Research Summary

    The mechanistic underpinnings of RNAi are broadly conserved across eukaryotes. Initial successes utilizing small RNAs to target oncogenic and viral mRNAs have generated excitement that small RNAs may eventually be utilized to treat human diseases. Prior to the rational use of small RNAs in therapeutics, it is essential to understand their biogenesis, specificity, transportation, and endogenous roles.

(1) The identification of nuclear RNAi in C. elegans.

    We are very interested in how small RNAs are transported and regulated, andhow they function in the nucleus in metazoan. To address these questions, we conducted a genetic screen to identify factors required for nuclear RNAi in the model organism C. elegans and have identified three new genes termed nuclear RNAi defective (NRDE)-1/2/3. We have also identified a bifurcation of the nuclear and cytoplasmic RNAi pathway, a novel small RNA transport pathway, and a novel nucleargene silencing mechanism.

    NRDE-3 is an Argonaute. Argonaute proteins are known to bind small RNAs recognizing complementary cellular RNAs via Watson-Crick base pairing and inhibit gene expression by a variety of mechanisms. Our work revealed that these Argonaute proteins can also escort small RNAs to their distinct subcellular compartments to silence target genes. In the absence of small RNAs, NRDE-3 resides in the cytoplasm. NRDE-3 transports small RNAs from the cytoplasm to the nucleus and associates with nascent transcripts generated by RNA polymerase II. This is the first demonstration that a specific Argonaute protein has transportation activity in addition to silencing .



(2) Nuclear RNAi mediates pre-mature transcription termination.

    Our recent work also established a novel nuclear gene silencing pathway in which small RNAs act in conjunction with these newly identified NRDE genes to terminate RNA polymerase II-mediated transcription by pausing the polymerase during its elongation phase. By combining a variety of genetic and biochemical assays, we have found a small RNA and NRDE-dependent silencing of pre-mRNAs 3’ to sites of RNAi, accumulation of RNA Polymerase II at genomic region targeted by RNAi, and decreases in RNA polymerase II occupancy and transcription activity 3’ to sites of RNAi. These experiments demonstrated that metazoan small RNAs elicit a co-transcriptional gene-silencing program, and can act as trans-acting terminators of RNA polymerase II.

       Taken together, this research indicates that metazoans use a different mechanism other than A. thaliana and S. pombe to silence gene expression in the nucleus. Further understanding how small RNAs function in the nucleus via NRDEs may permit more stable and specific inhibition of gene expression and facilitate advancement of both basic research and therapeutics.



(3) Nuclear RNAi-induced epigenetic modifications.

       Ribonucleoprotein comodelmplexes consisting of Argonaute-like proteins and small regulatory RNAs function in a wide range of biological processes. Many small regulatory RNAs are thought to function, at least in part, within the nucleus to regulate transcription and modify chromatin. We showed that dsRNA triggers H3K27 trimethylation in a sequence-dependent manner, which can be maintained and inherited to progenies for multiple generations. This modification requires the Nrde pathway in Caenorhabditis elegans. Endogenous small RNAs, including, but not limited to, NRDE-3 and HRDE-1-associated endo-siRNAs, induce H3K27me3 in an NRDE-dependent manner. Small RNA-mediated H3K9me3 and H3K27me3 have distinct genetic requirements and different roles in RNAi. The H3K27 methyltransferase, mes-2, is likely to be involved in small RNA-induced H3K27me3.



(4) Identificaiton of antisense ribosomal siRNA and the Susi pathway.

       For a long time, small fragments of ribosomal RNA have been widely considered by the research community as non-specific degradation products and neglected as garbage sequence. Little is known about their biological processes, functions and regurisiRNA_modellatory mechanisms. GUANG's team found that a new class of antisense ribosomal siRNAs (risiRNAs) downregulate pre-rRNA through a nuclear RNAi pathway. The risiRNAs are sensitive to environmental stimuli and gene mutations. When a gene SUSI-1(ceDis3L2) is mutated, risiRNAs are dramatically increased. Interesting, this SUSI-1(ceDis3L2) mutation is also identified in a human disease called Perlman's syndrome. Therefore, this study not only discovered a new mechanism to maintain RNA steady state in cells, but also is of great significance for the treatment of human Perlman's disease.



(5) The mechanism of transgenerational inheritance of RNAi.

     RNAi-elicited gene silencing is heritable and can persist for multiple generationRNAi inheritances after its initial induction in C. elegans. However, the mechanism by which parental-acquired trait-specific information from RNAi is inherited by the progenies is not fully understood. We identified a cytoplasmic Argonaute protein, WAGO-4, necessary for the inheritance of RNAi. WAGO-4 exhibits asymmetrical translocation to the germline during early embryogenesis, accumulates at the perinuclear foci in the germline, and is required for the inheritance of exogenous RNAi targeting both germline- and soma-expressed genes. WAGO-4 binds to 22G-RNAs and their mRNA targets. Interestingly, WAGO-4-associated endogenous 22G-RNAs target the same cohort of germline genes as CSR-1 and contain untemplated addition of uracil at the 3' ends. The poly(U) polymerase CDE-1 is required for the untemplated uridylation of 22G-RNAs and inheritance of RNAi. Therefore, we conclude that, in addition to the nuclear RNAi pathway, the cytoplasmic RNAi machinery also promotes RNAi inheritance.



(6) Functional proteomic approach identified USTC and PICS complexes requried for piRNA biogenesis.

     Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and non-self nucleic acids, maintain genome integrity, and are essential for fertility in a variety of organisms. In C. elegans most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units. While a few genes have been shown to be required for piRNA biogenesis the mechanism of piRNA transcription remains elusive. We used functional proteomics approaches to identify an upstream sequence transcription complex (USTC) and a piRNA and chromosomal segregation (PICS) complex that are essential for piRNA biogenesis. The USTC complex contains PRDE-1, SNPC-4, TOFU-4 and TOFU-5. The USTC complex form a unique piRNA foci in germline nuclei and coat the piRNA cluster genomic loci. USTC factors associate with the Ruby motif just upstream of type I piRNA genes. The PICS complex contains TOFU-6, PID-1, PICS-1, ERH-6 and TOST-1 that localize to the germline P-granules and engage in piRNA maturation.







(7) Developing CRISPR/Cas9 technologies for genome manipunation in C. elegans.

The CRISPR/Cas9 technology has been successfully applied for gene editing and chromosome engineering. In the past few years, We have developed the CRISPR/Cas9 techlogies to (a) delete large genomic fragments via dual sgRNAs; (b) induce chromsomal translocations using sgRNAs targeting different chromosomals; (c) induce chrosomal inversions covering thepairing centers by the combinational usage of CRISPR/Cas9 and Cre/LoxP means; and (d) generate a protocol to knock out essential genes in C. elegans.