Investigating the Roles of Nopp140 Isoforms in Drosophila melanogaster

This dissertation investigates the roles of two Drosophila Nopp140 isoforms: Nopp140-RGG and Nopp140-True in RNA polymerase I (Pol I) transcription and pre-rRNA processing. Nopp140 locates within nucleoli and Cajal bodies; it acts as a chaperone for small nucleolar ribonucleoproteins (snoRNPs) that modify pre-rRNA, but the exact mechanism(s) behind its involvement remain largely unexplored.\nChapter 1 provides a literature overview on RNA Pol I transcription and pre-rRNA processing. Chapter 2 explores experimentally how Nopp140 could interact with Pol I transcription machinery and pre-rRNA processing. For this, we established two distinct ectopic nucleolar models in living Drosophila cells that permit isolation and comparison of factors related to RNA Pol I transcription versus pre-rRNA processing. Two transgenic lines were constructed: the first transgene contains the entire rDNA intergenic spacer (IGS) sequence with the Pol I core promoter fused to the rDNA encoding the external transcribed spacer (ETS) and the 5’ portion of the 18S rRNA, followed by the bacterial DNA encoding chloramphenicol acetyl transferase (CAT). The second transgene contains the same IGS fused to only the CAT DNA. Both constructs were inserted into Drosophila’s third chromosome. The objective was to assess whether these transgenes could form ectopic nucleoli capable of recruiting RNA Pol I to transcribe either the ETS-18S-CAT RNA or the CAT RNA alone. Both transgenes produced ectopic nucleoli. Going further, both Nopp140 isoforms and RpL135, a large Pol I subunit, located to these ectopic nucleoli. Fibrillarin, a pre-rRNA processing component, co-localized to ectopic nucleoli expressing the ETS-18S-CAT RNA but not to those expressing only the CAT RNA. These findings suggest that both Nopp140-True and Nopp140-RGG associate with the Pol I transcription machinery regardless of the RNA transcribed. Hence, we conclude that Nopp140 is a bridge between Pol I transcription machinery and pre-rRNA processing. Chapter 3 describes the increase in R2 retrotransposon expression in Drosophila, particularly in the context of nucleolar stress conditions caused by mutations in the RNA Pol I transcription factor (udd), the deletion of the Nopp140 gene, or the over-expression of Nopp140-RGG, but not Nopp140-True. Chapter 4 concludes with a summary of findings and suggestions for future research.