Study On The Function Of Ribosomal Protein S29

Increased resistance to insecticides of disease-causing and carrying organisms is a growing public health concern worldwide. Insecticide resistance is the result of complex polygenic inheritance, and the mechanisms are not well understood. In previous studies, suppression subtractive hybridization (SSh) combined with cDNA microarray analysis identified a gene, rps29, encoding a ribosomal protein that is overexpressed in the insecticide-resistant strain of Culex pipiens pallens.In this study, tandem affinity purification was used to identify the proteins that can interact with RPS29. Among the candidate proteins, CYP6N3, a member of the CYP450superfamily, was identified, and binding to RPS29was confirmed in vitro and in vivo by GST-pull down and immunofluorescence and confocal laser scanning microscopy.To investigate the RPS29-CYP6N3interaction in relation to insecticide resistance, cell viability in the presence of the common insecticide DM was assessed over a wide range of DM concentrations (0-102.5mg/1) using the CCK-8assay. Cells transfected with pIB-RPS29were more susceptible to DM, and RPS29knockdown significantly enhanced cell viability. However, CYP6N3knockdown significantly reduced cell viability, while CYP6N3overexpression significantly enhanced cell viability. Furthermore, this enhancement was inhibited by RPS29overexpression and stimulated further by lowered RPS29expression. Additionally, cell viability in the siCYP6N3-transfected cells was lower than the control group and significantly inhibited by RPS29overexpression. Together, these results indicate that CYP6N3contributes to enhance the resistance of C6/36cells to DM, and RPS29could abrogate the contribution of CYP6N3to resistance.To further investigate the RPS29-CYP6N3interaction, cells were transiently co-transfected with constructs encoding GFP-RPS29and CYP6N3-MYC. Overexpression of RPS29resulted in a dose-dependent decrease in CYP6N3protein levels. However, RPS29expression was increased when CYP6N3was overexpressed. This alteration in CYP6N3and RPS29levels was likely due to post-translational regulation because the mRNA levels of both CYP6N3and RPS29were comparable to those of RPS29or CYP6N3transfected cells as determined by real-time PCR experiments. To determine whether the low protein level of CYP6N3was indeed due to decreased protein stability, cells were treated with MG132, a specific inhibitor of proteasomes. CYP6N3protein levels were clearly recovered in the MG132-treated cells expressing different amounts of RPS29, and the recovery was time-dependent.RPS29is a component of the40S subunit. It has been reported that RPS29, a member of the S14family, contains a zinc finger motif for DNA/RNA binding, which suggest that RPS29could be a potential transcription factor worthy of further investigation. Ribosome biosynthesis is a key process for all actively proliferated cells, with about80ribosomal proteins coordinately synthesized. Ribosomal protein genes (RPGs) are widely distributed across different loci within the genome. To date, little is known about how transcription of ribosomal protein genes (RPGs) is coordinately regulated.To investigate the role of ribosomal protein S29(RPS29) in ribosome biosynthesis, the expression of seventy RPGs was examined following depletion of RPS29by low density arrays in several human cell lines. The transcriptional level of most ribosomal proteins was decreased after RPS29depletion.According to data from LDA in the four cell lines, there are20ribosomal proteins down-regulated in four cell lines. In the20ribosomal proteins, we chose10ribosomal proteins randomly and detected the mRNA expression levels of these ribosomal proteins following depletion of RPS27. The results show that the mRNA expression levels of most ribosomal proteins have no change.The enhancer activities of several marginally significant motifs (MSMs) which are involved in RPGs and may have enhancer activities, such as MSM-d, were also decreased. In order to examine the impact of RPS29depletion on the enhancer activity of MSMs, forty eight hours after transfection with RPS29siRNA, pGL3p, pGL3p-MSM-a, pGL3p-MSM-b, pGL3p-MSM-c or pGL3p-MSM-d were separately co-transfected with pRL-SV40into cells. The data showed that the enhancer activity of all oligonucleotides was lower in cells in which RPS29had been depleted, compared with snc-RNA-transfected cells. The Fold differences in enhancer activity between the snc-RNA control and RPS29-depleted samples in each group were calculated. Then compared with pGL3p group, the enhancer activity of MSM-d was significantly decreased in all cell lines, whereas the enhancer activity of MSM-a was decreased in LTEP-a-2cell line, and MSM-b activity was decreased in A549.Taken together, these observations indicate that CYP6N3, a novel RPS29-interacting partner, could stimulate deltamethrin resistance in mosquito cells and RPS29overexpression targetted CYP6N3for proteosomal degradation, abrogating the CYP6N3-associated resistence to deltamethrin. Our findings provide a novel mechanism associated with CYP450s mediated DM resistance. Our results also demonstrated that RPS29could regulate the expression of RPGs through affecting enhancer activities of MSMs, which suggest that coordinated regulation of RPGs occurs at the level of transcription.