| dc.contributor.advisor | Eichele, Gregor Prof. Dr. | |
| dc.contributor.author | Joshi, Parth Devesh | |
| dc.date.accessioned | 2019-05-24T08:56:56Z | |
| dc.date.available | 2019-05-24T08:56:56Z | |
| dc.date.issued | 2019-05-24 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-002E-E64A-D | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-7463 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | Molecular characterization and functional analysis of a novel long noncoding RNA in the mouse | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Lührmann, Reinhard Prof. Dr. | |
| dc.date.examination | 2019-02-25 | |
| dc.description.abstracteng | Majority of mammalian transcriptomes encode long noncoding RNAs (lncRNAs) that are not translated into proteins. LncRNAs are mRNA-like transcripts that are longer than 200 nucleotides, transcribed by RNA-Pol-II and undergo post-transcriptional modifications and splicing. Although they are less evolutionary conserved than protein-coding genes, lncRNAs have been shown to regulate several biochemical and cellular processes at a transcriptional level. Yet to date, the mechanism of only a handful of them has been studied in mammalian organisms. Therefore, in order to understand the function of lncRNAs in the mouse, we have performed large-scale RNA-sequencing of whole E14.5 mouse embryos. Results obtained revealed ~7000 putative lncRNA transcripts expressed at this developmental stage. From this list, we focused our attention on a small group of divergent lncRNAs that are transcribed in close proximity to physiologically important protein-coding genes and that showed significant evolutionary conservation in mammals. Our RNA in situ hybridization (ISH) data in mouse embryos confirmed that a few of these divergent lncRNAs genes share similar expression profiles with their neighboring protein-coding genes.
From this list, we selected an uncharacterized and mammalian conserved divergent lncRNA Gm14204 that has an expression similar to its adjacent neurotransmitter transporter gene Slc32a1. Gm14204 is divergently transcribed with respect to Slc32a1 on the opposite strand with a distance of ~50 bp between the two genes. Keeping in mind the prevailing idea in the literature, we hypothesized that Gm14204 lncRNA might regulate Slc32a1 transcription. First, we performed an in-depth molecular characterization of Gm14204 expression using RNA-ISH and observed that it has a nervous system-specific expression pattern in the developing mouse embryos and an ubiquitous expression throughout the adult brain, which is broadly comparable to Slc32a1 mRNA expression. Next, using a series of RNA-FISH and single-molecule (sm) FISH experiments, we showed that Gm14204
lncRNA and Slc32a1 mRNA transcripts are co-expressed in a subset of GABAergic interneuron population in the adult mouse brain.
To investigate the function of Gm14204, we have generated the first genetic mouse model for Gm14204 by knock-in of transcription termination sequences into the first intron of this gene. LncRNA gene deletion was not advisable because Gm14204 and Slc32a1 genes are situated very close to each other and deleting a region of Gm14204 might disrupt cis-regulatory elements in that locus. Thus, our novel strategy prematurely terminated the transcription of all Gm14204 lncRNA isoforms, leaving an intact Slc32a1 gene locus. Furthermore, using quantitative RT-PCR analysis of embryonic mouse brains, we showed that the expression of Slc32a1 gene is not altered in the lncRNA mutants. Contrary to the general notion in the field that divergent lncRNAs regulate their adjacent genes, in this study, we provide evidence that the divergent Gm14204 lncRNA does not regulate Slc32a1 despite the fact that they share similar expression patterns. Additionally, this lncRNA also does not regulate other protein-coding genes in the Gm14204 genomic region. Moreover, our smFISH data in adult brain have clearly indicated that Gm14204 localizes to multiple locations inside the nucleus in a subset of neurons, suggesting a trans function of the lncRNA. Identification of these neurons with molecular markers might provide us with a deeper knowledge about the possible function of lncRNA.
Only a few lncRNA knockouts in mouse have been shown to be lethal whereas many others have reported the presence of subtler phenotypes. In our study, we found that genetic deletion of Gm14204 was not lethal. The mice were viable, healthy, showed no visible abnormalities and were born at a normal Mendelian ratio. We still search for a subtle phenotype in the nervous system where Gm14204 shows strong expression during mouse development and in the adult. | de |
| dc.contributor.coReferee | Wimmer, Ernst A. Prof. Dr. | |
| dc.subject.eng | Long noncoding RNA, Embryonic development, Mouse brain, lncRNA knockout | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-002E-E64A-D-5 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.identifier.ppn | 1666651583 | |