Periodic Reporting for period 3 - mRNP-PackArt (Nuclear mRNA Packaging and mRNP Architecture)
Periodo di rendicontazione: 2021-06-01 al 2022-11-30
One of the most fascinating questions in biology is how the immense complexity of life can arise from a simple DNA strand with just four letters that make up our genes. Thus, we want to uncover how this system of gene expression functions in cells and how the DNA is brought to life. First, the DNA is copied into mRNA, which needs to be packaged for transport through the cell: from the nucleus, where the DNA is stored, to the cytoplasm. Proteins package the mRNA by binding to it, and the resulting mRNA-protein complex is called an mRNP. This mRNP assembly is essential for gene expression. In addition, mRNP assembly is crucial for the regulation of gene expression. Thus, it is not surprising that mutations in one of the mRNP components are the cause for several diseases. In addition, the regulation of mRNP assembly is important during stress conditions including illness and viral infections.
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.
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.
The prerequisite for Aim 1 (Determination and quantification of the protein composition of a specific mRNP) and Aim 3 (Elucidation of the molecular architecture of native mRNPs) is the establishment of a purification protocol for a specific mRNP, i.e. mRNPs containing only one kind of mRNA, from our model organism S. cerevisiae. In the first reporting period we made good progress in the establishment of such a purification protocol based on antisense oligonucleotides (ASOs) targeting the mRNA CCW12. We greatly improved our protocol in terms of specificity and the amount of the mRNA purified. However, we still have a relatively high protein background, a problem we are currently addressing. In addition, we are working on an alternative purification protocol based on an aptamer sequence cloned into the gene encoding the mRNA to be purified. We tested different aptamers, and currently improve the purification with the most promising one.
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.
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.
The groundbreaking nature of this project is the elucidation of mRNP packaging on a mechanistic as well as structural level. During the past 2.5 years we have come closer to this aim by our structural analyses, the improvement of the protocol for the purification of specific mRNPs and especially by our functional studies elucidating the function of different mRNP components in the process of mRNP assembly (see “work performed” above).
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.
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.