Doctor of Philosophy (PhD)
Four nucleostemin-like proteins (NS1-4) were identified previously in Drosophila melanogaster. NS1 and NS2 are nucleolar proteins, while NS3 and NS4 are cytoplasmic. Here we show that NS2 is homologous to the human nucleostemin-like protein, Ngp1, and that endogenous NS2 and exogenous GFP-NS2 enriches within peripheral regions of nucleoli in larval polyploid midgut cells. Like NS1, depletion of NS2 in midgut cells blocked the release of the 60S ribosomal subunit as detected by the accumulation GFP-RpL11 within nucleoli, and this likely led to a general loss of 60S subunit proteins as shown by immunoblot analyses. At the ultrastructural level, nucleoli in midgut cells depleted of NS2 displayed enlarged nucleolar granular components (GCs) not only on the nucleolar periphery, but interspersed within dense fibrillar components (DFCs). Depletion of NS2 also caused nucleolar stress: larval midgut cells displayed prominent autophagy where mitochondria were found enclosed by isolation membranes derived from the rER. Larval imaginal wing disc cells depleted of NS2 responded by inducing apoptosis as marked by anti-caspase 3 labeling; loss of these cells resulted in defective adult wings. We conclude that nucleolar proteins NS1 and NS2 have similar but non-overlapping roles in the final maturation or nucleolar release of 60S ribosomal subunits. Nopp140, the nucleolar and Cajal body (CB) phosphoprotein of 140 kDa, is considered involved in several steps of ribosome biogenesis. In Drosophila, loss of Nopp140 causes redistribution of fibrillarin, the C/D small nucleolar ribonucleoprotein (snoRNP) methyltransferase; correspondingly, the 2’-O-methylation of ribosomal RNA (rRNA) was reduced. Nopp140 knockout also leads to a loss of cytoplasmic ribosomes and a significant drop in protein translation. Here we show that coilin, an essential component of CB, redistributed in the Nopp140-/- cells by immunofluorescence microscopy. Both immunofluorescence microscopy and Western blot analyses showed that the coilin protein level was elevated, and RT-PCR supported the results by showing coilin mRNA increased approximately two fold in Nopp140-/- larvae. NS1 and NS2 were also found to redistribute from the nucleolus upon Nopp140 gene knockout. In summary, the phenotypes described in Chapter 3 indicate that Nopp140 participates in several aspects of ribosome biogenesis, and it is essential to maintain nuclear homeostasis.
Document Availability at the Time of Submission
Secure the entire work for patent and/or proprietary purposes for a period of one year. Student has submitted appropriate documentation which states: During this period the copyright owner also agrees not to exercise her/his ownership rights, including public use in works, without prior authorization from LSU. At the end of the one year period, either we or LSU may request an automatic extension for one additional year. At the end of the one year secure period (or its extension, if such is requested), the work will be released for access worldwide.
Wang, Yubo, "Nucleolar Proteins are Essential for Ribosome Biogenesis and Nuclear Homeostasis in Drosophila melanogaster" (2015). LSU Doctoral Dissertations. 2194.