Ribonucleic acids (RNAs), single-stranded molecules, are essential parts of every living thing’s cells. By carrying the instructions for the synthesis of a protein in their own sequence, mRNAs, which function as “transcripts” of our genes, contribute to the translation of genetic information. Andreas Marx, a professor of organic and cellular chemistry at the University of Konstanz, points out that RNAs frequently require chemical modification after they are generated or repaired after damage in order to carry out their range of activities in the cell.
One chemical event that occurs during this process is the three-step joining (ligation) of two RNA strands at their respective opposing ends. Specialised enzymes called RNA ligases, which are present in all living things–including viruses, fungi, and plants–cause this process. These RNA ligases had not previously been identified in vertebrates, including humans. As the first human RNA ligase of this type, the protein C12orf29, which is part of a multidisciplinary research team from Konstanz, has now been found. The study’s results, which were published in Nature Communications, suggest that enzymes play a protective role in cells against cellular stress.
“We noticed C12orf29 during extensive studies of human lung carcinoma and kidney cells that we performed in search of proteins with a specific chemical signature and for which we used new chemical tools. It caught our attention because until then it was not understood what the protein’s functions were,” Marx said. The researchers, therefore, developed and used various protocols to purify and predict the structure of the unexplored protein and performed experiments to track down its chemical function. They were thus able to prove what was initially only a reasonable suspicion: C12orf29 links RNA strands using adenosine triphosphate (ATP).
The researchers were able to show in detail that this process follows a characteristic, three-step reaction pattern known from other RNA ligases of other life forms. To learn more about the function of C12orf29 at the cellular level, the researchers went one step further after elucidating the chemical mechanism. “We used the CRISPR/Cas gene scissors to generate a line of human kidney cells in which the gene encoding C12orf29 was knocked out. We were then able to compare these knockout (KO) cells with ‘normal’ kidney cells under varying experimental conditions,” Marx explained.
In particular, when treating the cells with menadione, a K vitamin, clear differences were observed between KO cells and the wild-type cells with functional RNA ligase: Comparatively low concentrations of menadione were sufficient to damage KO cells. In contrast, the wild-type cells were only damaged at significantly higher concentrations. Since menadione is known to cause oxidative stress, the researchers concluded from this result that C12orf29 protects against oxidative cellular stress. “We assume that a previously hidden human RNA repair mechanism underlies this biological function of C12orf29. We now need to examine this mechanism in further studies,” Marx said. (ANI)