In Caenorhabditis elegans, the Piwi-interacting small RNA (piRNA)-mediated germline surveillance system encodes more than 30,000 unique 21-nucleotide piRNAs, which silence a variety of foreign nucleic acids. What mechanisms allow endogenous germline-expressed transcripts to evade silencing by the piRNA pathway? One likely candidate in a protective mechanism is the Argonaute CSR-1, which interacts with 22G-small RNAs that are antisense to nearly all germline-expressed genes. Here, we use an in vivo RNA tethering assay to demonstrate that the recruitment of CSR-1 to a transcript licenses expression of the transcript, protecting it from piRNA-mediated silencing. Licensing occurs mainly at the level of transcription, as we observe changes in pre-mRNA levels consistent with transcriptional activation when CSR-1 is tethered. Furthermore, the recruitment of CSR-1 to a previously silenced locus transcriptionally activates its expression. Together, these results demonstrate a rare positive role for an endogenous Argonaute pathway in heritably licensing and protecting germline transcripts.

SummarySmall RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in Caenorhabditis elegans. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense.

Proper regulation of the germline transcriptome is essential for fertility. In C. elegans, germline homeostasis hinges on a complex repertoire of both silencing and activating small RNA pathways, along with RNA processing. However, our understanding of how fundamental RNA processing steps intersect with small RNA machineries in the germline remains limited. Here, we link the conserved intron binding protein, EMB-4/AQR/IBP160, to the CSR-1 and HRDE-1 nuclear 22G-RNA pathways in the C. elegans germline. Loss of emb-4 leads to distinct alterations in CSR-1- versus HRDE-1-associated small RNA and mRNA transcriptomes. Our transcriptome-wide analysis shows that EMB-4 is enriched along pre-mRNAs of nearly 8,000 transcripts. While EMB-4 complexes are enriched for both intronic and exonic sequences of HRDE-1 targets, CSR-1 pathway targets are enriched for intronic, but not exonic, sequences. These data suggest that EMB-4 could contribute to a molecular signature that distinguishes the targets of these two germline small RNA pathways.

Lymphocyte infiltration into epithelial tissues and proinflammatory cytokine release are key steps in autoimmune disease. Although cell-autonomous roles of lymphocytes are well studied in autoimmunity, much less is understood about the stromal factors that dictate immune cell function. Tissue inhibitor of metalloproteinases 3 (TIMP3) controls systemic cytokine bioavailability and signaling by inhibiting the ectodomain shedding of cytokines and their receptors. The role of TIMP3 in cytokine biology is emerging; however, its contribution to cellular immunology remains unknown. In this study, we show that TIMP3 produced by the hepatic stroma regulates the basal lymphocyte populations in the liver and prevents autoimmune hepatitis. TIMP3 deficiency in mice led to spontaneous accumulation and activation of hepatic CD4+, CD8+, and NKT cells. Treatment with Con A in a model of polyclonal T lymphocyte activation resulted in a greatly enhanced Th1 cytokine response and acute liver failure, which mechanistically depended on TNF signaling. Bone marrow chimeras demonstrated that TIMP3 derived from the stromal rather than hematopoietic compartment provided protection against autoimmunity. Finally, we identified hepatocytes as the major source of Timp3 in a resting liver, whereas significant Timp3 gene transcription was induced by hepatic stellate cells in the inflamed liver. These results uncover metalloproteinase inhibitors as critical stromal factors in regulating cellular immunity during autoimmune hepatitis.