QUANTIFICATION AND RNAi KNOCKDOWN PHENOTYPE OF RNP-4F ISOFORMS DURING CNS DEVELOPMENT IN DROSOPHILA
Abstract (Summary)The evolutionarily conserved Drosophila gene, rnp-4f, encodes a 943 amino acid protein that plays an important role in spliceosomal assembly. Preliminary studies have shown that while two differently sized rnp-4f mRNAs differing in size by 177-nt can be detected in early fly embryos via RT-PCR, the longer transcript predominates during central nervous system development. Using developmental Northerns, the presence of these two isoforms was confirmed and the relative frequencies of the individual isoforms were quantified. Notably, there is an apparent switch in relative isoform abundance during embryonic stages 6-12 h, when the longer ~3600-nt transcript becomes more abundant. The relative levels of total rnp-4f mRNAs (combined isoforms) across development were estimated by comparing the ratio of their band intensities to that of the constitutively expressed gene rp49. The results show that total rnp-4f mRNA levels dramatically decline during the mid-embryo stages of development to a low plateau and again increase somewhat during pupal and adult stages. Previous in situ localization studies reported that rnp-4f mRNAs concentrate to the dorsal roof of the ventral nerve cord during embryo stages of development, which may reflect the presence of an rnp-4f mRNA isoform having a unique function at that central nervous system location. These results were confirmed using in situ DIG-labeled RNA probe hybridization and extended using a probe specific to the longer isoform that retains the 177-nt intron 0 locus. The results show that the longer rnp-4f isoform localizes heavily to the dorsal roof of the ventral nerve cord, in addition to a broad homogenous tissue distribution. Dissection studies show that this localization closely resembles that of the midline glial cell-specific sim nuclear protein. An RNAi approach was utilized to specifically knock down the function of the longer isoform by constructing an isoform-specific duplex RNA molecule and microinjecting this into 1 h old embryos. The resulting phenotype showed disruption of the central nervous system, most commonly with a complete interruption in commissure formation towards the anterior end. This result shows that the longer rnp-4f isoform, which localizes to the dorsal roof of the ventral nerve cord, plays a role in the development of axon fiber tracts in the developing fly central nervous system.
School Location:USA - Ohio
Source Type:Master's Thesis
Date of Publication:01/01/2006