Periodic Reporting for period 4 - mRNP-PackArt (Nuclear mRNA Packaging and mRNP Architecture)
Reporting period: 2022-12-01 to 2024-05-31
One of biology’s key challenges is understanding how the complexity of life arises from just four nucleotide "letters" in the genetic code. Thus, this project aims to explore the mechanisms behind gene expression, the process by which this genetic information is brought to life. In the initial step of gene expression, the DNA is copied into messenger RNA (mRNA). Among other things, this mRNA is then packaged by proteins into a complex known as an mRNP (messenger ribonucleoprotein particle) and exported from the cell’s nucleus to its cytoplasm, where it directs protein synthesis. The assembly of mRNPs is not only essential for gene expression but also for the regulation this process. Disruptions in mRNP assembly are linked to a variety of diseases like neurological disorders (e. g. ALS), cancer and viral infections, such as HIV or influenza, highlighting its importance. Consequently, mRNP assembly is particularly important under stress, including illness or viral infection.
By deciphering the molecular mechanisms of mRNP assembly and how they change under stress, this project advances our understanding of gene regulation and provides a foundation for developing new therapies targeting mRNP-related diseases, offering significant societal and public health benefits.
During this project, we established the biochemical purification of a specific nuclear mRNP, i.e. a nuclear mRNP containing one specific mRNA, and initiated its structural analysis. We published the structure of the central complex TREX (doi: 10.1261/rna.079758.123) solved the structure of an mRNP component and analysed a trimeric mRNP subcomplex (to be published). Importantly, we revealed the functions of the mRNP component Npl3 (doi: 10.1093/nar/gkac1206) and of the Prp19C components Syf2 and Cwc15 (doi: 10.1261/rna.079944.124). In addition, we unravelled the function of three more mRNP components (to be published in three manuscripts) and analysed changes in mRNP assembly under stress (to be published). The importance of this project is also reflected by three review articles (dois: 10.1007/978-3-030-31434-7_1 10.1002/wrna.1582 10.3390/biom10091310) and one accepted review about mRNP assembly under stress (accepted in the Journal Molecular Cell, currently under embargo).
The overarching objective is to unravel mRNP assembly mechanisms and stress-related changes, providing the knowledge for future therapeutic developments.
By deciphering the molecular mechanisms of mRNP assembly and how they change under stress, this project advances our understanding of gene regulation and provides a foundation for developing new therapies targeting mRNP-related diseases, offering significant societal and public health benefits.
During this project, we established the biochemical purification of a specific nuclear mRNP, i.e. a nuclear mRNP containing one specific mRNA, and initiated its structural analysis. We published the structure of the central complex TREX (doi: 10.1261/rna.079758.123) solved the structure of an mRNP component and analysed a trimeric mRNP subcomplex (to be published). Importantly, we revealed the functions of the mRNP component Npl3 (doi: 10.1093/nar/gkac1206) and of the Prp19C components Syf2 and Cwc15 (doi: 10.1261/rna.079944.124). In addition, we unravelled the function of three more mRNP components (to be published in three manuscripts) and analysed changes in mRNP assembly under stress (to be published). The importance of this project is also reflected by three review articles (dois: 10.1007/978-3-030-31434-7_1 10.1002/wrna.1582 10.3390/biom10091310) and one accepted review about mRNP assembly under stress (accepted in the Journal Molecular Cell, currently under embargo).
The overarching objective is to unravel mRNP assembly mechanisms and stress-related changes, providing the knowledge for future therapeutic developments.
The overarching goal of this project is to understand the mechanisms of nuclear mRNP assembly, focusing both on its structural and functional aspects.
For Aim 1 (Determination of mRNP protein composition) and Aim 3 (Elucidation of native mRNP architecture), we developed two innovative purification methods based on antisense oligonucleotides and aptamers to isolate specific mRNPs in S. cerevisiae. While we successfully enriched target mRNAs and associated mRNP components, high protein background and heterogeneity in particle structure (as seen in electron microscopy) prompted us to discontinue this approach. However, these findings informed future refinements in mRNP purification techniques.
In Aim 3, we achieved a major success by determining the structure of the native S. cerevisiae TREX complex using XL-MS datasets and negative-stain electron microscopy. This work was published (doi: 10.1261/rna.079758.123) marking a critical milestone. Additionally, we expressed mRNP components from the thermophile C. thermophilum for structural studies, and we determined the structure of the TREX component Sub2, with ongoing efforts to resolve a trimeric complex involving Sub2 by cryo-EM.
For Aim 2 (Mechanism of mRNP packaging), we published groundbreaking discoveries on the roles of Npl3 and Prp19C in mRNP assembly and mRNA export (dois: 10.1093/nar/gkac1206 10.1261/rna.079944.124). Further analysis on the function of the TREX component Hpr1 and another two key mRNP component are in progress, with publication expected soon. We also extended this research to investigate mRNP assembly under stress, and our first manuscript on this topic is in preparation.
The significance of this work is underscored by three comprehensive reviews (dois: 10.1007/978-3-030-31434-7_1 10.1002/wrna.1582 10.3390/biom10091310) and an accepted review on mRNP assembly under stress conditions.
In conclusion, while the purification of specific mRNPs posed technical challenges, all other aspects of the project – including the structural elucidation and functional characterization of mRNP components – produced highly valuable results. These findings advance the field and are either already published or soon to be published.
For Aim 1 (Determination of mRNP protein composition) and Aim 3 (Elucidation of native mRNP architecture), we developed two innovative purification methods based on antisense oligonucleotides and aptamers to isolate specific mRNPs in S. cerevisiae. While we successfully enriched target mRNAs and associated mRNP components, high protein background and heterogeneity in particle structure (as seen in electron microscopy) prompted us to discontinue this approach. However, these findings informed future refinements in mRNP purification techniques.
In Aim 3, we achieved a major success by determining the structure of the native S. cerevisiae TREX complex using XL-MS datasets and negative-stain electron microscopy. This work was published (doi: 10.1261/rna.079758.123) marking a critical milestone. Additionally, we expressed mRNP components from the thermophile C. thermophilum for structural studies, and we determined the structure of the TREX component Sub2, with ongoing efforts to resolve a trimeric complex involving Sub2 by cryo-EM.
For Aim 2 (Mechanism of mRNP packaging), we published groundbreaking discoveries on the roles of Npl3 and Prp19C in mRNP assembly and mRNA export (dois: 10.1093/nar/gkac1206 10.1261/rna.079944.124). Further analysis on the function of the TREX component Hpr1 and another two key mRNP component are in progress, with publication expected soon. We also extended this research to investigate mRNP assembly under stress, and our first manuscript on this topic is in preparation.
The significance of this work is underscored by three comprehensive reviews (dois: 10.1007/978-3-030-31434-7_1 10.1002/wrna.1582 10.3390/biom10091310) and an accepted review on mRNP assembly under stress conditions.
In conclusion, while the purification of specific mRNPs posed technical challenges, all other aspects of the project – including the structural elucidation and functional characterization of mRNP components – produced highly valuable results. These findings advance the field and are either already published or soon to be published.
The groundbreaking nature of this project is the elucidation of mRNP packaging on a mechanistic as well as structural level.