3D/4D Dynamic of UBTF1 and UBTF2 during Nucleolar Inactivation at the Structural and the Ultrastructural Level

Author: Pavle Tchelidze
Co-authors: L. Rusishvili, D. Ploton
Annotation:

The membrane-less structure, known as nucleolus, is the largest and densest nuclear compartment, where the molecular and structural factories of ribosome biogenesis are properly associated and tightly coordinated. Posed as ribosomal RNA genes (r-genes) localization and transcription sites the nucleolus has become recognized as a unique model to study the spatial organization of actively transcribing mammalian genes in the functional and dynamic association with overall structure and compaction of interphase chromatin. Here, we focused on two, still enigmatic questions, concerning 4D dynamics of the nucleolus and NCs (step one) and ultrastructural 3D organization of r-chromatin and its reorganization during inactivation of r-genes transcription (step two). The precondition for step 1 was the ability of FCs and DFC to move and fuse within a crowded nucleolar volume, revealed by early EM studies on artificial inactivation of r-genes including the experiments where Actinomycin D (AMD) was first used as an inhibitor. Meanwhile, mechanisms underlying spatial displacement of these NCs at molecular and structural levels have not been elucidated so far. Our hypothesis was that the dynamics of nucleolar components during nucleolar segregation (NS) induced by AMD may be caused by a concerted gathering of nucleolar associated chromatin pulling FCs and DFC to the PCC shell. The precondition for step 2 was the existence of UBTF as two splicing variants, UBTF1 and UBTF2 which cannot be discerned with antibodies raised against UBTF. Thus, no data exist on their ultrastructural localization, their 3D organization/reorganization by inhibition of rRNA synthesis induced during mitosis or by drugs. Accordingly, we specifically identified each variant in transfected cells synthesizing GFP-tagged UBTF1 or UBTF2 by using anti-GFP antibodies. Pre-embedding nanogold strategy was chosen to obtain a strong signal to noise ratio and to perform electron tomography. In control cells, the two variants are mainly localized within FC but showed a different repartition. Electron tomography demonstrated that they are disposed as fibrils which are folded in loop-like structures. In contrast, after rRNA inhibition, their localization is identical and they remain organized as extended 3D loop-like structures. As UBTF is a useful marker to trace rDNA genes, we used these data to improve our previous model of rDNA gene 3D organization within FCs, using anti-RNA Polymerase I immunolabeling. Altogether, data obtained were used to formulate a dynamic model of the nucleolus, showing that: i) nucleoli remain independent units upon inhibition of rRNA synthesis, ii) in each nucleolus, structures containing UBTF gather during a sequential process and are always in contact with the condensing network of ICC and PCC. Successive time-lapse confocal microscopy and ultrastructural imaging of the same cells demonstrate that all FCs contain UBTF and are always in contact with condensing ICC that promotes the mobility of FC/DFC assembly. Our finding that FCs and ICC chromatin are contained within large NVs suggest that they are in continuity and belong to the same chromatin entity. At the same time UBTF1 and UBTF2 were similarly organized in the form of 10-25 nm fibrils folded in a loop-like manner both within control and AMD treated cells. We consider FC as a highly dynamic r-chromatin globule, emerging due to multistep folding of single rDNA loop