Periodic Reporting for period 3 - mRNP-PackArt (Nuclear mRNA Packaging and mRNP Architecture)
Periodo di rendicontazione: 2021-06-01 al 2022-11-30
Our understanding of the process of mRNP assembly serves as a basis to understand the mechanisms of the resulting diseases and thus to develop therapies to treat these diseases as well as approaches to prevent disease development. As mRNP assembly is implicated in many important diseases such as neurological disorders and cancer, the results of this project are important for society.
The overall objective of this project is to unravel the mechanism of mRNP assembly – including changed under a variety of stress conditions – and the molecular structure of the resulting mRNP.
For Aim 3 two XL-MS datasets of the TREX complex purified natively from S. cerevisiae and cross-linked with different cross-linkers were obtained. In addition, some negative stain data of the natively purified TREX complex from S. cerevisiae was obtained. Unfortunately, a cryo-EM structure of the human and the S .cerevisiae TREX complex expressed recombinantly was published in the journal “eLife” in November 2020 (doi: 10.7554/eLife.61503 and 10.7554/eLife.61467). In addition, a cryo-EM structure and XL-MS data of the S. cerevisiae TREX complex expressed recombinantly was recently published by bioXriv (doi: https://doi.org/10.1101/2021.01.04.425184). Thus, we are performing final experiments of our TREX complex purified from S. cerevisiae and prepare a manuscript with our data to be submitted soon. In addition to the work mentioned in the “Description of the action”, we expressed mRNP components including TREX subunits of the thermophile C. thermophilum in E. coli for structural analysis. Due to the thermophilic nature of this organism, its proteins are superbly suited for structure determination. For example, the structure of a Sub2-Tho1 dimer from C. thermophilum will be analyzed by crystallography soon.
For Aim 2 (Illumination of the mechanism of mRNP packaging) we analyzed the function of Prp9C components, Tho1 and Npl3 in nuclear mRNP assembly and nuclear mRNA export. Three manuscripts, each covering one of these proteins / protein complex, is in preparation. These analyses were done using “conventional” mutants as our structural data is not yet available. Furthermore, the analysis of the molecular function of Hpr1 (THO complex) and Nab2 is in progress.
In addition to the work described in the “Description of the Action” we are also analyzing the mechanism of mRNP assembly and nuclear mRNA export as well as the composition of mRNPs under various stress conditions.
Overall, the work is a bit behind schedule as it was very difficult to hire well-trained scientists and a technician (no technician could be hired so far) for this scientifically and technically very demanding project – even though all positions were advertised internally at our university, nationally and even internationally (in the journal “Nature”). The recruitment process was and is further hampered by the COVID-19 pandemic, which has made it extremely difficult to hire people.
Until the end of the project we will finish the purification of mRNPs (aims 1 and 3) and their biochemical by qMS (Aim 1) as well as structural analysis by XL-MS and electron microscopy (Aim 3). In addition, we will continue our functional analysis of mRNP assembly by assessing the function of further mRNP components and the three recruitment platforms (Aim 2) to obtain a consistent picture of all recruitment dependencies.