Living organisms portray diverse patterns of growth and developmental regulation. The entire process, beginning from a single cell to the formation of tissues and organ systems and the final culmination in a form that characterizes a fully grown organism is closely guarded by numerous molecular pathways. Like a master conductor, the genetic material of the cell which is stored in its DNA (deoxyribonucleic acid), determines the fate of each individual cell and demarcates its developmental direction. Although in an organism, this genetic material is normally identical in every cell, there are differences in the ways cells respond to them. For example, skin cells behave differently from muscle cells and their developmental processes are highly variable in space and time.
It is now known that based on intrinsic or extrinsic environmental cues, the information passed on from DNA is converted into a functional protein product through an RNA (ribonucleic acid) intermediate. Thus, different genes fire at different times leading to diverse patterns of developmental regulation among cells. However, there exists a stark contrast between the size of the genome (DNA), the transcriptome (transcribed RNAs) and the proteome (functional protein products) inside a cell. While there are abundant RNA molecules that are transcribed from the DNA, only few give rise to proteins.
The search for function of RNAs that do not code for proteins is a relatively new topic in molecular biology. With advancements in sequencing methodologies, there is a rapid surge in the discovery of such molecules but due to the nonavailability of systematic tools to study them, the functional characterization of these RNAs has been relatively slow. Even among the non coding RNAs, there exists small and long varieties of which the long non coding RNAs (lncRNAs) have more heterogenous functional attributes. The roles that these lncRNAs play in development is only recently emerging, especially in the field of embryonic stem (ES) cell biology.
ES cells are of particular interest to researchers due to their properties of replicating indefinitely in culture and giving rise to all the germ layers that eventually constitute an organism. These unique abilities make them perfect models to study essential cellular developmental processes and also contribute to the understanding of the molecular pathways that ultimately lead to diseases like like other processes, is orchestrated by a host of different factors in which lncRNAs are slowly emerging as important players. Although there are thousands of lncRNAs identified, only a few have been implicated in pluripotency. I reasoned that there should be more such candidates and to study them one needs to develop a strategy to functionally investigate several lncRNAs simultaneously.
Loss-of-function screens have been extremely successful for dissecting the functions of protein coding genes. Among the triggers for conducting such screens, endoribonuclease-prepared small interfering RNAs (esiRNAs) have been demonstrated as effective mRNA depletion agents with minimum silencing of non-intended targets. Since these RNA interference (RNAi) agents had not been comprehensively tested on lncRNAs, I used them for conducting a screen to discover lncRNAs involved in pluripotency. Using a combination of RNAi and localization strategies, I here report the discovery of a novel lncRNA called Panct1 which through interaction with other factors takes part in the ES cell pluripotency programme. In the process of characterization of Panct1, I have also identified and partially characterized a potential DNA binding protein called CXORF23 which might emerge as an important player in the determination of stem cell fate. These discoveries hint towards the presence of more such lncRNA protein interactions and further widen our understanding of stem cell biology.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa.de:bsz:14-qucosa-134658 |
| Date | 09 July 2014 |
| Creators | Chakraborty, Debojyoti |
| Contributors | Technische Universität Dresden, Fakultät Mathematik und Naturwissenschaften, Prof. Dr. Frank Buchholz, Prof. Dr. Francis Stewart, Prof. Dr. Frank Buchholz |
| Publisher | Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/pdf |
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