In C. elegans, 22G-RNA (22G), a.k.a. secondary siRNA, is the effector molecule of RNAi, while primary siRNA, 26G-RNA, and 21U-RNA serve as the trigger for initiating 22G. Our previous studies have demonstrated that all the germline genes in C. elegans are more or less regulated by 22Gs in two major pathways: the WAGO pathway and CSR-1 pathway. In the former, 22G binds multiple WAGO Argonautes to regulate non-functional genes and transposons, and the biogenesis is dependent on EKL-1, DRH-3 and RNA-dependent RNA polymerase (RdRP), MUT-7, and RDE-3; in the latter, 22G binds CSR-1 Argonaute to regulate many functional genes, and the biogenesis is dependent on EKL-1, DRH-3 and RdRP but not on MUT-7 and RDE-3. In both pathways, 22Gs are mapped to the whole length of the target transcripts. We have identified a new 22G pathway, which specifically targets the 3' UTRs of thousands of germline transcripts, most of which are also targeted by CSR-1 but along the whole length. These 3' UTR-associated 22Gs binds some WAGOs but not CSR-1 or the CSR-1 paralog C04F12.1. However, the biogenesis is not dependent on RDE-3, suggesting this is not a typical WAGO or CSR-1 pathway. These 3'UTR 22Gs bind WAGO-9 but not WAGO-1 since: 1) these 22Gs are highly enriched in WAGO-9 IP in the
csr-1 mutant and weakly enriched in the WAGO-9 IP in the WT worm, but not in WAGO-1 IP in either strain; 2) some of these 22Gs are depleted in the
wago-9 csr-1 mutant but not in
wago-1 csr-1 mutant. Other WAGOs may also play roles in binding these 22Gs. The 3' UTR targeting suggests that the biogenesis may be associated with protein translation since cells may only recognize the 3' UTR using protein translation. Since CSR-1 also binds 22Gs mapped to the whole size of these target transcripts, the CSR-1 22Gs may represent a biogenesis process irrelevant to protein translation. These 22Gs may play an important role in regulating germline genes since the
wago-9 and
csr-1 mutant has a much more severe sterile phenotype (no eggs at all) than the
csr-1 alone mutant (~30 eggs/worm). Among the target mRNAs regulated most by these CSR-1/WAGO-9 22Gs, ~1/3 are also targeted by the
mir-58 family, which may also primarily regulate its targets by inhibiting translation. In summary, this is a new small RNA pathway which specifically targets the 3'UTRs of functional germline genes and its functions and biogenesis may be associated with translation.