The effects of blebbistatin, an NMMII inhibitor, were compared with the effects of reversine

extracts was sufficient to complement splicing of the Ftz reporter pre-mRNA similar to the recombinant SRSF1. The appearance of splicing products was correlated with the increasing concentration of the recombinant PfSR1, similar to the effect observed with the recombinant SRSF1. Similar results were obtained with the b-globin reporter pre-mRNA. We concluded that PfSR1 by itself could mediate splicing of pre-mRNA and demonstrate that PfSR1 is a functional splicing factor. PfSR1 is an AS factor with similar activity to SRSF1 The fact that PfSR1 by itself could complement splicing of pre-mRNA in an in vitro S100 complementation assay encouraged us to further investigate whether PfSR1 can also function as AS factor in living cells. Toward this aim we applied two mini-gene constructs successfully used to demonstrate the AS activity of the mammalian SRSF1. In these systems, HEK 293T cells are cotransfected with an expression vector that over-expresses a candidate AS factor together with another vector that carries an artificial mini-gene. The E1A mini-gene contains several competing 50 -ss and could potentially give rise to a few different alternative spliced mRNA products. Following transfection, the cotransfected cells are harvested for further analyses of protein expression and detection of differential AS isoforms of pre-mRNA of the mini-gene. Our initial attempts to express PfSR1 in the HEK 293 T cells had very poor protein yield. We reasoned that since the P. falciparum genome is AT rich, its codon usage might not be optimal for protein translation in mammalian cells. To determine whether PfSR1 can influence the choice of different 50 -ss and thus give rise to alternatively spliced isoforms we D-α-Tocopherol polyethylene glycol 1000 succinate expressed PfSR1 in the presence of either the E1A or the b-globin mini-genes and found that for both mini-gene constructs PfSR1 acts as AS factor. In cells that were cotransfected with the adenovirus E1A mini-gene and an empty pcDNA3 vector, the putative AS machinery gave rise to three differentially spliced isoforms of E1A that are detected by RTPCR. Remarkably, in cells in which either PfSR1 or SRSF1 are coexpressed with the mini-gene, there is a clear PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19816210 change in the ratio of the different mRNA isoforms compared with the cells that were transfected with the empty pcDNA3 vector. In both PfSR1 and SRSF1 over-expressing cells there is an increase in the amount of the distal and proximal isoforms S13 and S9 while the amount of the intermediate isoform S12 decreases . These results show that ectopic expression PfSR1 promotes the proximal 50 -ss selection and modulate the AS regulation of the E1A mini-gene similar to SRSF1. Similarly, when PfSR1 is expressed in the presence of the b-globin D50 -ss mini-gene the balance between the distal and proximal 50 -ss selection is affected, indicating that PfSR1 strongly promotes the proximal 50 -ss. In cells that were transfected with the empty pcDNA3 vector the dominant spliced mRNA product is the 586-bp isoform that corresponds with splicing at the distal ss. However, in cells in which PfSR1 is expressed the 810-bp isoform, which corresponds with the proximal ss, becomes significantly more abundant than in the cells containing the empty pcDNA3 vector. This splicing pattern is similar to the results obtained in the SRSF1 expressing cells. Altogether, these results demonstrate that PfSR1 regulate ss selection resulting in AS patterns which are similar to those that are obtained with SRSF1 Nucleic Acids Research