Function and Regulation of hClp1 RNA kinase

hClp1 is an RNA kinase that phosphorylates RNA at the 5?-end. It is heavily involved in the metabolism of various forms of RNA including mRNA, tRNA, and small RNAs like siRNA and miRNA. Since RNA is vital in gene expression and regulation, the function of hClp1 needs to be optimal for a cell to survive. The function and regulation of hClp1 is still not properly understood, as such much work needs to be done to understand how hClp1 functions within the cell. A two-hybrid screening was previously performed to identify novel protein interactors of hClp1, and protein kinase inhibitor beta (PKIB) had been found to interact with hClp1. Here, we set out to investigate the effect PKIB has on hClp1 kinase activity. The aims of this project are to (1) generate recombinant hClp1, recombinant PKIB, and MCF7 cell lysate, (2) develop a protocol for an RNA kinase assay that can be used to observe and analyse kinase activity, and (3) to investigate the effect PKIB has on hClp1 kinase activity. Recombinant PKIB was successfully generated using BL21 Escherichia coli, however recombinant hClp1 was not. MCF7 cell lysate was successfully extracted, but no kinase activity was detected from the samples. An RNA kinase assay was developed that can successfully detect kinase activity. Due to unsuccessful hClp1 production, the effect of PKIB on hClp1 was not investigated. Further research should include optimisation of hClp1 expression and MCF7 cell lysate extraction, so that the novel interaction between PKIB and hClp1 can be studied.