Periodic Reporting for period 1 - RNA Smuggling (Mechanistic analysis of piRNA precursor nuclear export)
Período documentado: 2022-01-01 hasta 2023-12-31
The export of messenger RNA (mRNA) from the nucleus to the cytoplasm is a key step in eukaryotic gene expression. mRNAs are transcribed by RNA Polymerase II as pre-mRNAs, which subsequently undergo a series of processing steps such as capping, splicing and poly-adenylation to generate a mature mRNA. Each of these processing steps is accompanied by the deposition of proteins on the mRNA, serving as ‘processing marks’, leading to the establishment of nuclear ribonucleoprotein complexes (mRNPs). To discriminate mature mRNPs form pre-mRNAs and other RNAs in the nucleus these processing marks must be recognized by the mRNA export machinery Despite the fact that key components of mRNA export have been discovered in genetic screen over two decades ago we still lack a mechanistic understanding of this essential process.
piRNAs are 23-30nt short RNAs which are key for the silencing of transposable elements, and thus for the maintenance of genome integrity in the germline. piRNAs are processed from a long precursor transcript in the cytoplasm. Unlike mRNAs, this precursor does not undergo processing in the nucleus and thus lacks key features of mRNPs. However, the precursor has to be exported to the cytoplasm to to evade nuclear degradation and to allow for the generation of piRNAs. Interestingly, previous research had uncovered a piRNA precursor export pathway in the Drosophila germline that hijacks key mRNA export components, namely the THO complex and the DExD ATPase Uap56, and combines them with pathway specific factors, such as Bootlegger and Nxf3, a homolog of the mRNA export factor Nxf1.
Here I aimed at dissecting the export of piRNA precursor transcripts in mechanistic detail. Understanding this specialized pathway held promise to gain new insights into the function of enigmatic mRNA export factors, such as the THO complex and UAP56, thus generating an entry point for the study of mRNA export. This approach turned out to be fruitful and I could uncover a mechanistic model for the nuclear export of messenger RNA, presumably conserved in all eukaryotes from yeast to human.
piRNAs are 23-30nt short RNAs which are key for the silencing of transposable elements, and thus for the maintenance of genome integrity in the germline. piRNAs are processed from a long precursor transcript in the cytoplasm. Unlike mRNAs, this precursor does not undergo processing in the nucleus and thus lacks key features of mRNPs. However, the precursor has to be exported to the cytoplasm to to evade nuclear degradation and to allow for the generation of piRNAs. Interestingly, previous research had uncovered a piRNA precursor export pathway in the Drosophila germline that hijacks key mRNA export components, namely the THO complex and the DExD ATPase Uap56, and combines them with pathway specific factors, such as Bootlegger and Nxf3, a homolog of the mRNA export factor Nxf1.
Here I aimed at dissecting the export of piRNA precursor transcripts in mechanistic detail. Understanding this specialized pathway held promise to gain new insights into the function of enigmatic mRNA export factors, such as the THO complex and UAP56, thus generating an entry point for the study of mRNA export. This approach turned out to be fruitful and I could uncover a mechanistic model for the nuclear export of messenger RNA, presumably conserved in all eukaryotes from yeast to human.
In my work I first used the piRNA precursor export pathway in the Drosophila germline to test multiple hypothesis on the function and interplay of mRNA / piRNA precursor export components. To my surprise, the results I obtained suggested that most of these hypotheses were incorrect and that UAP56 and the THO complex would function differently than anticipated based on the current literature.
This allowed me then to develop a new model for mRNA export, which I tested combining biochemistry, in silico protein interaction screening, cellular assays and structural biology. My new mRNA export model suggests that the DeXD box ATPase UAP56 itself is another ‘maturation mark’ for mRNAs. Binding of UAP56 to the RNA leads to the remodeling of a nuclear THO-bound mRNP, yielding mature, export competent mRNPs. UAP56 mediates then the docking of the mRNP at the nuclear pore complex anchored where the hand over to the mRNA nuclear export factor NXF1 likely happens (Hohmann et al, BioRxiv 2024, https://doi.org/10.1101/2024.03.24.586400(se abrirá en una nueva ventana)).
With the new mRNA export model in hand, I can again focus on the piRNA precursor export. Here I aim at understanding how the mRNA export machinery can be ‘tricked’ into exporting a transcript that violates mRNA quality control hallmarks and would otherwise be funneled into degradation. My results rationalize (1) how the export machinery is recruited directly to the piRNA precursor transcription site and (2) what the nature of a piRNA ribonucleoprotein, piRNP, is. These results further suggest a finely tuned balance in the evolution of the piRNA precursor nuclear export pathway where selectively parts of the mRNA export pathway are co-opted while others are actively avoided.
I have been able to present my research at several international conferences. My work on the nuclear export of mRNA is under review in a scientific journal and has been published on BioRxiv (https://doi.org/10.1101/2024.03.24.586400(se abrirá en una nueva ventana)). The content of this publication has additionally been highlighted on Twitter (for example: https://twitter.com/HohmannUlrich/status/1773234210486075577(se abrirá en una nueva ventana)).
This allowed me then to develop a new model for mRNA export, which I tested combining biochemistry, in silico protein interaction screening, cellular assays and structural biology. My new mRNA export model suggests that the DeXD box ATPase UAP56 itself is another ‘maturation mark’ for mRNAs. Binding of UAP56 to the RNA leads to the remodeling of a nuclear THO-bound mRNP, yielding mature, export competent mRNPs. UAP56 mediates then the docking of the mRNP at the nuclear pore complex anchored where the hand over to the mRNA nuclear export factor NXF1 likely happens (Hohmann et al, BioRxiv 2024, https://doi.org/10.1101/2024.03.24.586400(se abrirá en una nueva ventana)).
With the new mRNA export model in hand, I can again focus on the piRNA precursor export. Here I aim at understanding how the mRNA export machinery can be ‘tricked’ into exporting a transcript that violates mRNA quality control hallmarks and would otherwise be funneled into degradation. My results rationalize (1) how the export machinery is recruited directly to the piRNA precursor transcription site and (2) what the nature of a piRNA ribonucleoprotein, piRNP, is. These results further suggest a finely tuned balance in the evolution of the piRNA precursor nuclear export pathway where selectively parts of the mRNA export pathway are co-opted while others are actively avoided.
I have been able to present my research at several international conferences. My work on the nuclear export of mRNA is under review in a scientific journal and has been published on BioRxiv (https://doi.org/10.1101/2024.03.24.586400(se abrirá en una nueva ventana)). The content of this publication has additionally been highlighted on Twitter (for example: https://twitter.com/HohmannUlrich/status/1773234210486075577(se abrirá en una nueva ventana)).
The nuclear export of mRNA is a key step in eukaryotic gene expression. Despite key players in this pathway being discovered over 20 years ago, we lack a mechanistic understanding of the process. My work proposes a model for how nuclear mRNA export functions, closing this major gap in molecular biology textbooks. This model has broad implications not only for understanding mRNA export regulation but also for the study of mRNA quality control.
Besides the key players implicated in our novel model for mRNA nuclear export, which are conserved throughout eukaryotes, many additional proteins have been linked to mRNA export. Their precise function is however mostly unknown. Importantly, many of these proteins, as well as core mRNA export factors, have been found involved in various forms of cancer. My work does not directly address the impact of any of these factors in human medicine, but with the novel mechanistic model for mRNA export proposed here we are now able to study their function and to potentially understand their role in disease.
Besides the key players implicated in our novel model for mRNA nuclear export, which are conserved throughout eukaryotes, many additional proteins have been linked to mRNA export. Their precise function is however mostly unknown. Importantly, many of these proteins, as well as core mRNA export factors, have been found involved in various forms of cancer. My work does not directly address the impact of any of these factors in human medicine, but with the novel mechanistic model for mRNA export proposed here we are now able to study their function and to potentially understand their role in disease.