Characterization of the effect of the V67 point mutation in dRNase Z in Drosophila melanogaster on gene expression




Chong Gao

Introduction

Human ELAC2 is isolated by genome analysis in high-risk prostate cancer families and was grouped as a putative prostate cancer susceptibility gene. [1] In vitro biochemical studies showed that ELAC2 is a tRNA processing endonuclease, the RNase Z enzyme, which generates the mature 3’-end of tRNA molecules by removal of the 3’-trailer elements of pre-tRNAs. [2] The Drosophila genome contains a single ortholog of human ELAC2 in the 47A1 region on the 2R chromosome, which was previously identified as Juvenile hormone-inducible protein 1, JhI-1. Silencing of JhI-1 in vivo by RNAi in Drosophila S2 cells disrupts tRNA maturation and triggers accumulation of pre-tRNA molecules with 3’ extensions. Therefore it’s renamed as dRNase Z.

Previous studies revealed ELAC2 is involved in TGF-β/Smad signaling mediated growth arrest as an important transcriptional scaffold protein. Furthermore, the DNA microarray data suggested that it is likely participate in the p53 signaling pathway and apoptosis. [1] Therefore in this project, target genes ECR, Wg, USP of TGF-β/Smad pathway were selected to be the candidate genes studies on mRNA expression in V67/ED24 mutant and control larvae.
In order to understand in vivo function of dRNase Z, a library of diverse mutant alleles in Drosophila melanogaster was generated. Most point mutations result phenotypes similar to flies with RNAi deletion on dRNase Z, which is early lethal in the beginning of 1st instar. Only V67, which exhibit an Arg to Glu mutation at a conserved region in front of the functioning His-motif, can live until pupation. However, the mutant eventually dies before pupation because of the deregulated ectopic differentiation of lamellocyte (autoimmune reaction), which gives rise to melanotic tumors at different part of the larva.

dRNase Z located on the 2nd chromosome. ED24 represents an dRNase Z gene truncated allele, therefore only one mutated allele can be produced in V67/ED24 strain, Cyo, {w+, GFP} is a wildly commercialized allele with a fluorescent protein inserted in. (Figure 1) Daughter strains are early lethal with two Cyo, {w+, GFP} allele or exhibiting the same normal phenotype as heterozygotes, which present one copy of the normal dRNase Z allele, with either one dRNase Z absence allele, one dRNase Z point mutated allele. Both two heterozygotes have no effect on other proteins’ expression. Our study strain has one copy of the dRNase Z truncated allele and one copy of dRNase Z point mutated allele. Point mutation locates in front of the conserved region of a known functioning His – motif and this one basepair change leads to Arg – Glu amino acid mutation.

Genomic-wide microarray analysis has revealed common targets for the tkv and babo pathways and provided new insights into downstream genes of the two distinct TGF-β-like pathways, which is mostly novel and functioning in growth control. Among the genes regulated by both pathways is ultraspiracle (USP), which further connects TGF-β with neuronal remodeling. [2] ECR was selected due to its key regulatory role in introducing Drosophila into pupation stage and its ability of binding ecdysone is greatly stimulated by the addition of USP as a partner in heterodimerization. Furthermore, candidate gene Wg was chosen not only for its key role in Drosophila development, but also due to previous studies which demonstrated that inhibition of Drosophila Wg signaling involves competition between Mad and Armadillo/β-Catenin for dTcf binding.[6]

Figures


Figure 1-Schematic diagram of study strain and control strains, V67 represents the point mutation of dRNase Z on the second chromosome. ED24 is the null mutation of dRNase Z, which due to chromosome deficiency, cannot produce dRNase Z.


Figure 2-Expression of ECR (A) and Wg (B) in Mutant (V67/ED24) and Control (V67/GFP & ED24/GFP) larvae. RT-PCR products were fractionated on a 1% agarose gel. Internal control RP49 (C) was utilized to monitor the amount of mRNA present in the samples. Band intensity was quantified by Bio-Rad Quantity One and RP49 normalized relative ECR and Wg mRNA expression are shown on the right.


Figure 3-Expression of USP in Mutant (V67/ED24) and Control (V67/GFP & ED24/GFP) larvae. RT-PCR was performed on DNase-treated RNA samples (A). Internal control RP49 (C) was utilized to monitor the amount of mRNA present in the samples. PCR using USP primers on DNase-treated RNA samples (B) was performed to detect possible DNA contamination. RT-PCR and PCR products were fractionated on a 1% agarose gel. Band intensity was quantified by Bio-Rad Quantity One. RP49 normalized relative USP mRNA expression are shown on the right.


Figure 4-Schematic diagram of dpp/BMP (left) and Activin (right) pathways. Both MAD and dSmad2 are R-Smad, MED is the Co-Smad. MAD and dSmad2 are phosphorylated and form dimmers with MED, respectively, in order to continue the signaling. Dpp/BMP pathway inhibits the expression of Wg through the MAD/MED dimer, whereas ECR expression level is promoted by Activin signaling through the dSmad2/MED dimer.


TGF-β superfamily members, which is a pivotal antimitogenic factor in the normal prostate play a critical role in many cellular processes such as proliferation, differentiation, development, apoptosis, and cancer. [3] (Figure 5) Seven ligands in Drosophila are roughly grouped into two major TGF-β pathways, the dpp/BMP and activin pathways, mainly signaling through thick veins (tkv) and baboon (babo).

Although it has been revealed that both TGF-β/BMP and Wnt/Wingless (Wg) pathways play important roles in organismal patterning and growth and their deregulation can give rise to oncogenesis, fibrosis, immune- and vascular disorders in mammalian, [4] whether they are regulated in the same way in Drosophila was not fully understood. dpp/BMP and Activin pathway compete for the same co–smad called MED. [8]

In this study, downstream target genes of TGF-β pathway ECR, Wg were down-regulated, This observation suggests that dRNase Z in Drosophila elicits TGF-β/Smad induced transcriptional responses by promoting the interaction with activated dSmad2. Disruption of dRNase Z leads to a negative impact on Activin pathway which then causes ECR down-regulated. Since ECR is master regulator of molting in Drosophila development, low level expression of ECR not only leads to underdeveloped larva, but also causes pupation failure, which is consistent with V67/ED24 phenotype. Moreover, it has been reported that Wg expression is antagonized by dpp/BMP signaling. In vivo, high levels of activated dpp/BMP receptor and its effecter Mad can inhibit the expression of Wg. Conversely, loss of mad can induce Wg expression. [5] Function defect of dRNase Z in Activin pathway leaves more MED available for dpp/BMP pathway which gives rise to Wg deregulated. Lacking of Wg leads to a long period of developmental stage in mutant compare to the normal phenotype, which was also consistent in V67/ED24 strain.

However, USP, which is regulated by both dpp/BMP and Activin pathways, is up-regulated in Mutant. This interesting observation may indicates dpp/BMP pathway has a higher efficiency on USP expression than Activin, so when there are more MED available for dpp/BMP, USP is up-regulated; Or, suggests that dRNase Z has other biological unidentified functions that would affect USP regulation. Moreover, the strong impact of dRNase Z on Wg and ECR might also suggests that there would be possible regulatory mechanism dRNase Z involved to mediate these genes.

Full Paper

Acknowledgments

I would like to thank Bo Liu and Leleesha Samaraweera for their continuous help through this project. Special thanks to Dr. Dubrovsky and Xie Xie for the larvae and valuable discussions. Sincere thanks to Dr. Berish Rubin for his guidance and providing the opportunity to do the present project.


This document was last modified 05/12/2010.
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