Search for the Argonaute protein that governs miRNA regulation in Dictyostelium discoideum

• Main Author: Åström, Miranda
• Format: Others
• Language: English
• Published: Uppsala universitet, Institutionen för biologisk grundutbildning 2021
• Subjects: Argonaute proteins; RNA interference; miRNA; 3xFLAG-tag; Microbiology; Mikrobiologi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-432967

MicroRNAs are small non-coding RNAs that regulate gene expression through RNA interference. These small RNAs enact gene silencing by forming a RNA-inducing silencing complex together with the effector protein Argonaute. The function of the Argonautes in the social amoeba Dictyostelium discoideum is not yet fully understood. In this study, we look closer at Argonaute B by investigating if it is possible to extract the protein from the cells by the addition of a polypeptide protein tag called 3xFlag. At the same time, we also look into if Argonaute B is important for cell growth. Sequences of the 3xFlag tag with or without the Argonaute B gene (agnB) attached had previously been cloned into a vector and transformed into Dictyostelium discoideum cell. The 3xFlag::agnB sequence was confirmed in wild type and agnB knock-out strains through polymerase chain reaction. We then verified the expression of the fusion protein in the cells by western blot. The cell growth was measured by how the number of cells changed over time. The experiment suggested that Argonaute B is important for growth. Our result show that the construct 3xFlag::agnB sequenced had correctly been transformed into the strains and is highly expressed under tested conditions. We could also see that Argonaute B is an important factor in cell growth.