Abstract:
The nucleolus is a nuclear sub-organelle that lacks a membrane. It is the site of
ribosome biogenesis within the nucleus bearing RNA Polymerase I and associated transcription
factors. In addition to its role in ribosome biogenesis, the “multifunctional” nucleolus is
involved in cellular stress sensing, cell cycle regulation, DNA damage repair, senescence and
apoptosis. Nucleoli assemble on ribosomal DNA (rDNA) present on the p-arms of human
chromosomes 13, 14, 15, 21 and 22, at the end of mitosis. Cancer cells show an increase in
nucleolar numbers required for protein synthesis. However, the factors that regulate nucleolar
numbers and morphology in cancer cells, is unclear. Here, we have assessed the role of nuclear
Lamins in regulating nucleolar morphology. Lamins are type V intermediate filament proteins
that maintain the mechanical integrity of the nucleus. We show a prominent role of Lamin B2
in modulating nucleolar structure in colon cancer cells. We found that in addition to its
association with the nuclear envelope, a subpopulation of Lamin B2 localizes to the border of
the nucleolus. The nucleolar subpool of Lamin B2 associates with the nucleolar proteins
Nucleolin and Nucleophosmin (B23). Furthermore, we found that the head domain of Lamin
B2 is essential for maintaining nucleolar morphology, whereas the tail domain is required to
maintain an intact nuclear morphology. Interestingly, Lamin B2 depletion is accompanied by
the upregulation of pre-rRNA and intergenic RNAs. Nucleolin speckles formed upon inhibition
of RNA Pol I, persisted for a much longer duration upon Lamin B2 depletion. We found that
localization of non-coding IGS RNA is enhanced in Nucleolin speckles in Lamin B2 depleted
cells. Thus, Lamin B2 impinges on structure and function of the nucleolus. We also found a
regulatory feedback between Lamins and the nucleolar rRNA methyltransferase - Fibrillarin.
Fibrillarin depletion showed invaginated nuclei with decrease in Lamin A/C and B2 expression
levels. Furthermore, Fibrillarin depleted cells showed Actin accumulation adjacent to the
nucleus. We surmise that a combined effect of a weakening of the nuclear lamina and altered
cytoskeletal arrangement impinges on nuclear morphology in Fibrillarin depleted cells. Finally,
we investigated mechanisms by which proteins are shuttled and retained in the nucleolus. We
found a predominant role for Nucleolin in mediating nucleolar localization of H2B. The Nterminal
and the RNA binding domains of Nucleolin are required for the compartmentalization
of H2B into the nucleolus in an RNA dependent manner. Taken together, we have investigated
into the mechanisms that regulate morphology, function and dynamics of the nucleolus.
