Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - Human Rpc5 (RNA Polymerase III Rpc4/Rpc5 subcomplex and Selenocysteine tRNA transcription)

Teaser

To a great extent, the development of human diseases such as cancer are caused by the modification of some function performed by our cells. If the cells cannot correctly execute that function (or they execute it in excess), the whole body is affected and, in the end, the...

Summary

To a great extent, the development of human diseases such as cancer are caused by the modification of some function performed by our cells. If the cells cannot correctly execute that function (or they execute it in excess), the whole body is affected and, in the end, the disease progresses. The execution of these function is mediated by molecular machines (or enzymes) present inside our cells. One of these machines, known as RNA Polymerase III, participates in a process known as transcription, which is the conversion of our DNA genes into a “readable” molecule known as RNA. This is an essential function that takes part in all the cells of our body and its alteration causes catastrophic consequences. In order to correctly perform this function, the enzyme needs to be bound to the DNA and it requires the participation of other factors known as Transcription Factors. To understand how the disease appears and progresses we first need to understand how the basic cellular machines work. In the case of the RNA polymerase III, we still don’t know how it gets bound to the DNA and, given its direct relevance in cancer development, understanding this process might have a profound impact in biomedical research.
The overall objective of the project is to study the hypothetical interaction between a part of the RNA Polymerase III known as Rpc5 and a transcription factor known as Brf2, whose levels are altered in several cancers.

Work performed

In order to understand how the binding to the DNA happens, we need to learn about the organization and structure of the proteins involved in the process. Given the difficulty of assessing the transcription function directly in the cell (by in-vivo experiments) we decided to study it in a test tube using in vitro approaches. The RNA Polymerase III enzyme is formed by several parts or subunits. In this project, we focused in one of them known as Rpc5. The first step in the project was the production of enough amount of the proteins of interest. Once the Rpc5 protein was produced, the next step was to confirm if the binding to the Brf2 protein was happening. Using several different methods, the binding activity could be unequivocally confirmed for the first time.
Next, with the objective of understanding how the mechanism of action works we decided to study the “shape” of Rpc5 alone and how it changes after binding to Brf2. This approach provided the “structure” of several parts of the Rpc5 protein. The arrangement of the protein is very similar to that observed previously in other factors that bind to human genes. This result confirms the importance of the Rpc5 protein in the binding of the RNA polymerase III to the DNA.
These results open the possibility of creating specific drugs that could control the process of transcription during cancer progression. The fine regulation of this enzyme could help in the fight against tumor development.
During the progress of this project, the obtained results and used methods have been presented and discussed in several conferences and meetings including the “CSHL Meeting (2015)-Mechanisms of Eukaryotic Transcription” or the “80th Harden Conference (2016)” among others. Additionally, it has been presented in more informal events such as the “Science Bites” Initiative or the “Happy Poster Hour”.

Final results

The prevalence of cancer has a profound impact in all levels of the society. An increasing number of research groups have focused their efforts in understanding the mechanisms underlying tumor development. Among the diversity of cellular functions that become altered during cancer, the analysis of DNA-dependent mechanisms has traditionally focus most of the attention. In particular, the transcription process by RNA Pol III generates molecules that are essential for the cellular function and survival and, therefore, they have a direct effect in cancer progression. Unfortunately, the molecular mechanisms behind RNA transcription are not completely known.
The results obtained in this project have provided some of the first clues about the organization of human RNA Pol III. This project also provided novel insights into the binding of the RNA Pol III enzyme to specific DNA genes. Given the link between the expression of these genes and cancer development, the identification of the factors responsible of RNA Pol III binding to the DNA might be a keystone for the future discovery of anti-tumoural drugs.
The evolution of cancer is a highly intricate process consisting on the activation and deactivation of multiple cellular mechanisms and functions. Identifying the molecular basis of the processes affected during tumorigenesis is essential for the future understanding of the disease. In this regard, this project sheds some light into the basic process of transcription which, in the future, might have an influence in the battle against cancer and its impact in society.

Website & more info

More info: http://www.icr.ac.uk/our-research/research-divisions/division-of-structural-biology/vannini-group.