What drives the RAN translation and why do we get neurodegenerative disease when it goes wrong?

Project Information

Prot-RAN

Grant agreement ID: 101003385

DOI

10.3030/101003385

Project closed

EC signature date 3 April 2020

Start date 1 July 2020

End date 24 March 2023

Funded under

• SPREADING EXCELLENCE AND WIDENING PARTICIPATION

Total cost

€ 149 625,60

EU contribution

€ 149 625,60

Coordinated by

UNIWERSYTET IM. ADAMA MICKIEWICZA WPOZNANIU
Poland

Periodic Reporting for period 1 - Prot-RAN (What drives the RAN translation and why do we get neurodegenerative disease when it goes wrong?)

Reporting period: 2020-07-01 to 2022-06-30

Summary of the context and overall objectives of the project

The project “Prot-RAN: What drives the RAN translation and why do we get neurodegenerative disease when it goes wrong?” studies the regulation of non-AUG initiated protein translation. This phenomenon is based on the observation that the expanded short tandem repeats in RNA can trigger the production of mutant proteins, without the canonical AUG initiation codon, which is usually used for protein translation. This aberrant RAN translation was initially described in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy (DM1), and to date was linked to many neurodegenerative diseases, e.g. fragile X-associated tremor/ataxia syndrome (FXTAS) or Huntington’s Disease (HD).
The short tandem repeats, usually three to eight base pairs long, are common in human genome. They are also genetically unstable and their uncontrolled expansion may lead to inherited disorders. For example, in the 5’UTR of fragile X mental retardation 1 (FMR1) gene, healthy individuals possess typically between 5 and 54 CGG trinucleotide repeats, while premutation expansions (55-200 CGG repeats) causes FXTAS. Symptoms of FXTAS include intention tremor, gait ataxia, parkinsonism and cognitive decline, amongst others.
The pathogenesis of FXTAS remains unclear, and to date, various pathogenesis models have been proposed. One of the possible mechanism is the repeat associated non-AUG initiated (RAN) translation. Resulting aberrant proteins- FMRpolyG containing polyglycine tracts- accumulate in nuclear inclusions in the brain of FXTAS patients, leading to neuronal death.
Despite emerging reports about the possible mechanisms driving RAN translation, still little is known about this process. The main goal of Prot-RAN project was to identify proteins regulating RAN translation. This is needed to understand the disease mechanisms and find potential drug targets for neurodegenerative disease- FXTAS. In parallel, the other dimension of the MSCA Individual Fellowship was to ensure the transfer of knowledge between the host and the researcher (and vice versa), in order to ensure successful career advancement during the fellowship.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

To identify proteins playing role in RAN translation, we bridged cutting-edge proteomics with RNA biology techniques. Briefly, we prepared biotin-tagged RNA molecules harboring 99CGG repeats in the context of 5’UTR of FMR1, which were then incubated with cellular extracts. Next, the proteins which bound to the RNA of interest were identified by high resolution mass spectrometry. The contribution of selected candidate modifiers to RAN translation efficiency was further analysed using silencing and overexpression approach in cells expressing FMRpolyG protein. This allowed us to identify the insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), and its paralogs (IGF2BP1 and IGF2BP2), as modifiers of FMRpolyG level in FXTAS cell models. A part from gene silencing approach, we also used small molecule inhibitors of IGF2BP3. Using these drugs, we were able to decrease the expression of both IGF2BP3 and FMRpolyG in cells. A part from IGF2BP3, we also identified another protein- EFTUD2 as promising RAN translation modifier. Although, due to time restriction, we were not able to investigate its mechanism of action, this protein will be further studied even after the MSCA fellowship (in ongoing collaboration between the Fellow and the Host lab).
During this work, I supervised two Master Students (one thesis defended in 2021, another one ongoing project started in December 2022) and one Bachelor (ongoing project started in December 2022). I also received a Sonata research grant for young investigators from National Science Centre (Poland), which enables me to pursue my research on RAN translation after the end of MSCA fellowship and to co-supervise one PhD student.
I have received training in RNA biology and microscopy (via collaboration during project in Host lab), attended a series of on-site courses on student supervision and research management (organized by the Host Institution). I also collaborated with researchers from AMU Faculty of Chemistry to develop a MS-based quantitative analysis of FMRpolyG and other RAN proteins.
The scientific background of our project was described in 1 review paper, and results of our research will be reported in 3 papers (two manuscripts in preparation, one need more supporting data). This work has been presented at two conferences (one poster, one talk), and popularized during AMU Biology Night, workshop organized for PhD students in Polish Academy of Sciences, Poznan and visit in pre-school class. Our activities were posted at Web (https://annbau1-protran.web.amu.edu.pl/) Twitter (@UAM_IBMiB) and LinkedIn (my personal account).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

In MSCA-funded Prot-RAN project, we aimed to identify novel modifiers of RAN translation in FXTAS using multidisciplinary techniques. Innovative aspects included using high resolution mass spectrometry to identify proteins binding to 5’UTR of FMR1 RNA containing expanded CGG repeats. The obtained data were then used to decipher new RAN translation modifiers in FXTAS. Our research clearly demonstrated that IGF2BP3 and its paralogs influence the level of toxic FMRpolyG protein and thus may play a role in FXTAS pathogenesis.
The results were disseminated during international conferences, and will be published as open access articles in peer-reviewed journals. Additionally, after publication, the mass spectrometry data will be shared in the open-access servers to enable other scientists to perform their in-silico analysis.
What is more, the presentation of our MSCA project during Biology Night and workshop for PhD students helped to increase awareness about FXTAS and R AN translation in society.

Looking at FMRpolyG inclusions under microscope

Developing quantitative mass spectrometry method for FMRpolyG

Presenting my work at 5th Intermational Conference on FMR1 premutation