siRNA Biogenesis and Silencing
RNA interference is a conserved biological response to double-stranded RNA that regulates gene expression. The response is mediated by small interfering RNAs (siRNAs), which guide the sequence-specific degradation of cognate messenger RNAs (mRNAs). Our group has structurally characterized and mechanistically defined events associated with processing of long double-stranded RNAs into siRNAs by the endonuclease activity of Dicer and guide-strand-mediated cleavage of target RNAs by Argonaute (Ago), the key component exhibiting slicer activity, within the RNA-induced silencing complex (RISC).
We have written a review with John van der Oost (Wangeningen University) and Eugene Koonin (NIH) on a comparison between eubacterial and eukaryotic RNAs (Swarts et al. 2014).
Swarts, D. C. et al., Koonin, E. V., Patel, D. J. & van der Oost, J. (2014). The evolutionary journey of Argonaute proteins. Nat. Struct. Mol. Biol. 21, 743-753.
Dicer Enzymes. In a collaborative study with the David Bartel lab (MIT), we demonstrated that budding yeast Dicers initiate processing in the interior and work outwards in contrast to canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini (Weinberg et al. 2011).
Weinberg, D., Nakanishi, K., Patel, D. J. & Bartel, D. P. (2011). The inside-out mechanism of Dicers from budding yeasts. Cell 146, 262-276.
In another study in collaboration with the Narry Kim lab (Seoul National University), we demonstrated that 5’-phosphorylated end recognition by Dicer is important for precise and effective biogenesis of miRNAs (Pqrk et al. 2011; Tian et al. 2014).
Park, J. E., Heo, I., Tian, Y., Simanshu, D. K., Chang, H., Jee, D., Patel, D. J. & Kim, V. N. (2011). Dicer recognizes the 5’-end of RNA for efficient and accurate cleavage. Nature 475, 201-205.
Tian, Y., Simanshu, D. K., Ma, J. B., Park, J-E, Heo, I., Kim, V. N. & Patel, D. J. (2014). A phosphate-binding pocket within the platform-PAZ-connector helix cassette of human Dicer. Mol. Cell 53, 606-616.
Eubacterial Argonaute Enzymes. Ago proteins constitute a key component of the RNA-induced silencing complex (RISC), contributing to both the architectural and catalytic functionalities associated with siRNA guide strand selection within the RISC loading pathway, and subsequent guide strand-mediated cleavage of complementary mRNA by catalytically competent RISC. Our structure-function studies on Agos undertaken in collaboration with the Thomas Tuschl lab (Rockefeller University) established that eubacterial Agos are bonafide guide DNA strand-mediated site-specific RNA endoribonucleases (Yuan et al. 2005), a conclusion subsequently verified in collaboration with the John van der Oost lab (Wangeningen University) by the demonstration that these ssDNA guides are plasmid derived and function in host defense by DNA-guided DNA interference (Swarts et al. 2014).
Yuan, Y. R., et al., Tuschl, T. & Patel, D. J. (2005). Crystal structure of Aquifex aeolicus Argonaute provides unique perspectives into the mechanism of guide strand-mediated mRNA cleavage. Mol. Cell 19, 405-419.
Swarts, D. C. et al., Patel, D. J., Berenguer, J., Brouns, S. J. & van der Oost, J. (2014). DNA-guided DNA interference by prokaryotic Argonaute. Nature 507, 258-261.
Further studies demonstrated anchoring of the 5’-phosphorylated and 2-nt 3’-ends in the MID-PIWI and PAZ pockets respectively (Ma et al. 2015), while positioning bases 2 to 6 of the DNA guide strand for nucleation-mediated pairing with the mRNA target (Ma et al. 2014).
Ma, J-B., Ye, K. & Patel, D. J. (2004). Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429, 318-322.
Ma, J. B., Yuan, Y. R., Meister, G., Pei, Y., Tuschl, T. & Patel, D. J. (2005). Structural basis for 5’-end-specific recognition of the guide RNA strand by the A. fulgidus PIWI protein. Nature 434, 666-670.
Notably, we observed pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3’-end of the guide from the PAZ pocket (Wang et al. 2008, 2009).
Wang, Y., Sheng, G., Juranek, S., Tuschl, T. & Patel, D. J. (2008). Structure of the guide-strand-containing argonaute silencing complex. Nature 456, 209-213.
Wang, Y., Juranek, S., Li, H., Sheng, G., Wardle, G. S., Tuschl, T. & Patel, D. J. (2009). Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes. Nature 461, 754-761.
Sheng, G., Zhao, H., Wang, J., Rao, Y., Tian, W., Swarts, D. C, van der Oost, J., Patel, D. J. & Wang, Y. (2014). Structure-based cleavage mechanism of T. thermophiles Argonaute DNA guide strand-mediated DNA target cleavage. Proc. Natl. Acad. Scis. USA. 111, 652-657.
Eukaryotic Argonaute Enzymes. In a collaborative effort with the David Bartel lab (MIT), we demonstrated that activation of Ago for target RNA cleavage requires repositioning of loop, thereby inserting an invariant glutamate into the catalytic pocket for formation of a catalytically competent tetrad alignment (Nakanishi et al. 2012). Finally, higher resolution structures of Ago complexes have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg2+ cations and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for Ago-mediated target cleavage by a RNase H-type mechanism (Sheng et al. 2014).
Nakanishi, K., Weinberg, D. E., Bartel, D. P. & Patel, D. J. (2012). Structure of yeast Argonaute with guide RNA. Nature 486, 368-374.
The role of Ago phosphorylation was highlighted in the demonstration that EGFR modulated miRNA maturation in response to hypoxia (Shen et al. 2013).
Shen, J. et al., Patel, D. J. & Hung, M. C. (2013). EGFR modulates miRNA maturation in response to hypoxia through phosphorylation of Ago2. Nature 497, 383-387.
Viral Suppressors. Viruses have organized a counter-defense strategy whereby their genomes encode proteins that specifically suppress siRNA-mediated degradation. Our studies on the p19 viral suppressor from tombusvirus establish that a pair of a-helical ‘reading heads’ project from opposite ends of the p19 homodimer and position pairs of tryptophans for stacking over the terminal base pairs, thereby measuring and bracketing both ends of the siRNA duplex (Ye et al. 2003). In a collaborative study championed by the Nam-Hai Chua lab (Rockefeller University), it was demonstrated that that the Cucumber mosaic virus-encoded 2b protein blocks Ago1 cleavage activity to inhibit miRNA pathways, attenuate RNA silencing and counter host defense (Zhang et al. 2006).
Ye, K., Malinina, L. & Patel, D. J. (2003). Recognition of siRNA by a viral suppressor of RNA silencing. Nature 426, 874-878.
Zhang, X., Yuan, Y-R., Pei, Y., Tuschl, T., Patel, D. J. & Chua, N-H. (2006). Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis AGO1 cleavage activity to counter plant defense. Genes Dev. 20, 3255-3268.
Germline-specific piRNAs and Piwi proteins play a critical role in genome defense against transposable elements, thereby protecting the genome against transposon-induced defects in gametogenesis and fertility. Current efforts are addressed at understanding the role of proteins and associated RNAs in mechanisms underlying piRNA biogenesis and transposon suppression.
Ping-Pong Cycle. In collaborative projects championed by the Alexei Aravin lab it has been shown that Krimper interacts directly with Aubergine and Ago3 to coordinate the assembly of the ping-pong piRNA processing complex (Webster et al. 2015) and that Cutoff, a part of the RDC complex, composed of Rhino, Deadlock and Cutoff, prevents premature termination of piRNA precursor transcription, by suppressing cleavage of pre-miRNA at canonical polyA sites (Chen et al. 2016).
Webster, A. et al., Patel, D. J. and Aravin, A. A. (2015). Aub and Ago3 are recruited to nuage through two mechanisms to form a ping-pong complex assembled by Krimper. Mol. Cell 59, 564-575.
Chen, Y-C. A. et al., Patel, D. J., Smibert, C. A., Lipshitz, H. D., Toth, K. F. and Aravin, A. A. (2016). Cutoff suppresses RNA polymerase II termination to ensure expression of piRNA precursors. Mol. Cell 63, 97-109.
SFiNX Complex. In collaborative projects championed by the Julius Brennecke lab (IMBA-Vienna), it was shown that SFiNX (silencing factor interacting nuclear export variant), an interdependent protein complex composed of Nxf2-Nxt1 and Panaromix is required for Piwi-mediated cotranscriptional silencing in Drosophila (Batki et al 2019). Further, dynein light chain protein Cutup mediates SFiNX dimerization and forms molecular condensates in a nucleic acid-stimulated manner (Schnabl et al. 2021). In addition, Piwi-mediated stabilization of the corepressor complex SFiNX on chromatin leads to SUMOylation of its Panoramix subunit (Andreev et al. 2022).
Batki, J. et al., Patel, D. J. and Brennecke, J. (2019). The SFINX complex licenses piRNA-guided heterochromatin formation. Nat. Struct. Mol. Biol. 26,720-731.
Schnabl, J. et al., Patel, D. J. & Brennecke, J. (2021). Molecular principles of Piwi-mediated cotranscriptional silencing through the dimeric SFiNX complex. Genes Dev. 35, 392-409.
Andreev, V. I. et al., Patel, D. J. & Brennecke, J. (2022). Panaromix SUMOylation on chromatin connects the piRNA pathway to the cellular heterochromatin machinery. Nat. Struct. Mol. Biol. 29, 130-142.
RNAs undergo chemical modifications that can affect their activity, localization and stability. The current challenge championed by the Narry Kim lab (Seoul National University) has been to understand the molecular enzymatic machineries and biological functions of uridylation (U-tail) by TUTases (Lim et al. 2015) and adenylation (A-tail) by Wispy at the 3’-end of RNA (Lee et al. 2015).
Lim, J., Ha, M., Chang, H., Kwon, S. C., Simanshu, D. K., Patel, D. J. & Kim, V. N. (2015). Uridylation by TUT4 and TUT7 marks mRNA for degradation. Cell 159, 1365-1376.
Lee, M. et al., Patel, D. J. & Kim, V. N. (2015). Adenylation of maternally inherited microRNAs by Wispy. Mol Cell 56, 696-707.