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Title: Studies on the long non-coding RNA, MRHL in mouse embryonic stem cells
Authors: Rao, M.R.S.
Neha, C.V.
Keywords: Stem cells
Mouse embryo system
Issue Date: 2017
Publisher: Jawaharlal Nehru Centre for Advanced Scientific Research
Citation: Neha, C.V. 2017, Studies on the long non-coding RNA, MRHL in mouse embryonic stem cells, MS thesis, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru
Abstract: LncRNAs have emerged as sentinels in the regulation of various cellular processes and hence, it is not surprising that the number of long non-coding RNA genes correlate with developmental complexity over the course of evolution. The versatility of RNA as a biomolecule has been instrumental in its participation in diverse cellular contexts. Meiotic recombination hotspot locus (Mrhl) is a long non-coding RNA that was discovered in our laboratory. It showed a tissue specific expression pattern and was highly expressed in testis, liver, spleen and kidneys (38). Owing to its high expression in adult testis, the initial characterization of the RNA was carried out in a B-type mouse spermatogonial cell line GC1-spg. In these cells, Mrhl was seen to be a nuclear restricted RNA that was processed to a 80 nucleotide transcript by Drosha machinery (39). Further, it was shown to be a chromatin associated RNA with a role in regulating gene expression of several loci (some of which were exerted by its physical association) (42). Functional analysis of the RNA revealed its role in the regulation of one of the important signaling pathway, Wnt, which is crucial all along the course of development. Wnt signaling in turn negatively regulates Mrhl, forming a feedback loop (40). This interplay between Mrhl and Wnt has been extensively characterized in our laboratory and has been found to be a key step in meiotic commitment of mouse spermatogonial cells (41). Since long non-coding RNAs exhibit tissue specific and context specific function, the present study involves the basic characterization of Mrhl in mouse embryonic stem cells. We find that similar to mouse spermatogonial cells, Mrhl is a nuclear restricted and chromatin associated RNA. However, unlike the case in Gc1-spg cells, Mrhl does not interact with p68, a DEAD box helicase. This interaction of Mrhl with p68 is instrumental in its regulation of Wnt- signaling. Hence, it was not surprising to find that in mouse embryonic stem cells, down regulation of Mrhl does not result in the nuclear translocation of beta- catenin, which is a hallmark of canonical Wnt signaling activation (unpublished work; Iyer, D.).To understand the function exerted by Mrhl in mouse embryonic stem cells, we resorted to two approaches. Firstly, upon down regulation of the RNA, we find no change in the pluripotency markers indicating that Mrhl is not directly involved in the maintenance of pluripotency. Further insights into the functions of the RNA will be obtained by global gene expression profiling upon down regulation of Mrhl. Additionally, preliminary results suggest that Mrhl is up regulated as mouse embryonic stem cells undergo differentiation. The second approach employed was to determine the interacting protein partners of the Mrhl by RNA pull-down followed by mass spectrometric analysis. It will be interesting to know the repertoire of interacting partners the RNA can have and the list of processes it might be regulating in this context. Since Mrhl is chromatin bound in mouse embryonic stem cells too, it will be enlightening to map the genome wide occupancy of this RNA in this system, which is epigenetically quite unique. Also, these studies will also provide us with a comparative analysis of the common and exclusive roles Mrhl can carry out in these two different systems.
Description: Open access
Appears in Collections:Student Theses (MBGU)

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