The Nucleolar Ultra-structure And Pre-rRNA Biogenesis

The nucleolus is an important nuclear membrane-less organelle in mammalian cells that is primarily associated with ribosome biogenesis as well as additional functions such as regulation of mitosis,cell-cycle progression,proliferation and many forms of stress response.The function of nucleolus is highly coupled with its ultrastructure,however,due to the limitation of research tools,the ultrastructure of the nucleus is not well understanded,which greatly limits the research of the nucleolar functions.In this doctoral dissertation,we investigated the ultrastructure of human nuclei and the regulation of the biological production of ribosomal RNA precursors using a variety of advanced imaging techniques combined with biophysical and molecular biological experimental methods.In this dissertation,we optimized the fixation method in imaging in order to promote the study of the ultrastructure in the nucleolus and the arrangement patterns of the nucleolar RNA.With the introduction of glyoxal,we improved the contrast and signal background ratio of the RNA signal in fluorescence in situ hybridization and enabled the combination of multiple imaging methods without affecting the ultrastructure of the nuclear membrane-less organelles.Using optimized imaging methods,we resolved the ultrastructure of the nucleolus in human live cells under super resolution microscopy and found that the DFC region was non-uniform but consisted of 18-24 clusters formed by processing factors.We found that the DDX21-ring structure located in the DFC region can be regulated by the long noncoding RNA SLERT and associated with Pol I transcription.We used super resolution microscopy to identify r DNA transcription sites in situ in human cells,the fine localization of pre-r RNAs,and found that pre-r RNAs have directional translocation within the FC/DFC transcription unit which named nascent pre-r RNAs sorting.By screening,we demonstrated that the nucleolar protein Fibrillarin plays a critical role in the process of nascent pre-r RNAs sorting to the DFC,and the length of its intrinsically disordered region determines the strength of FBL self-association thus directly affecting the pre-r RNA sorting ability of FBL,which further facilitated the assembly of the DFC region of the nucleoli.This work systematically resolved the ultrastructure of human nuclei,investigated the regulation of nucleolar structure and pre-r RNA transcription by long non-coding RNAs,proposed a model in which phase separation promotes the directed translocation of newly generated r RNA precursors,and found that the process of nascent pre-r RNAs sorting can promote the assembly of the DFC in human nucleoli.