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ABSTRACTThe ribosomal RNA genes (rDNA) of Drosophila melanogasterreside within centromere-proximal nucleolar organizers on both the X and Y chromosomes. Each locus contains between 200–300 tandem repeat rDNAunits that encode 18S, 5.8S, 2S, and 28S ribosomal RNAs (rRNAs) necessary for ribosome biogenesis. In arthropods like Drosophila, about 60% of the rDNAgenes have R1and/or R2retrotransposons inserted at specific sites within their 28Sregions; these units likely fail to produce functional 28S rRNA. We showed earlier that R2expression increases upon nucleolar stress caused by the loss of the ribosome assembly factor, Nucleolar Phosphoprotein of 140 kDa (Nopp140). Here we show that R1expression is selectively induced by heat shock. Actinomycin D, but not α-amanitin, blocked R1expression in S2 cells upon heat shock, indicating that R1elements are transcribed by Pol I. A series of RT-PCRs established read-through transcription by Pol I from the 28Sgene region into R1. Sequencing the RT-PCR products confirmed the 28S-R1 RNA junction and the expression of R1elements within nucleolar rDNArather than R1elements known to reside in centromeric heterochromatin. Using a genome-wide precision run-on sequencing (PRO-seq) data set available at NCBI-GEO, we show that Pol I activity on R1elements is negligible under normal non-heat shock conditions but increases upon heat shock. We propose that prior to heat shock Pol I pauses within the 5ʹ end of R1where we find a consensus “pause button”, and that heat shock releases Pol I for read-through transcription farther into R1.

Mammalian nucleostemin (NS) is a nucleolar guanosine triphosphate-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogaster contains a four-member nucleostemin family (NS1–4). NS1 is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed green fluorescent protein (GFP)-NS1 enrich within the nucleolar granular regions as expected, whereas overexpressed GFP-NS1 localized throughout the nucleolus and nucleoplasm, and to several transcriptionally active interbands of polytene chromosomes. Severe overexpression correlated with the appearance of melanotic tumors and larval/pupal lethality. Depletion of 60% of NS1 transcripts also lead to larval and pupal lethality. NS1 protein depletion>95 correlated with the loss of imaginal island (precursor) cells in the larval midgut and to an apparent block in the nucleolar release of large ribosomal subunits in terminally differentiated larval midgut polyploid cells. Ultrastructural examination of larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes and a concomitant appearance of cytoplasmic preautophagosomes and lysosomes. We interpret the appearance of these structures as indicators of cell stress response.

Mammalian nucleostemin (NS) is a nucleolar guanosine triphosphate-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogastercontains a four-member nucleostemin family (NS1–4). NS1 is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed green fluorescent protein (GFP)-NS1 enrich within the nucleolar granular regions as expected, whereas overexpressed GFP-NS1 localized throughout the nucleolus and nucleoplasm, and to several transcriptionally active interbands of polytene chromosomes. Severe overexpression correlated with the appearance of melanotic tumors and larval/pupal lethality. Depletion of 60% of NS1 transcripts also lead to larval and pupal lethality. NS1 protein depletion>95 correlated with the loss of imaginal island (precursor) cells in the larval midgut and to an apparent block in the nucleolar release of large ribosomal subunits in terminally differentiated larval midgut polyploid cells. Ultrastructural examination of larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes and a concomitant appearance of cytoplasmic preautophagosomes and lysosomes. We interpret the appearance of these structures as indicators of cell stress response.

Nopp140 associates with small nucleolar RNPs to chaperone pre-rRNA processing and ribosome assembly. Alternative splicing yields two isoforms in DROSOPHILA: Nopp140-True is homologous to vertebrate Nopp140 particularly in its carboxy terminus, whereas Nopp140-RGG contains a glycine and arginine-rich (RGG) carboxy terminus typically found in vertebrate nucleolin. Loss of ribosome function or production at critical points in development leads to Minute phenotypes in Drosophila or the Treacher Collins syndrome (TCS) in humans. To ascertain the functional significance of Nopp140 in Drosophila development, we expressed interfering RNA using the GAL4/UAS system. Reverse transcription-PCR showed variable losses of Nopp140 mRNA in larvae from separate RNAi-expressing transgenic lines, whereas immunofluorescence microscopy with isoform-specific antibodies showed losses of Nopp140 in imaginal and polyploid tissues. Phenotypic expression correlated with the percent loss of Nopp140 transcripts: a ≥50% loss correlated with larval and pupal lethality, disrupted nuclear structures, and in some cases melanotic tumors, whereas a 30% loss correlated with adult wing, leg, and tergite deformities. We consider these adult phenotypes to be Minute-like and reminiscent of human craniofacial malformations associated with TCS. Similarly, overexpression of either isoform caused embryonic and larval lethality, thus indicating proper expression of Nopp140 is critical for normal development.

The Nopp140 gene of Drosophila maps within 79A5 of chromosome 3. Alternative splicing yields two variants. DmNopp140 (654 residues) is the sequence homolog of vertebrate Nopp140. Its carboxy terminus is 64% identical to that of the prototypical rat Nopp140. DmNopp140-RGG (688 residues) is identical to DmNopp140 throughout its first 551 residues, but its carboxy terminus contains a glycine/arginine-rich domain that is often found in RNA-binding proteins such as vertebrate nucleolin. Both Drosophila variants localize to nucleoli in Drosophila Schneider II cells and Xenopus oocytes, specifically within the dense fibrillar components. In HeLa cells, DmNopp140-RGG localizes to intact nucleoli, whereas DmNopp140 partitions HeLa nucleoli into phase-light and phase-dark regions. The phase-light regions contain DmNopp140 and endogenous fibrillarin, whereas the phase-dark regions contain endogenous nucleolin. When coexpressed, both Drosophila variants colocalize to HeLa cell nucleoli. Both variants fail to localize to endogenous Cajal bodies in Xenopus oocyte nuclei and in HeLa cell nuclei. Endogenous HeLa coilin, however, accumulates around the periphery of phase-light regions in cells expressing DmNopp140. The carboxy truncation (DmNopp140DeltaRGG) also fails to localize to Cajal bodies, but it forms similar phase-light regions that peripherally accumulate endogenous coilin. Conversely, we see no unusual accumulation of coilin in cells expressing DmNopp140-RGG.

The Nopp140gene of Drosophilamaps within 79A5 of chromosome 3. Alternative splicing yields two variants. DmNopp140 (654 residues) is the sequence homolog of vertebrate Nopp140. Its carboxy terminus is 64% identical to that of the prototypical rat Nopp140. DmNopp140-RGG (688 residues) is identical to DmNopp140 throughout its first 551 residues, but its carboxy terminus contains a glycine/arginine-rich domain that is often found in RNA-binding proteins such as vertebrate nucleolin. Both Drosophilavariants localize to nucleoli in DrosophilaSchneider II cells and Xenopusoocytes, specifically within the dense fibrillar components. In HeLa cells, DmNopp140-RGG localizes to intact nucleoli, whereas DmNopp140 partitions HeLa nucleoli into phase-light and phase-dark regions. The phase-light regions contain DmNopp140 and endogenous fibrillarin, whereas the phase-dark regions contain endogenous nucleolin. When coexpressed, bothDrosophilavariants colocalize to HeLa cell nucleoli. Both variants fail to localize to endogenous Cajal bodies inXenopusoocyte nuclei and in HeLa cell nuclei. Endogenous HeLa coilin, however, accumulates around the periphery of phase-light regions in cells expressing DmNopp140. The carboxy truncation (DmNopp140ΔRGG) also fails to localize to Cajal bodies, but it forms similar phase-light regions that peripherally accumulate endogenous coilin. Conversely, we see no unusual accumulation of coilin in cells expressing DmNopp140-RGG.