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Published on: September 25, 2013
RNAi knockdown of Nopp140 induces Minute-like phenotypes in Drosophila
Zhengfang Cui1, Patrick J DiMario
1Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
Abstract:
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 >or=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.
Insights
Nopp140 protein is crucial for ribosome biogenesis and development. Its disruption in Drosophila causes lethality and deformities, mirroring human Treacher Collins syndrome and Minute phenotypes.
Area of Science:
- Molecular Biology
- Developmental Biology
- Genetics
Background:
- Nopp140 protein is essential for ribosome biogenesis and pre-rRNA processing.
- Two Drosophila isoforms, Nopp140-True and Nopp140-RGG, exist due to alternative splicing.
- Disruptions in ribosome production are linked to developmental disorders like Treacher Collins syndrome (TCS) and Minute phenotypes.
Purpose of the Study:
- To investigate the functional role of Nopp140 in Drosophila development.
- To understand the consequences of Nopp140 loss-of-function and overexpression on development.
Main Methods:
- Utilized the GAL4/UAS system for RNA interference (RNAi) to reduce Nopp140 expression.
- Employed reverse transcription-PCR (RT-PCR) to quantify Nopp140 mRNA levels.
- Used immunofluorescence microscopy with isoform-specific antibodies to assess protein localization and loss.
- Observed and documented phenotypic outcomes in larvae, pupae, and adults.
Main Results:
- Variable reduction in Nopp140 mRNA levels was observed across different RNAi lines.
- Loss of Nopp140 protein was detected in imaginal and polyploid tissues.
- Significant Nopp140 loss (>=50%) resulted in larval/pupal lethality, nuclear abnormalities, and melanotic tumors.
- Moderate Nopp140 loss (30%) led to adult deformities resembling Minute phenotypes.
- Overexpression of either Nopp140 isoform caused embryonic and larval lethality.
Conclusions:
- Nopp140 plays a critical role in Drosophila development, influencing ribosome production and tissue integrity.
- Developmental phenotypes resulting from Nopp140 disruption are analogous to human TCS and Minute phenotypes.
- Precise regulation of Nopp140 expression levels is vital for normal embryonic and larval development.
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