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J Am Chem Soc ; 145(34): 18968-18976, 2023 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-37596976


We report the development of a self-renewable tag (srTAG) for protein fluorescence imaging. srTAG leverages the "on-protein" fluorophore equilibrium between the fluorescent zwitterion and non-fluorescent spirocyclic form and the reversible fluorescence labeling to enable self-recovery of fluorescence after photobleaching. This small-sized srTAG allows 2-6 times longer imaging duration compared to other commonly used self-labeling tags and is compatible with fluorophores with different spectral properties. This study provides a new strategy for fine tuning of self-labeling tags.

Corantes Fluorescentes , Imagem Óptica , Ionóforos , Fotodegradação
Cell ; 186(13): 2865-2879.e20, 2023 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-37301196


Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.

RNA , Retroelementos , RNA/metabolismo , Clivagem do DNA , DNA Polimerase Dirigida por RNA/metabolismo , Transcrição Reversa
Cell ; 186(15): 3245-3260.e23, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37369203


Terrestrial organisms developed circadian rhythms for adaptation to Earth's quasi-24-h rotation. Achieving precise rhythms requires diurnal oscillation of fundamental biological processes, such as rhythmic shifts in the cellular translational landscape; however, regulatory mechanisms underlying rhythmic translation remain elusive. Here, we identified mammalian ATXN2 and ATXN2L as cooperating master regulators of rhythmic translation, through oscillating phase separation in the suprachiasmatic nucleus along circadian cycles. The spatiotemporal oscillating condensates facilitate sequential initiation of multiple cycling processes, from mRNA processing to protein translation, for selective genes including core clock genes. Depleting ATXN2 or 2L induces opposite alterations to the circadian period, whereas the absence of both disrupts translational activation cycles and weakens circadian rhythmicity in mice. Such cellular defect can be rescued by wild type, but not phase-separation-defective ATXN2. Together, we revealed that oscillating translation is regulated by spatiotemporal condensation of two master regulators to achieve precise circadian rhythm in mammals.

Relógios Circadianos , Camundongos , Animais , Relógios Circadianos/genética , Ritmo Circadiano/fisiologia , Núcleo Supraquiasmático/metabolismo , Processamento de Proteína Pós-Traducional , Mamíferos