Periodic Reporting for period 3 - RNApaxport (Mechanisms of human mRNA packaging and export)
Période du rapport: 2023-11-01 au 2025-04-30
Messenger RNA (mRNA) is the genetic carrier of information from DNA to protein. In eukaryotes, mRNA production in the nucleus is separated from mRNA translation in the cytoplasm. This separation requires mechanisms for selective mRNA transport, to ensure that only fully matured mRNAs are transported into the cytoplasm and yield functional proteins. Despite its importance, the molecular basis for how mature mRNAs are recognised, discriminated from immature precursors, and prepared ('packaged') for nuclear export remains poorly understood. Further, defects in mRNA maturation and packaging are frequently linked with disease, such as cancer. A molecular understanding of these key gene expression steps may therefore aid in the therapeutic treatment of disease. In this project, we combine structural and functional studies to study how the essential and conserved transcription-export export complex selects and packages human mRNA-protein complexes for productive nuclear export. Further, we aim to understand how human mRNPs organize in three-dimensions, which is important to understand mRNA regulation. Towards these goals, we defined three objectives to (1) determine the three-dimensional structure of the transcription-export complex, (2) of parts of mRNA-protein complexes, and (3) of complete maturing human mRNPs. Combined with functional validation of the structural data, we expect that these findings will make a major contribution to our understanding of human mRNA packaging and export. In the current reporting period, we have completed aim 1, and are making progress towards aims 2 and 3. Our current results are summarised in two publications (Puehringer et al., eLife, 2020; Pacheco-Fiallos et al., Nature, 2023), which provide novel mechanisms for how human mRNPs are recognised and packaged for mRNA nuclear export.
In the current reporting period, work has been done on all three project aims (1-3). The main results of this period are summarised in two publications (Puehringer et al., eLife, 2020; Pacheco-Fiallos et al., Nature, 2023), which mark the completion of aim 1 and partial progress in aims 2 and 3. On the performed work performed and main results: For aim 1, we have produced the recombinant human core transcription-export complex and determined its cryo-EM structure. Biochemical validation and an endogenous purification illustrated that the unexpected multimeric state of the core transcription-export complex is adopted both in vitro and of complexes obtained from human cells. These results indicated that multivalent interactions between the transcription-export complex and mRNP components may be important for mRNP recognition and packaging. These findings marked the completion of aim 1. We have further, towards aim 2 b, reconstituted two minimal mRNA-bound exon junction in complex with partial or complete transcription-export complex subunits and determined their cryo-EM structures. Together with biochemical validation and the findings from aim 1, the data indicate that specific multivalent protein-protein and non-sequence specific protein-mRNA interactions are the basis for the selective recognition of mRNPs for nuclear export licensing. Towards aim 3, we have established tools for the purification of 'average' human mRNPs engaged with the complete transcription-export complex from human cells, and have subjected these to cryo-EM, cryo-ET, crosslinking mass spectrometry, and biochemical characterisation. These results show that human mRNPs for three-dimensional globules, which are coated on their surface by transcription-export complexes. These findings have major implications on the mechanisms of human mRNA biogenesis and regulation.
The results obtained in the current reporting period have led to exciting and new hypothesis in mRNA biology, which go substantially beyond the state of the art. Specifically, our results show that human mRNA-protein complexes (mRNPs) form compact three-dimensional globules that are coated on their surface by transcription-export complexes. We provide a possible mechanism based on in vitro reconstitution of mRNPs for the recognition and packaging of mRNA through the transcription-export complex. In technical terms, we go beyond the state-of-the-art and illustrate how cryo-electron microscopy single particle analysis, cryo-electron tomography, high-end protein-protein crosslinking coupled to mass spectrometry, and biochemistry can be combined to study heterogenous and low-abundance human protein nucleic acid complexes. In the next reporting period, we expect additional insights on how mRNPs form and are regulated by mRNP binding proteins that enable mRNA export. This goals will be achieved as parts of aims 2 and 3, and are realistic to achieve until the end of the project.