Biologically Characterization Of Human IWS1 (hIWS1) In Vivo

During the transcription of eukaryotic genes performed by RNA polymeraseâ…¡(RNAPâ…¡),transcription elongation is a complex phase,which needs a set of factors to regulate the catalytic activity of RNAPâ…¡. Up to date, lots of transcription elongation factors have been determined, while it is still required to probe into other unknown ones. Here, in human cells, we have identified and characterized a factor, hIWS1 (human IWS1, hIWS1), a novel RNAPâ…¡transcription elongator. On the other hand, PRMT5 is one major PRMTs that generates either monomethylarginines (MMA) or symmetric dimethylarginines (sDMA). Recently, some research has demonstrated that PRMT5 would be involved in RNAPâ…¡transcriptional regulation. So we probed into the relationship between this putative RNAPâ…¡transcription elongator hIWS1 and PRMT5.1. Cloning,expression and Identification of hIWS11.1 Total RNA is extracted from human 293T cells, which is subsequently reverse transcribed into first strand cDNA. Then this first strand cDNA is used as a template for PCR amplification of hIWS1 cDNA. PCR is carried out with primers designed based on the predicted cDNA sequence in GenBank (Accession No. AL834178). Using the sequence-specific primers for the putative hIWS1 gene, we have amplified the cDNA coding both N-terminal (hIWS1-N391) and C-terminal (hIWS1-C297) fragments of hIWS1 gene, respectively, and the correction of which is verified by sequencing. The full length cDNA of hIWS1` would be generated by using both of these achieved fragments, and the full length cDNA of hIWS1 is consensus with AL834178.This suggests the existence of matured mRNA of hIWS1 in human cells, which splicing mechanism is consistent with the predicted sequence in GenBank.1.2 According to the predicted ORF of AL834178, the PCR product hIWS1-N391 is cloned into the vector pET-22b to generate expression plasmids, pET22b-N391. E.coli strain BL21 (DE3)...