mRNA and natural endogenous antisense RNA in Dictyostelium discoideum

Abstract: The aim of this Master's Thesis was to investigate the presence and regulation of RNAi in the model organism Dictyostelium discoideum. The background for the project is a cDNA library representing 18-25nt RNAs (9000 clones) from Dictyostelium discoideum that recently was constructed. The result revealed a number of small RNAs with antisense complementary to mRNAs and from these we chose three genes for further investigation; hatA, rsmF, and DDB0230011 (kielin). We also studied two "relatives" to hatA, namely hatB and hatC to see if the genes are similar to each other in the setup of both an mRNA and asRNA. The first aim was to find both mRNA and asRNA for the genes which was done with RT-PCR. We found that all genes have both an mRNA and an asRNA, except for hatC which lacks an asRNA. By performing a Northern blot analysis of hatA mRNA and asRNA during development and in some RNAi knockout strains we found that both RNAs are is down regulated during development and also in some of the knockout strains. hatA has previously been reported to have a role in osmotic stress. We decided to investigate this effect in two normal Dictyostelium (Ax4:4 and Ax2) strains and two knockout strains, rrpC- and drnA- (done in Ax4:4 and Ax2 respectively). The results revealed that the asRNA and mRNA is down regulated due to stress and that it also differs in gene expression between untreated cells and cells treated with a starvation buffer. The theory based on our results is that the RNA expression goes down during development which is initiated by the starvation buffer and when the hyper osmotic buffer is added the expression goes down even more. The mRNA predicted to encode a Kielin homolog has previously been proven to be located in the tip of structure that has been allowed to develop for 16 hours and therefore we wanted to study the location of both mRNA and asRNA in this type of structure, also known as a slug. By using Fluorescent In situ hybridization we were able to detect both mRNA and asRNA, where the mRNA seems to be located in the entire structure while the majority of the asRNA can be found in the outer cell layer in one end of the slug.