PCR products confirmed trans-splicing. b, Expression of the trans-splicing reporter in ASK. c, Western blot on animals expressing the trans-splicing reporter. A GFP antibody was used. The slower-migrating product (of the expected weight of ERI-6::GFP) is presumably ERI-6::GFP. The faster-migrating product may be GFP expressed using an alternative start codon. d, DiO fills five pairs of sensory neurons. e, ERI-6/7::mCherry is expressed in one amphid neuron. f, Merged image identifies ASK as the ERI-6/7-expressing neuron. d, e, f,

Figure 4. ERI-6/7 is required for endogenous RNAi

Shown are northern blots probed for two different endogenous siRNAs, targeting the genes T01A4.3 and K02E2.6. Mutants of additional genes known to be required for endogenous RNAi (dcr-1, drh-3 and rde-4) are shown as controls. 5S rRNA is shown as a loading control.