The Director of the Institute of Human Genetics, Polish Academy of Sciences in Poznan (IHG PAS) announces an open competition for the position of an adjunct at the Department of Nucleic Acid Function (DNAF) of the IHG PAS
The competition is open to persons who meet the conditions set out in the Act of 30 April 2010 on the Polish Academy of Sciences (Journal of Laws of 2016, item 572, as amended) and the Regulations for conducting competitions for scientific positions at the Institute of Human Genetics, Polish Academy of Sciences in Poznan.
The project is carried out within the OPUS18 grant from the National Science Centre, Poland, project leader is prof. Jadwiga Jaruzelska.
Project title: „NANOS1 RNP-interactome: structure and dynamics during specification/early stages of human germ cell development - significance for human reproduction”. Infertility affects 15% of couples world-wide. Genetic defects account for 15-30% of male infertility cases and is a risk factor for testis germ cell tumour (TGCT). NANOS is posttranscriptional gene expression regulator, contains highly conserved RNA-binding domain composed of (CCHC)2 zinc-finger and interacts with few proteins. We have identified v-NANOS1 protein variant in association with infertility in patients. Therefore, the general objective is to get comprehensive insight into structure and dynamics of NANOS1 ribonucleoprotein interactome during specification/early stages of human male germ cell development. We will use human models upon stable wt- and v-NANOS1 overexpression: 1/ hESCs in vitro differentiation towards specification and early development of primordial germ cells (hPGCLCs) and 2/ TCam-2 cell line originating from seminoma and representing slightly later PGC developmental stage. Specific objectives are: 1. Identification of target RNAs bound by wt- and v-NANOS1 at different developmental stages applying protein-crosslinked RNA extraction (XRNAX) and eCLIP. The anti-FLAG antibody will serve to co-IP NANOS1-bound RNAs. Then, total eCLIP isolated RNA using RiboZero will be analyzed applying RNA-Seq. Databases and bioinformatic tools will be used for gene identification. 2. Screening for wt and v-NANOS1 protein binding partners by using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) followed by LC-MS/MS mass spectrometry and validation by co-IP followed by western. 3. Identification of specific motifs in target RNAs recognized by wt- and v-NANOS1 protein partners containing RNA-binding domains employing bioinformatic tools and validate RNA-protein interactions applying luciferase reporter assays. Ribonucleoprotein networks will be assembled by implementing pertinent bioinformatic tools/platforms such as CytoScape. We expect these networks being at least partially different for wt- and v-NANOS1 and will differ in specific developmental stages. 4. Most interesting interactions (RNA/protein and protein/protein) in wt-NANOS1 associated network, missing from the v-NANOS1 ones, will be validated applying phenotypic assays for functional dependence on NANOS1. For that purpose, endogenous NANOS1 silencing will be followed by measurement of the level of NANOS1 targets by real time qPCR/western. We anticipate that the level of target RNAs/protein will change upon NANOS1 silencing. If so, we will test whether it influences apoptosis, cell cycle, proliferation or other parameters that could explain phenotype of the patients and TCam-2 cells.
The project ‘NANOS1 RNP-interactome: structure and dynamics during specification/early stages of human germ cell development - significance for human reproduction’ is carried out within OPUS18 grant from the National Science Centre Poland.
A list of documents that the candidate should attach to the competition application:
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