Different ribosomal proteins play an individual role in dendritic complexity in Drosophila melanogaster larvae

Different ribosomal proteins play an individual role in dendritic complexity in Drosophila melanogaster larvae.

Title: Different ribosomal proteins play an individual role in dendritic complexity in Drosophila melanogaster larvae.

Authors: Vaishnavi Veeranki, Erin N Lottes, Shatabdi Bhattacharjee, Emily A Wilde, Daniel N Cox

Faculty Sponsor: Dr. Daniel N Cox, Neuroscience Institute, Georgia State University. 

Introduction: Ribosomes are macromolecular machines that are responsible for complex protein synthesis mechanisms at the cellular level and enable proper coding of amino acids during translation. Eukaryotic ribosomes are composed of small and large subunits consisting of 79 ribosomal proteins (RPs) and 4rRNAs. They are in clustered structures called polysomes, attached to membrane-bound organelles like endoplasmic reticulum or as free cytosolic ribosomes promoting protein synthesis. Specific RPs of the ribosomes play an active role in translation regulation. Over the last 30 years studies have shown that dendrites have their own set of ribosomal machinery, including RPs and mRNAs carrying out translation and facilitating local protein synthesis. These RPs participate in ribosome assembly and in protein synthesis and play important roles in dendritic growth, which is crucial in establishing synapses with other neurons. In several neurodegenerative disorders dysregulation of ribosomal proteins has been identified as a biomarker of advanced disease conditions. 

Purpose of the study: We conducted our studies to investigate the functional role of individual role of ribosomal proteins in the development of gross dendritic morphology. Experiments were performed in across 31 different ribosomal proteins in class IV neurons of Drosophila melanogaster larvae. This study is important because, it enhances our understanding about how ribosomes regulate translation of gene post transcription. Therefore, it helps in understanding the underlying cellular and molecular mechanisms of different neurodegenerative disorders. 

Methods: The genes that encode for the expression of the RPs were silenced using RNAi which interrupts the translation by cleaving mRNA.  We silenced specific RPs genes in Class IV neurons using GAL-4/UAS Binary system. To visualize the neurons, we marked the cell membrane with GFP using the same GAL-4/UAS binary system. We knocked down 31 different ribosomal proteins and examined the effects on the total dendritic length. We further investigated the localization of ribosome using a GFP-tagged RPL10Ab construct. Non-parametric Kruskal-Wallis was performed with Dunn’s multiple comparison post-hoc to determine significantly changed total dendritic length in RP knockdowns. tSNE analysis was employed taking multiple parameters like total dendritic length, branch number, branch density to determine ribosomal proteins that are greatly affected.

Results: In our screen of individual RP knockdowns in Class IV md neurons, we found about 16 different RPs showing significant decline in total dendritic length (TDL) from controls. Five distinct groups were recognized which directed us to look at several RPs from different groups. We also found that the ribosomal proteins with greater decline in TDL restricted the RpL10Ab expression to the soma. 

Conclusion: Our results indicate that ribosomal proteins play an important role in driving the development of dendritic complexity. In Class IV multidendritic neurons, knockdown of ribosomal protein results in a dramatic reduction in total dendritic length compared to wild-type neurons.