Rabbit anti-Arabidopsis thaliana AGO2 (N-term) Polyclonal Antibody

Regulatory factors function by modulating a variety of cascade mechanisms in cells. RBM4 is a multifunctional RNA-binding protein in post-transcriptional gene regulation. Cytoplasmic RBM4 interacts with Ago2 to regulate inflammatory responses by affecting mRNA decay and cap-dependent translation. However, it is unclear whether RBM4 functions in inflammation regulation by its splicing factor role. Here, the cell biology, gene expression profile and alternative splicing pattern of HeLa cells with RBM4 overexpression (RBM-OE) were compared with the control. The results showed that RBM4-OE inhibited proliferation. RBM4-OE extensively affects the transcriptional level of genes involved in cell surface receptor signalling pathway, inflammatory responses and the response to lipopolysaccharide. RBM4 broadly regulated the alternative splicing of hundreds of genes with functions of protein binding, helicase activity, DNA binding and transcription co-activator. RBM4-regulated splicing of these genes plays an important role in apoptotic process and gene transcription regulation. As an example, exon inclusion of TNIP1 mediated by RBM4 affects the expression of its targets in inflammatory pathways. These results indicated that RBM4 can mediate the inflammatory response via splicing regulation, which adds to the understanding of the critical role of RBM4 in cancer complicated by inflammation. In conclusion, this study indicated a mechanism in which the dysregulation of alternative splicing can influence cellular biology and lead to various immune-related diseases.

Argonaute proteins play important roles in gene regulation with small RNAs (sRNAs) serving as guides to targets. Argonautes are believed to bind sRNAs in a sequence non-specific manner. However, we recently discovered that Argonautes selectively load endogenous single-stranded (ss) RNAs, suggesting that Argonaute loading may conform to sequence specificity. To identify sequences preferred for Argonaute loading, we have developed HIgh-throughput Sequencing mediated Specificity Analysis (HISSA). HISSA allows massively parallel analysis of RNA binding efficiency by using randomized oligos in in vitro binding assays and quantifying RNAs by deep-sequencing. We chose Drosophila as a model system to take advantage of the presence of two biochemically distinct Argonautes, AGO1 and AGO2. Our results revealed AGO2 loading to be strongly favored by G-rich sequences. In contrast, AGO1 showed an enrichment of the GAC' motif in loaded species. Reanalysis of published sRNA sequencing data from fly tissues detected enrichment of the GAC motif in ssRNA-derived small RNAs in the immunopurified AGO1-complex under certain conditions, suggesting that the sequence preference of AGO1-loading may influence the repertoire of AGO1-bound endogenous sRNAs. Finally, we showed that human Ago2 also exhibited selectivity in loading ssRNAs in cell lysates. These findings may have implications for therapeutic ssRNA-mediated gene silencing.