Untersuchung der Dynamik von Riboproteonuklein-Komplexen an naszenter mRNA mit dem RNA-Interactome-Capture-Verfahren
Zusammenfassung der Projektergebnisse
Messenger RNA (mRNA) serves as the intermediary that carries genetic information, encoded in the nucleus, to the protein synthesis machinery located in the cytoplasm. From the moment of transcription, mRNAs associate with numerous RNA-binding proteins to form ribonucleoprotein complexes (RNPs), which help to coordinate RNA processing activities and protect the mRNA from degradation. RNP remodeling helicases facilitate the dynamic re-arrangement and release of RNA-binding proteins associated with RNPs and are essential for proper gene expression, ensuring that newly synthesized mRNAs are correctly processed, exported, and ultimately translated. A major subgroup of helicases that can actively drive RNP remodeling are DEAD-box ATPases. This project aimed to identify early RNP remodeling events mediated by DEAD-box ATPases in fission yeast. To track the evolution of the RNP coat on newly transcribed mRNAs, we initially attempted a combination of pulse-chase labeling, kinetic crosslinking, and enrichment of RNA - protein crosslinks. However, in our hands these methods were not selective enough to yield data with comprehensive coverage with the short labeling times required. As an alternative strategy, we purified nuclear RNPs by affinity purification of tandem-affinity tagged components of the nuclear cap-binding complex. This approach revealed that depleting the essential DEAD-box ATPase Dbp2 increases the association of nuclear RNPs with structural components of chromatin - such as histones - suggesting a delay in RNP release from chromatin after mRNA production. Results from this project contributed to identifying Dbp2 as the active component of an RNP remodeling checkpoint, where release of RNPs from chromatin serves as a crucial regulatory output. According to our model, Dbp2 is essential for releasing and recycling components of the cleavage and polyadenylation complex - a key component of the RNA 3'-end processing machinery - from the fully processed mRNA, which otherwise blocks its export. Moreover, comparative proteomics data indicated that chromatin-retained RNPs interact with chromatin remodeling complexes that decondense chromatin to activate transcription, suggesting transcriptional feedback when RNP assembly is disrupted. In summary, our study underscores the important role of RNP remodeling helicases, particularly Dbp2, in coordinating RNA processing and export, establishing chromatin retention of RNPs as a crucial layer of nuclear RNA quality control that ensures aberrant transcripts are effectively retained and controlled.
Projektbezogene Publikationen (Auswahl)
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DEAD-box ATPase Dbp2 is the key enzyme in an mRNP assembly checkpoint at the 3’-end of genes and involved in the recycling of cleavage factors. Nature Communications, 15(1).
Aydin, Ebru; Schreiner, Silke; Böhme, Jacqueline; Keil, Birte; Weber, Jan; Žunar, Bojan; Glatter, Timo & Kilchert, Cornelia
