Small non-coding RNA扮演調控基因，影響轉譯的重要角色。miRNA (micro RNA)已知結合在mRNA的3’UTR，阻止mRNA轉譯或使其直接降解，抑制基因表現。piRNA (piwi-interacting RNA)主要存在生殖腺，本研究分析線蟲中的piRNA，長度為21nt，開頭序列為U (簡稱21U-piRNA)。此類21U-piRNA所結合的mRNA分為兩類，分別是WAGO target mRNA及CSR-1 target mRNA，當結合至WAGO target mRNA，會招募RdRP (RNA-dependent RNA polymerase)至結合位點周遭，誘發22G-RNA (長度22~23，開頭為G的小RNA)，並與WAGO蛋白形成複合物，使WAGO target mRNA降解或在細胞核即無法轉錄；CSR-1 target mRNA則不受piRNA結合影響，仍能正常表達。
本研究探討piRNA是否如miRNA一樣偏好結合在mRNA的3’UTR區域，或是有其他偏好結合區域；並更進一步探討其在WAGO target mRNA及CSR-1 target mRNA誘發22G-RNAs的分布，藉此給予piRNA調控機制更好的詮釋，提供給生物學家明確的研究方向及實驗設計。
分析piRNA結合位點分布以及22G-RNAs分布，需使用CLASH實驗資料、iCLIP實驗資料以及small RNA-seq實驗資料，並搭配本研究室自行開發之工具，從定序資料中擷取出small RNA-mRNA交互對以及結合位點分布資訊。再經由工具一:結合位點/22G-RNA密度觀察mRNA三區域 (5’UTR、CDS、3’UTR)中的piRNA位點及22G-RNA分布情形；以及工具二:以單核苷酸解析度觀察start/stop codon周遭，piRNA位點與22G-RNA的實際累積分布情形。
經以上兩種工具分析，miRNA偏好結合在目標mRNA的3’UTR區域，與已知符合，因此強化對這兩種工具的信心程度。piRNA不論對於WAGO target mRNA與CSR-1 target mRNA，都偏好結合在CDS區域，與miRNA截然不同。在WAGO target mRNA上，也在CDS誘發最多WAGO 22G-RNA。
然而，尚有更多發現，本研究在WAGO target mRNA中亦發現CSR-1 22G-RNA，並且也在CDS區域分布最多；在CSR-1 target mRNA中，發現WAGO-1 22G-RNA跟WAGO-9 22G-RNA都有被產生，亦分布在CDS較多，但是密度較在WAGO target mRNA中低，CSR-1 22G-RNA則是分布在3’UTR最多。
因此推論piRNA結合在WAGO target mRNA中能誘發WAGO-1、WAGO-9、CSR-1 22G-RNA，且CDS區域最多；piRNA結合在CSR-1 target mRNA中亦能誘發WAGO-1、WAGO-9 22G-RNA，且CDS區域最多，而CSR-1 22G-RNA則是因EGO-1在3’UTR誘發，所以在3’UTR分布最多，推測若將EGO-1破壞，則可能就在CDS分布最多。綜合以上，若具備足夠實驗資料及分析結果，滿足上述假設，本研究提出模型:piRNA應不論WAGO target mRNA或CSR-1 target mRNA都偏好結合至CDS區域，且結合後也誘發WAGO-1、WAGO-9、CSR-1 22G-RNA，mRNA受到調控、被降解與否的關鍵在piRNA誘發WAGO-1、WAGO-9的能力差別，以及是否有如EGO-1機制或其他機制誘發更多CSR-1 22G-RNA保護CSR-1 target mRNA。

Small non-coding RNA plays an important role in RNA interference (RNAi), which regulating genes and affecting translation. miRNA (micro RNA) is known to bind to the 3’UTR of mRNA to inhibit gene expression. piRNA (piwi-interacting RNA) mainly expressed in germline cell P-granule. In C. elegans, piRNA bind two kinds of mRNA, namely WAGO target mRNA and CSR-1 target mRNA. When piRNA bind to WAGO target mRNA, it will degrade WAGO target mRNA. On the other hands, CSR-1 target mRNA is protected by CSR-1 protein so as to normally express. piRNA preferential binding region (5’UTR, CDS, 3’UTR) remains unknown. This study discover that piRNA preferentially bind to CDS both on WAGO target mRNA and CSR-1 target mRNA, which is completely different from miRNA. And on the WAGO target mRNA, piRNA also induce most WAGO 22G-RNAs at CDS. However, there are more findings. On WAGO target mRNA, CSR-1 22G-RNA was also found and distributed most at CDS. On CSR-1 target mRNA, WAGO-1 22G-RNA and WAGO-9 22G-RNA are discovered and also distribute most as CDS, CSR-1 22G-RNA distribute most at 3’UTR. Therefore, we infer that piRNA have the most binding sites at CDS on WAGO target mRNA, and can also induce most WAGO-1, WAGO-9 and CSR-1 22G-RNA. As for CSR-1 target mRNA, piRNA have the most binding sites at CDS, and can also induce most WAGO 22G-RNA, while CSR-1 22G-RNA is enriched at 3’UTR. The CSR-1 22G-RNA enrichment at 3’UTR mostly induced by EGO-1 protein, if we mutant the EGO-1 protein, we suspect that CSR-1 22G-RNA might distribute most at CDS.

[1] D. Moazed. Small RNAs in transcriptional gene silencing and genome defence, Nature, vol. 457, no. 7228, pp. 413-20, 2009.
[2] A. S. Pickford and C. Cogoni. RNA-mediated gene silencing, Cell Mol Life Sci, vol. 60, no. 5, pp. 871-82, 2003.
[3] M. Tijsterman, R. F. Ketting, and R. H. Plasterk. The genetics of RNA silencing, Annu Rev Genet, vol. 36, no., pp. 489-519, 2002.
[4] M. Wakiyama, K. Takimoto, O. Ohara, and S. Yokoyama. Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system, Genes Dev, vol. 21, no. 15, pp. 1857-62, 2007.
[5] A. J. Pratt and I. J. MacRae. The RNA-induced silencing complex: a versatile gene-silencing machine, J Biol Chem, vol. 284, no. 27, pp. 17897-901, 2009.
[6] D. H. Kim and J. J. Rossi. Strategies for silencing human disease using RNA interference, Nat Rev Genet, vol. 8, no. 3, pp. 173-84, 2007.
[7] David P Bartel, MicroRNAs: Genomics, Biogenesis, Mechanism, and Function, Cell, vol. 116, no. 2, pp. 281-297, 2014.
[8] Shen EZ, Chen H, Ozturk AR, Tu S, Shirayama M, Tang W, Ding YH, Dai SY, Weng Z, Mello CC. Identification of piRNA Binding Sites Reveals the Argonaute Regulatory Landscape of the C. elegans Germline, Cell, vol. 172, no. 5, pp. 937-951.e18 ,2018.
[9] Singh, M., Cornes, E., Li, B. et al. Translation and codon usage regulate Argonaute slicer activity to trigger small RNA biogenesis, Nat Commun, vol. 12, no. 1, pp. 3492, 2021.
[10] G. Kudla, S. Granneman, D. Hahn, J. D. Beggs, and D. Tollervey. Cross-linking, ligation, and sequencing of hybrids reveals RNA-RNA interactions in yeast, Proc Natl Acad Sci U S A, vol. 108, no. 24, pp. 10010-5, 2011.
[11] J. Ule, K. B. Jensen, M. Ruggiu, A. Mele, A. Ule, and R. B. Darnell. CLIP identifies Nova-regulated RNA networks in the brain, Science, vol. 302, no. 5648, pp. 1212-5, 2003.
[12] D. D. Licatalosi, A. Mele, J. J. Fak, J. Ule, M. Kayikci, S. W. Chi, T. A. Clark, A. C. Schweitzer, J. E. Blume, X. Wang, J. C. Darnell, and R. B. Darnell. HITS-CLIP yields genome-wide insights into brain alternative RNA processing, Nature, vol. 456, no. 7221, pp. 464-9, 2008.
[13] Helwak A, Kudla G, Dudnakova T, Tollervey D. Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding, Cell, vol. 153, no. 3, pp. 654-665, 2013.
[14] Mückstein U, Tafer H, Hackermüller J, Bernhart S, Stadler P, Hofacker I. Thermodynamics of RNA-RNA binding, Bioinformatic, vol. 22, no. 10, pp. 1177–1182, 2006.
[15] Wei-Sheng Wu, Wei-Che Huang, Jordan S Brown, Donglei Zhang, Xiaoyan Song, Hao Chen, Shikui Tu, Zhiping Weng, Heng-Chi Lee, pirScan: a webserver to predict piRNA targeting sites and to avoid transgene silencing in C. elegans, Nucleic Acids Research, vol. 46, no. W1, pp. W43-W48, 2018.
[16] John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. Human MicroRNA targets, PLoS Biol, vol. 2, no. 11,pp. e363, 2004.