Periodic Reporting for period 1 - RNAfate (RNAfate- revealing regulation of cellular “noise”.)
Okres sprawozdawczy: 2019-09-01 do 2021-08-31
RNA is an essential element in the flow of genetic information in the cell. Recent development of genome-wide technologies revealed multiple novel RNA transcripts, mostly non-coding (nc) for proteins. Their biological role in the cell is still poorly understood and at first they were considered as “noise”. During the recent decade, research has revealed growing evidence for the importance of ncRNAs in organism development and disease. Thus, further clarification of their functions remains a pressing need in the field. Among the ncRNA group, a subset of RNAs of at least 200 nucleotides, called long non-coding (lnc)RNAs, are of mayor interest. LncRNAs share many features with messenger RNA, but they cannot be translated into proteins. In some cases, even small changes in amount of some of the lncRNA in the cell can have a big impact on a cell or an entire organism. Thus, regulation of their levels is crucial to prevent undesired effects. After ncRNA synthesis the next key processes modulating amount of RNA in the cell is degradation. This ultimate step of RNA regulation is the focus of this work. Most of key proteins responsible for lncRNA removal in nucleus in human cells are already characterized. However cytoplasmic fate of lncRNAs remain less understood. Therefore, taking advantage of the advancement of high throughput RNA sequencing technology and novel genetic tools for the modification of genes in human cells, this project focused on defining the key exonucleolytic enzymes responsible for regulation of lncRNA levels in the cytoplasm of human cells, as well as identifying hidden or previously not detected lncRNAs.
During the course of this project, I created a genetically modified cell line in which the protein XRN1 can be removed by simple addition of a drugs to cell culture. XRN1 is a protein involved in the degradation of RNA from its head to tail. A second cell line, in which it is possible to remove DIS3, a protein that degrades RNA in reverse direction, was provide by our collaborator (S. West, University of Exeter, UK). Furthermore, after removal of these proteins in the respective cell lines, cell compartments were separated and cytoplasmic RNA content was evaluated through RNA sequencing. Using our custom inhouse bioinformatic pipelines for data analysis we identified which of the enzymes play major role in regulation of lncRNAs in cytoplasm. This results were also supported by analysis of already published data with other systems and cell lines. The produced data as well as the list of the known and novel transcripts identified to be regulated by XRN1 or DIS3 in the cytoplasm will be made available in publicly accessible Gene Expression Omnibus database upon publication of the research results in scientific journal (manuscript in preparation).
This work as first presents direct involvement of XRN1 and DIS3 in dynamic regulation of cytoplasmic lncRNAs in human cells. Cells use multiple ways to regulate levels of their RNAs. Previously used techniques required long lagging times during which cells could compensate for the lack of one protein, making impossible to evaluate the direct impact of protein of interest in the degradation of particular ncRNAs. In system applied in my study, the removal of a regulatory protein is much faster, providing more relevant information about the role of the investigated proteins. Additionally, DIS3 gene mutations were found in a fraction of patients with multiple myeloma, thus we are further exploiting relevant clinical data from our collaborator (Claudia Haferlach, MLL Münchner Leukämielabor GmbH, Munich, Germany) to see if I can find a link between patients samples with DIS3 mutation and our molecular mechanisms. Recently, a role of lncRNAs as transcripts producing peptides (a short chain of amino acids) starts to be appreciated, even though lncRNAs were thought to be non-coding. Some of this short peptides were shown to have a molecular function. Thus, some of the lncRNAs accumulating after removal of DIS3 or XRN1 could be source of such peptides and they could have some relevance for the cells. Identification of such short peptides that could have functional relevance can be a perspective study, further exploring results obtained during this MSCA fellowship. Moreover, if some of the lncRNA derived peptides would be presented on the cell surface and specific to cancer cells, they could be also explored as potential targets for immunotherapy.