Effects Of Alus On Gene Expression And Bioinformatics Analysis Of Centromere Sequences In C Group Chromosomes

Objective: The effects of RNAs on gene expression mainly include two aspects of inhibiting and activating gene expression. The effects of RNAs on gene expression have attracted attention from biologists for decades, and evidence for RNA-activated genes is accumulating.To study the roles of repetitive sequences in activating genes, in this study, we used different combined Alu repetitive sequences, for example, two Alu genes locating in same plasmid or different plasmids; sense Alus and antisense Alu combination; plasmids transiently or stably transfected into He La cells. The expression of EGFP reporter gene was observed using fluorescence microsope or flow cytometry. Using bioinformatics analysis, we analyzed the simulative RNA binding strengths of centromere regions and their flanking sequences of C group chromosomes. Through the above methods we studied the roles of repetitive sequence in RNA activation and inferred the mechanism of RNA activation.Methods:1 The construction of reporter plasmids and modulator plasmids2 The construction of PSI-rTetR plasmid3 Cell transfectionCell transfection contained transient transfection and stable transfection. The detailed methodology is written in Text.4 Heat shock treatment5 Determination of EGFP-positive cells with fluorescence microscopeThe expression of the EGFP protein was assessed by inspecting the He La cells stably or transient transfected with expression vectors using fluorescence microscope.6 Determination of EGFP-positive cells with flow cytometry7 Sequence data and softwareWe obtained data from the NCBI human genome database. The search software used to analyze the frequency values of 7-nucleotide strings was written by our team.8 The algorithm for the binding strength of the RNA populationThe algorithm used to assess the binding strength of the RNA population was calculated based on the principle that RNA and DNA sequences with greater complementarily would result in greater binding between RNA and DNA. The detailed algorithm is written in Text.9 Tet-on systemsHe La cells were transfected with Mini C1 reporter plasmids. The transfected cells were grown in 10% FCS-DMEM containing 1 mg/ml G418 and transfected with PSI-rTetR plasmid and then treated with Puro to receive G418 and Puro double resistance He La cells. EGFP gene expression was observed after these He La cells treating with Dox or without Dox.10 Statistical analysisBetween-group comparisons were made using mean ± S.D. and t-test. Statistical significances was accepted at p<0.05. At least three independent experiments were performed for all the studies. Images shown are representative of at least 3 independent experiments.Results:1 The construction and identification of expression vectorsFive categories expression vectors were constructed in this study:1.1 Reporter plasmids containing EGFP reporter gene were derived from p EGFP-C1(Fig. 1A, Table 1).1.2 Modulator plasmids were derived from pcDNA3.1. These modulator plasmids were used for transient transfection(Fig. 1B, Table 1).1.3 PSI modulator plasmids were derived from PSI plasmid. These plasmids were used for stable transfection(Fig. 1C, Table 1).1.4 Mini-C1 reporter plasmids were derived from mini-C1 plasmid(Fig. 1D, Table 1).1.5 PSI-rTetR plasmids for stable transfection(Fig. 1E, Table 1).2 Combined sense Alu and antisense Alu activated EGFP reporter gene2.1 The effects of combined sense Alu and antisense Alu on EGFP gene expressionFig. 2 suggest that combined sense-antisense Alu repetitive sequences activated the EGFP reporter gene when stably transfected. However, combined sense-antisense repetitive sequences did not activate the EGFP reporter gene when transient transfection. In addition, two genes were located in different plasmids; combined sense-antisense repetitive sequences did not promote significantly EGFP gene expression regardless of transient or stable transfection(Table 4-Table 7). These results show that combined sense-antisense repetitive sequences activating gene needs stable transfection and these sequence located in same plasmid.2.2 The effects of Alu homologous sequence(Alu JB) on EGFP geneC1-Alu-sense-Alu JB-sense, C1-Alu-sense-Alu JB-antisense plasmids(Table 1) were stably transfected into He La cells. Fig.3 shows that EGFP gene expression quantities were no significant difference between those two plasmids and they were lower than that of C1-Alu-sense-antisense. These results suggest that C1-Alu-sense-Alu JB-antisense plasmid did not activate EGFP reporter gene.3 The effects of heat shock on EGFP reporter geneHe La cells stably transfected with plasmids were heated at 45 for 30 ?min. EGFP gene expression was observed using fluorescence microscope at different times after heat shock. Fig. 4 shows that heat shock significantly increased EGFP gene expression quantity of C1-Alu-sense-Alu JB-antisense plasmid(Alu-sense-Alu JB-antisense). The EGFP gene expression quantity reached peak at 2-4 days after heat shock and then decreased quickly and reached the same level as before heat shock at 12 days. Heat shock also significantly increased the EGFP gene expression of Alu-sense-antisense plasmid. The role of heat shock to Alu-sense-antisense plasmid maintained much longer time than that in Alu-sense-Alu JB-antisense though the increase was less than the plasmid of Alu-sense-Alu JB-antisense. EGFP gene expression quantity still kept at higher level at 20 days after heat shock in Alu-sense-antisense plasmid.Hela cells were stably transfected with other control plasmids and then treated at 45 for 30 min. Fig. 5?-Fig. 6 show that heat shock did not increase EGFP gene expression in these control plasmids.4 Tet-on systemsThese results furtherly proved that combinated sense Alu and antisense Alu repetitive sequences activated EGFP reporter gene.5 The binding strengths of RNA populations at centromeres in C group chromosomes are lowWe used bioinformatics analysis to simulate the binding strengths of RNAs to centromere sequences and their flanking sequences. The RNA population binding strength at centromere sequences is significantly lower than that of the flanking sequences(Fig. 8 and Table 8).Conlusion:1 Plasmid transfection experiments demonstrated that transcribed repetitive sense-antisense sequences activated the EGFP reporter gene upon stable transfection. Heat shock increased significantly the EGFP gene expression in both Alu-sense-antisense and Alu-sense-Alu JB-antisense plasmids. However, the roles of heat shock in Alu-sense-antisense kept much longer time than in Alu-sense-Alu JB-antisense plasmid.2 The RNA population binding strength at centromere sequences is significantly lower than that of the flanking sequences. The results of low RNA population binding strengths at centromeres are consistent with the experimental results that suggest that RNAs activate genes.