Nucleolar ribosomal RNA synthesis continues in differentiating lens fiber cells until abrupt nuclear degradation required for ocular lens transparency.

Abstract

Cellular differentiation requires highly coordinated action of all three transcriptional systems to produce rRNAs, mRNAs and various 'short' and 'long' non-coding RNAs by RNA Polymerase I, II and III systems, respectively. RNA Polymerase I catalyzes transcription of about 400 copies of mammalian rDNA genes, generating 18S, 5.8S and 28S rRNA molecules. Lens fiber cell differentiation is a unique process to study transcriptional mechanisms of individual crystallin genes as their very high transcriptional outputs are directly comparable only to globin genes in erythrocytes. Importantly, both terminally differentiated lens fiber cells and mammalian erythrocytes degrade their nuclei through different mechanisms. In lens, the generation of the organelle-free zone (OFZ) includes the degradation of mitochondria, endoplasmic reticulum, Golgi apparatus and nuclei. Here, using RNA fluorescence in situ hybridization (FISH), we evaluated nascent rRNA transcription, located in the nucleoli, during the process of mouse lens fiber cell differentiation. Lens fiber cell nuclei undergo morphological changes including chromatin condensation prior to their denucleation. Remarkably, nascent rRNA transcription persists in all nuclei that are in direct proximity of the OFZ. Additionally, changes in both nuclei and nucleoli shape were evaluated via immunofluorescence detection of fibrillarin, nucleolin, UBF and other proteins. These studies demonstrate for the first time that highly condensed lens fiber cell nuclei have the capacity to support nascent rRNA transcription. Thus, we propose that 'late' production of rRNA molecules and consequently of ribosomes increases crystallin protein synthesis machinery within the mature lens fibers.