DDX21 interacts with nuclear AGO2 and regulates the alternative splicing of SMN2

ABSTRACT AGO2 is the only member of mammalian Ago protein family that possesses the catalytic activity and plays a central role in gene silencing. Recently researches reported that multiple gene silencing factors, including AGO2, function in the nuclei. The molecular mechanisms of the gene silencing factors functioning in nuclei are conducive to comprehend the roles of gene silencing in pretranslational regulation of gene expression. Here, we report that AGO2 interacts with DDX21 indirectly in an RNA-dependent manner by Co-IP and GST-Pulldown assays and the 2 proteins present nuclei foci in the immunofluorescence experiments. We found that DDX21 up-regulated the protein level of AGO2 and participated in target gene, SNM2, alternative splicing involved in AGO2 by the indirect interaction with AGO2, which produced different transcripts of SMN2 in discrepant expression level. This study laid important experiment foundation for the further analysis of the nuclear functions of gene silencing components.

Multiple gene silencing in STAT pathway in K562 cells

Context: Chronic myeloid leukemia (CML) is characterized by the presence of a fusion oncoprotein BCR-ABL. This mutation imparts a constitutive phosphorylation activity of tyrosine residues in the cellular proteins. One of the targets of BCR-ABL is the STAT5 protein, which when phosphorylated induces gene expression of antiapoptotic proteins such as BCL-XL. The STAT pathway has been targeted in the past by disrupting any one protein only. A multiple gene silencing has never been done in this pathway. Aim: The aim of this study was to compare the effects of downregulation of BCR-ABL, STAT5A, STAT5B, and BCL-XL, individually and simultaneously, in human CML cell line (K562 cells) through RNA interference (RNAi). Further, gene expression, inhibition of proliferation, and apoptosis induction were assessed in K562 cells. Materials and Methods: K562 cells were transfected with various combinations of small iRNA (siRNA) and the expressions of aforesaid genes were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. K562 cell proliferation and apoptosis were analyzed using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and flow cytometry, respectively. The results were compared through one-way analysis of variance. Results: qPCR and western blotting results post-siRNA transfection confirmed the targeted gene suppression and protein reduction in K562 cells. The cell proliferation assay and apoptosis assay revealed that simultaneous gene silencing of BCR-ABL, STAT5A, STAT5B, and BCL-XL had the highest killing effect on K562 cells as compared to knocking down these genes individually or in any other combinations. Conclusions: This was the first time it was shown that multiple gene silencing in STAT pathway in CML cell line K562 was better as compared to individual gene silencing.

A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

ABSTRACTWe describe here the construction of a series of 71 vectors to silence central carbon metabolism genes inEscherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared toin silicopredictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that anyE. colistrain can be used and multiple gene silencing is easily possible in any combination.