David William Timme
The purpose of my research was to investigate what role ribosomal protein L41 (RPL41) has in regulating transcriptional activities of the Collagen 11a2 (Col11a2) gene. Prior to the ORCA grant, I had researched identifying possible transcription factors which bound to the B/C enhancer region of the Col11a2 gene, possibly regulating transcription. This work revealed that RPL41 could bind to the B/C enhancer in a eukaryotic (yeast) system. Since a protein binding to DNA could be relevant to transcriptional activity, unrelated to transcriptional regulation, or merely an artifact from the investigational method, it was important to examine the specific role RPL41 might play on regulation of the Col11a2 gene. This investigation was performed through use of transient transfections into rat chondrosarcoma (RCS) cells.
Transfections were prepared such that the effect of RPL41 on transcription could be observed. RCS cells were transfected with various plasmids which provided controls and were used for reporting transcriptional activity. If addition of the RPL41 reporter plasmid influenced transcription of the luciferase reporter positively or negatively, compared to control cells lacking the RPL41 plasmid, then RPL41’s effect could be realized. A luciferase reporter gene was under control of the B/C or D/E enhancer. Cells were also transfected with a ß-galactosidase reporter plasmid as an internal control for transfection efficiency. Results were calculated as luciferase units/ ß-galactosidase. The control cells’ transcription levels of the luciferase reporter were normalized to 100%, and the RPL41 transfected cells’ luciferase levels were compared to that. Transfections were performed in triplicate, and three independent trials were conducted. The results were reported as the average of these three trials. This method allowed for measurement of transcription in eukaryotic cells with and without the presence of RPL41 with statistical relevance.
The results showed that RPL41 could downregulate transcription of the luciferase gene under control of the B/C enhancer (Fig. 1) to 61% (p<.05), but had no transcriptional effect with the D/E enhancer (Fig. 2). Both of these genetic regions are known to be partially controlled by the protein SOX9, and I had supposed that other protein regulators would similarly act in a concerted fashion with both of the enhancers. The absence of an effect at both enhancer sites indicates that RPL41 acts in a specific manner, and is not a general suppressor.
RPL41 is a unique protein to study in the manner of a transcriptional regulator. It is very small, only 25 amino acids, and is a ribosomal protein that has a previously very under classified role. In a recent published review of the functions of ribosomal proteins, RPL41 is not even listed as a protein whose effect in the ribosome is known. South Korean researchers found that in a yeast two-hybrid system, RPL41 interacted with a protein kinase which phosphorylated a DNA topoisomerase, but this relationship is too tentative to suppose a causal relationship to DNA activity, and it would be foolish to extrapolate from this information that RPL41 has a protein-DNA relationship. However, this finding does hint that RPL41 could have an extra role outside of the actual ribosomal complex. RPL41 is made up of 18 basic amino acid residues, and so it could be that ionic interactions between the negatively charged DNA and positively charged RPL41 could be responsible for its binding characteristics. However, if a general ionic interaction were responsible for binding, then it seems likely that RPL41 would not distinguish between the B/C and D/E enhancers, as it appears to do in transfection experiments. Possibly, RPL41 can bind to DNA sequences in a site specific manner to regulate transcription in a previously undocumented manner.
The results indicate that RPL41 does not act as a positive transcription factor, but acts to inhibit transcription. One possible mechanism would be to bind to the enhancer site, and prevent binding of other proteins which are necessary for proper assembly of the transcription machinery. Assembly of the transcription initiation machinery requires a concerted effort of proteins bending, folding, and looping DNA, recruiting various proteins, and providing a favorable environment. If some of the proteins are missing, or their binding is prevented, then the environment for transcription initiation can be disrupted. Perhaps the binding of RPL41 to the B/C enhancer area is preventing another protein’s binding, disrupting the favorable environment, and causing the transcription levels to drop to 61% of the control.
Further experiments which I planned to conduct, but did not have the time to accomplish, were transfections using enhancer mutants. These experiments would help reveal what portion of the B/C enhancer sequence RPL41 binds to. Another researcher in the lab has been working to confirm binding of RPL41 to the B/C or D/E enhancer sequences in an in vitro system using electrophoretic mobility shift assays. As of this date, results of these experiments have not been obtained. An experiment of this type would be necessary before my work could be published in a journal.