基礎生物學門？醫工/骨科/幹細胞學門？ IACUC 實驗設計 編列研究助理 國外差旅: GRC, EV Asia conference, foreign scholars **延攬研究人員表格區
Aim 1
(Phenotypic characterization) Granule property: with or without DNMT3L_AT FRAPAim 2
Omics profilingAim 3
Spermiogenesis (or stress/ injury response) - Injury repair: working cell model—liver?12152024
G-quadruplex structures within the 3’UTR of LINE1 elements stimulate retrotransposition Sahakyan, A., Murat, P., Mayer, C. et al. Nat Struct Mol Biol 24, 243–247 (2017). https://doi.org/10.1038/nsmb.3367
- L1-originated quadruplex sequences (LQS): the most frequent LQS representative, referred to as LQS^ref, stemmed from the L1PA3 family d(GGGGACTGTTGTGGGGTGGGGGGAGGGGGGAGGG); r(GGGGACUGUUGUGGGGUGGGGGGAGGGGGGAGGG)
- “G-rich sequences are a hallmark of young L1 retrotransposons”
- “Mutations in 3′-UTR G4 motifs are coupled with L1 speciation”
- “G4-motif alteration modulates L1Hs mobility in cultured cells”
- “Stabilization of the 3′-UTR G4 motif stimulates L1Hs mobility”
- “Notably, L1 subfamilies harboring more stable G4s had higher genomic copy numbers, consistent with a link between G4 stability and L1 retrotransposition activity”
- Other: L1Hs-DNA-G4 d(GGGGACTGTGGTGGGGTCGGGGGAGGGGGGAGGG); r(GGGGACUGUGGUGGGGUCGGGGGAGGGGGGAGGG)
Visualizing liquid-liquid phase transitions https://doi.org/10.1101/2023.10.09.561572
- RNA G-quadruplex (rG4) recognized by G3BP1
Spinach RNA Aptamer sequence from PDB DOI: https://doi.org/10.2210/pdb4KZD/pdb NAKB: 4KZD (mouse) Spinach is an in vitro-selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence, with an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif
List of DNMT3L_AT-binding RNAs by prediction from catRAPID <-> rQ4 database <-> piRNA cluster database
- Through its interaction with the RNA G4 structure, DNMT3L_AT becomes indispensable for spermatogenic cells to circumvent genome-hazardous transposable elements (TEs) during meiosis by forming membrane-less germ granules where TE transcripts are sequestered, translation stalled, and subsequently processed into piRNAs.
- master regulator of xxx differentiation… functions to directly enhance the expression of the lineage-committed transcription factors required for the xxx cell subtypes.
- The response to different stressors converges at the induction of the integrated stress response (ISR), a common adaptive molecular pathway (Harding et al., 2003; Pakos-Zebrucka et al., 2016) that induces phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) to inhibit translation initiation and decrease global protein synthesis (Hinnebusch, 1994). In turn, mRNAs exiting the translational pool get localized in stress granules (SGs), membraneless organelles that are formed upon stress when translation of mRNAs gets arrested (Panas et al., 2016; Protter and Parker, 2016).
- SGs comprise several different types of biomolecules, particularly mRNAs and RNA-binding proteins, but their precise functions in mRNA storage or decay are not yet fully understood (Anderson and Kedersha, 2008; Marcelo et al., 2021). For spermatocytes (Meiosis, particularly homologous recombination, induces cellular stress?) (Chromatin opening? Dysregulated transcription, including TEs?) responding to the dual stressors coming from dysregulated transcription and de-repressed TE, germ cells may adapt a strategy to converge piRNA pathway into a specialized membrane-less organelle, the germ granule.
Is rG4 constitutional for D3L_AT germ granule formation? Will removal or destabilization of G4 structure disintegrate the IMC? Will addition of G4 structure containing RNA into recombinant D3L_AT condensate
Does a membrane platform need to underpin D3L_AT IMC? Or specific lipid metabolites derived from mitochondrial membranes stimulate condensation, e.g. PA catalyzed by mitoPLD from cardiolipin? - palmitoylation engineered D3L_AT to ectopically express at plasma membrane? - PalmGRET construction
title: DNMT3L_AT functions as a RNA-binding scaffold for germ granules assembly associated with mitochondria dynamics
title: DNMT3L_AT facilitates mitochondria-associated granule formation, essential for timely transcriptional and translational regulation throughout meiosis.
標題：無膜胞器新成員DNMT3L_AT對減數分裂中RNA和蛋白質表達及降解時機的精準調控
DNMT3L_AT binding to RNAs nucleates germ granule formation
G3BP2:CAPRIN (stress granule formation; polysome dissembled) <=> G3BP:USP10 (polysome reformed)
PABPN1 EIF4A:EIF4G:EIF4E EIF2-α (EIF2S1):EIF2-β (EIF2S2): EIF2-γ (EIFS3) complex for translation initiation; phosphorylation of eIF2alpha by HRI, GCN2, or PKR inhibits protein synthesis, leading to stress response; p-eIF2 is de-phosphorylated by PP1, which can be inhibited by PPP1R; therefore, PPP1R2 (Protein Phosphatase 1 Regulatory Inhibitor Subunit 2; or inhibitor 2, R2) stimulates stress granule formation??
CYFIP1-EIF4E-FMR1 complex binds to the mRNA cap for translational repression. (CYFIP1, NUFIP2, FXR1 <-> DNMT3L proximal)
CELF1 (CUGBP Elav-like family member 1) Component of an EIF2 complex at least composed of CELF1/CUGBP1, CALR, CALR3, EIF2S1, EIF2S2, HSP90B1 and HSPA5. Associates with polysomes *Inactivation of CUG-BP1/CELF1 Causes Growth, Viability, and Spermatogenesis Defects in Mice Kress, C., Gautier-Courteille, C., Osborne, H. B., Babinet, C., & Paillard, L. (2007). Molecular and cellular biology, 27(3), 1146–1157. https://doi.org/10.1128/MCB.01009-06 *RNA CUG-binding protein 1 increases translation of 20-kDa isoform of CCAAT/enhancer-binding protein beta by interacting with the alpha and beta subunits of eukaryotic initiation translation factor 2. Timchenko, N. A., Wang, G. L., & Timchenko, L. T. (2005). JBC 280(21), 20549–20557. https://doi.org/10.1074/jbc.M409563200
PNBP-2 (PNBPN1): Polyadenylate-binding protein 2
*A Translation-Activating Function of MIWI/piRNA during Mouse Spermiogenesis. Dai, P., Wang, X., Gou, L. T., … & Liu, M. F. (2019). Cell, 179(7), 1566–1581.e16. https://doi.org/10.1016/j.cell.2019.11.022
https://pubmed.ncbi.nlm.nih.gov/39333531/ Rojas-Ríos P, Chartier A, Enjolras C, et al. piRNAs are regulators of metabolic reprogramming in stem cells. Nat Commun. 2024;15(1):8405. Published 2024 Sep 27. doi:10.1038/s41467-024-52709-4 https://pubmed.ncbi.nlm.nih.gov/39312580/ Wei C, Yan X, Mann JM, et al. PNLDC1 catalysis and postnatal germline function are required for piRNA trimming, LINE1 silencing, and spermatogenesis in mice. PLoS Genet. 2024;20(9):e1011429. Published 2024 Sep 23. doi:10.1371/journal.pgen.1011429 https://pubmed.ncbi.nlm.nih.gov/39271704/ Ramat A, Haidar A, Garret C, Simonelig M. Spatial organization of translation and translational repression in two phases of germ granules. Nat Commun. 2024;15(1):8020. Published 2024 Sep 13. doi:10.1038/s41467-024-52346-x https://academic.oup.com/biolreprod/article/99/4/773/4985832 Yaoyao Wu, Kaibiao Xu, Huayu Qi, Domain-functional analyses of PIWIL1 and PABPC1 indicate their synergistic roles in protein translation via 3′-UTRs of meiotic mRNAs, Biology of Reproduction, Volume 99, Issue 4, October 2018, Pages 773–788, https://doi.org/10.1093/biolre/ioy100 https://pubmed.ncbi.nlm.nih.gov/31835033/ Dai P, Wang X, Gou LT, et al. A Translation-Activating Function of MIWI/piRNA during Mouse Spermiogenesis. Cell. 2019;179(7):1566-1581.e16. doi:10.1016/j.cell.2019.11.022