Deletion of Drosophila Nopp140 induces subcellular ribosomopathies.
The nucleolar and Cajal body phosphoprotein of 140 kDa (Nopp140) is considered a ribosome assembly factor, but its precise functions remain unknown. To approach this problem, we deleted the Nopp140 gene in Drosophila using FLP-FRT recombination. Genomic PCR, reverse transcriptase-PCR (RT-PCR), and immunofluorescence microscopy confirmed the loss of Nopp140, its messenger RNA (mRNA), and protein products from all tissues examined. Nopp140-/- larvae arrested in the second instar stage and most died within 8 days. While nucleoli appeared intact in Nopp140-/- cells, the C/D small nucleolar ribonucleoprotein (snoRNP) methyltransferase, fibrillarin, redistributed to the nucleoplasm in variable amounts depending on the cell type; RT-PCRs showed that 2'-O-methylation of ribosomal RNA (rRNA) in Nopp140-/- cells was reduced at select sites within both the 18S and 28S rRNAs. Ultrastructural analysis showed that Nopp140-/- cells were deficient in cytoplasmic ribosomes, but instead contained abnormal electron-dense cytoplasmic granules. Immunoblot analysis showed a loss of RpL34, and metabolic labeling showed a significant drop in protein translation, supporting the loss of functional ribosomes. Northern blots showed that pre-RNA cleavage pathways were generally unaffected by the loss of Nopp140, but that R2 retrotransposons that naturally reside within the 28S region of normally silent heterochromatic Drosophila ribosomal DNA (rDNA) genes were selectively expressed in Nopp140-/- larvae. Unlike copia elements and the related R1 retrotransposon, R2 expression appeared to be preferentially dependent on the loss of Nopp140 and not on environmental stresses. We believe the phenotypes described here define novel intracellular ribosomopathies resulting from the loss of Nopp140.
Department of Biological Sciences, Louisiana State University, 202 Life Sciences Building, Baton Rouge, LA, 70803-1715, USA.
Fang
F
He
Allison
A
James
Himanshu
H
Raje
Helya
H
Ghaffari
Patrick
P
DiMario
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D020541:Coiled Bodies / Q000235:genetics / Q000378:metabolism; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D017353:Gene Deletion; D023281:Genomics; D008297:Male; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012335:RNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification*; D016601:RNA-Binding Proteins; D020718:Ribonucleoproteins, Small Nucleolar / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000473:pathology*
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D029721:Drosophila Proteins; C453465:Nopp140 protein, Drosophila; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D020718:Ribonucleoproteins, Small Nucleolar; C047458:fibrillarin