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ZNHIT6: a new SUMO E3 ligase regulating RNA metabolism

Periodic Reporting for period 1 - SUMO-ZNHIT6-snoRNPs (ZNHIT6: a new SUMO E3 ligase regulating RNA metabolism)

Reporting period: 2016-10-01 to 2018-09-30

This project is focused on answering a basic biological question by performing a series of well controlled and designed experiments. My host laboratory is interested in the role of a small ubiquitin-like modifier (SUMO) in various cellular processes, which can be studied by a combination of biochemical, biological and global proteomic approaches.

The overall objective of the project was to investigate the role of ZNHIT6, a putative SUMO E3 ligase in the regulation of RNA metabolism. In particular we wanted to investigate a role for this protein in the regulation of small nucleolar RNA-protein complexes (snoRNPs) that mediate the modification of rRNA nucleotides and processing of rRNA precursors. It has been previously established that SUMO-mediated protein modifications play a crucial role in the regulation of snoRNP functions and this project would therefore expand our current understanding of this important biological process. It has a potentially wider benefit to society as snoRNAs have been shown to play a role in cancer and neurodegenerative and viral diseases.

During the fellowship, we have widened our research interest to also determine the biological role of ZNHIT6 in stem cell differentiation, due to its importance in general translation regulation. That might have numerous implications in regenerative medicine, cancer treatments, and our understanding of the role of ZNHIT6 and the wider SUMO network in early human development.
In this project, we have been asking two main questions. First, we wanted to determine if ZNHIT6 could be a new SUMO E3 ligase. By employing rigours biochemical tests, we demonstrated that despite being a very good SUMOylation substrate, ZNHIT6 is not a SUMO ligase. The second question was aimed at understanding the role of ZNHIT6 in regulation of RNA metabolism. For this, we have managed to characterize the expression and localization of ZNHIT6 as well as the consequences of its constitutive knock-out, which provides us with some information about its biological role. In addition, we have performed a number of interesting experiments, through which we wanted to assess the role of ZNHIT6 and SUMO in the regulation of a biologically crucial process of stem cell differentiation. As such, we have generated a unique dataset that is completely novel in the stem cell field.

I will aim to publish all my research data in open-access journals as soon as possible. I have also decided to deposit all my proteomics data sets in PRIDE repository managed by EBI, which I believe will be a valuable resource for the research community as we have created unique and novel data sets that could give rise to interesting projects in the future. So far, I have presented my data as a poster during 2017 EMBO Conference on Ubiquitin and SUMO: From molecular mechanisms to systemwide responses in Cavtat in Croatia and during our yearly Department meetings. A paper describing the SUMO proteome of human iPS cells and how it changes during differentiation is in preparation. “Dramatic changes to the SUMO proteome during differentiation of human induced pluripotent stem cells”

See Figure 1. Schematic representation of the progress of the project incorporating research questions and obtained results [Figure 1 is attached to the Summary for Publication].
In this project, I have managed to shed light on the biological role of ZNHIT6 beyond what was known about this protein so far. By the use of state-of-the-art genome-engineering technologies and global proteomic approaches I was able to demonstrate that ZNHIT6 is an essential protein that is implicated in the regulation of various processes. Growing amount of evidence suggest that SUMOylation is a crucial regulatory mechanism in various cellular processes. Based on our recent findings, we hope to be amongst the first groups to demonstrate the complexity of this regulatory network during differentiation. The main impact of these research project is the discovery of new roles of ZNHIT6 and SUMOylation in the regulation of important biological processes such as differentiation. Beyond that, we have gathered a number of large datasets that will be made public and thus can be used by various research groups around the world to help in their research. I believe that these data can give rise to novel interesting project that would be of great benefit in a wider socio-economic sense.