Final Report Summary - AS-ARK-JV-GD (Regulation of coupling between transcription and splicing during cell cycle progression and UV irradiation)
Our project objectives were: 1/ to understand why the hyperphosphorylated RNA Polymerase II presented a slow elongation rate, whereas a phosphorylated polymerase is associated with active transcription; and 2/ to study the role of chromatin modifications on alternative splicing during cell cycle progression.
To tackle the first aim of the project, we have decided to follow two different approaches. The first one was to perform an RNA Polymerase II immunoprecipitation in presence or absence of UV irradiation and camptothecin treatment, that are responsible of its hyperphosphorylation, to know if a variation in the partner composition could explain the slowdown of elongation. This work was performed in collaboration with Dylan Taatjes (Boulder, Colorado, USA), a recognized expert in proteomics. The RNA polymerase II has been immunoprecipitated using three different antibodies (8WG16 that recognizes both non-phosphorylated and phosphorylated isoforms of the polymerase, PS2 and PS5 that recognize the phospho-serine 2 and 5, respectively), in two different cell compartments (nuclear extract and chromatin bound fraction). Out if the many identified partners, we looked for proteins that were present in the non-treated samples and absent in the treated ones, or vice versa. Then we decided to select proteins that present the same profile with the three antibodies. Among these proteins, one candidate specially called our attention and we are currently working on the role of this protein in RNA Polymerase II elongation during DNA damage.
The second approach was to study more deeply the phosphorylation patterns of the polymerase. In fact, the carboxy terminal domain, CTD, of the largest subunit (Rpb1) of the RNA polymerase II complex is the key domain that regulates its activity and is the target of multiple post-translational modifications, such as phosphorylation. The CTD is composed by 52 repetitions of the heptapeptide, which consensus sequence is Tyr-Ser-Pro-Thr-Ser-Pro-Ser, where 5 out of 7 residues are phosphorylable, meaning that the number of phosphorylation patterns is incredibly high however only one band, named RNA Pol II O, is detectable by western blot analysis. As the Rpb1 subunit weight is between 200 and 240 kDa, we decided to cut out the CTD, using an N-protease that does not cut into it, to decrease its size and facilitate its analysis. As the CTD is extremely unstable alone, we had to immunoprecipitate the entire Rpb1 subunit with an antibody directed against the phospho-CTD and, after an extensive wash, to digest it with the protease. Surprisingly, the CTD alone was still associated to the cross-linked antibody/bead complex, what allowed us to isolate it easily. Finally, by western blot analysis we have identified a new CTD post-translational pattern after UV irradiation and camptothecin treatment. The sequencing of this cut CTD is quite complicated and we are, together with CRG proteomic facility, trying to get it successfully done. Moreover, we are using this novel technique of CTD isolation to perform far western-blot to study the role of the phosphorylation patterns on the proteomics candidate binding.
While working on the aim 1 of the project, we found that the kinetic model of coupling between transcription and splicing developed by the outgoing lab was not complete and that some alternative splicing events are regulated on the opposite manner than the described mechanism. We found this discovery of the utmost importance and decided to add it to the main project. This work, that describes the mechanism of how slow elongation rate could also favor exon skipping, has been published in Molecular Cell (Dujardin et al., 2014).
For the aim two of the project we decided to synchronize cells, by using either Nocodazole or a double thymidine block and, while the cells were released, to check for modification of alternative splicing events and/or of RNA Polymerase II phosphorylation patterns. Unfortunately we did not identify any alternative splicing or RNA Polymerase II phosphorylation modification during cell cycle progression so far. We have decided to change our approach by searching for specific interactions between the general splicing factor U2AF65 and histone marks. For this objective, U2AF65 full length, U2AF65-RRM1-2 and SRSF1 have been purified from mammalian cells to keep the post-translational modifications that could be required for a proper interaction. SRSF1 is our control as it has been demonstrated that this protein could interact with some histone marks (Loomis et al., 2009). If U2AF65 can interact with some histone modifications, it should be through its RS domain, implicated in protein-protein interaction and not through its RNA Recognition Motif (RRM). For this I developed a mutant of U2AF65 containing only the RRM1 and RRM2 that will help us to discriminate non-specific interactions. After the purification of the three proteins, they were incubated on a histone peptide array (Active Motif) and then the arrays were developed with ECL. We have identified several histone modifications bound specifically by U2AF65 full length and we are now trying to find a correlation with splicing regulation. For instance, we have started to analyze the strength of the 3' splice sites of exons where these marks are found.
All the results obtained during the development of the aim 1 of the project will allow us to better understand alternative splicing regulation after DNA damage. The fact that a slowdown in transcription elongation can favor exon skipping, a finding that has been confirmed by the lab of David Bentley (Fong et al., 2015), is extremely important to understand co-transcriptional splicing regulation. This is true especially for cancer treatment, such as camptothecin, where genome-wide splicing should be analyzed. We moreover have identified a specific RNA Polymerase II partner that is released upon DNA damage and which could explain the general reduction in elongation rate of the polymerase. The discovery is really important to understand how splicing can be affected through transcription regulation. For the second aim, the fact that the general splicing factor U2AF65 is able to interact with several histone marks could lead to a better understanding of the mechanism of splicing regulation by chromatin structure, which is still unclear.
To tackle the first aim of the project, we have decided to follow two different approaches. The first one was to perform an RNA Polymerase II immunoprecipitation in presence or absence of UV irradiation and camptothecin treatment, that are responsible of its hyperphosphorylation, to know if a variation in the partner composition could explain the slowdown of elongation. This work was performed in collaboration with Dylan Taatjes (Boulder, Colorado, USA), a recognized expert in proteomics. The RNA polymerase II has been immunoprecipitated using three different antibodies (8WG16 that recognizes both non-phosphorylated and phosphorylated isoforms of the polymerase, PS2 and PS5 that recognize the phospho-serine 2 and 5, respectively), in two different cell compartments (nuclear extract and chromatin bound fraction). Out if the many identified partners, we looked for proteins that were present in the non-treated samples and absent in the treated ones, or vice versa. Then we decided to select proteins that present the same profile with the three antibodies. Among these proteins, one candidate specially called our attention and we are currently working on the role of this protein in RNA Polymerase II elongation during DNA damage.
The second approach was to study more deeply the phosphorylation patterns of the polymerase. In fact, the carboxy terminal domain, CTD, of the largest subunit (Rpb1) of the RNA polymerase II complex is the key domain that regulates its activity and is the target of multiple post-translational modifications, such as phosphorylation. The CTD is composed by 52 repetitions of the heptapeptide, which consensus sequence is Tyr-Ser-Pro-Thr-Ser-Pro-Ser, where 5 out of 7 residues are phosphorylable, meaning that the number of phosphorylation patterns is incredibly high however only one band, named RNA Pol II O, is detectable by western blot analysis. As the Rpb1 subunit weight is between 200 and 240 kDa, we decided to cut out the CTD, using an N-protease that does not cut into it, to decrease its size and facilitate its analysis. As the CTD is extremely unstable alone, we had to immunoprecipitate the entire Rpb1 subunit with an antibody directed against the phospho-CTD and, after an extensive wash, to digest it with the protease. Surprisingly, the CTD alone was still associated to the cross-linked antibody/bead complex, what allowed us to isolate it easily. Finally, by western blot analysis we have identified a new CTD post-translational pattern after UV irradiation and camptothecin treatment. The sequencing of this cut CTD is quite complicated and we are, together with CRG proteomic facility, trying to get it successfully done. Moreover, we are using this novel technique of CTD isolation to perform far western-blot to study the role of the phosphorylation patterns on the proteomics candidate binding.
While working on the aim 1 of the project, we found that the kinetic model of coupling between transcription and splicing developed by the outgoing lab was not complete and that some alternative splicing events are regulated on the opposite manner than the described mechanism. We found this discovery of the utmost importance and decided to add it to the main project. This work, that describes the mechanism of how slow elongation rate could also favor exon skipping, has been published in Molecular Cell (Dujardin et al., 2014).
For the aim two of the project we decided to synchronize cells, by using either Nocodazole or a double thymidine block and, while the cells were released, to check for modification of alternative splicing events and/or of RNA Polymerase II phosphorylation patterns. Unfortunately we did not identify any alternative splicing or RNA Polymerase II phosphorylation modification during cell cycle progression so far. We have decided to change our approach by searching for specific interactions between the general splicing factor U2AF65 and histone marks. For this objective, U2AF65 full length, U2AF65-RRM1-2 and SRSF1 have been purified from mammalian cells to keep the post-translational modifications that could be required for a proper interaction. SRSF1 is our control as it has been demonstrated that this protein could interact with some histone marks (Loomis et al., 2009). If U2AF65 can interact with some histone modifications, it should be through its RS domain, implicated in protein-protein interaction and not through its RNA Recognition Motif (RRM). For this I developed a mutant of U2AF65 containing only the RRM1 and RRM2 that will help us to discriminate non-specific interactions. After the purification of the three proteins, they were incubated on a histone peptide array (Active Motif) and then the arrays were developed with ECL. We have identified several histone modifications bound specifically by U2AF65 full length and we are now trying to find a correlation with splicing regulation. For instance, we have started to analyze the strength of the 3' splice sites of exons where these marks are found.
All the results obtained during the development of the aim 1 of the project will allow us to better understand alternative splicing regulation after DNA damage. The fact that a slowdown in transcription elongation can favor exon skipping, a finding that has been confirmed by the lab of David Bentley (Fong et al., 2015), is extremely important to understand co-transcriptional splicing regulation. This is true especially for cancer treatment, such as camptothecin, where genome-wide splicing should be analyzed. We moreover have identified a specific RNA Polymerase II partner that is released upon DNA damage and which could explain the general reduction in elongation rate of the polymerase. The discovery is really important to understand how splicing can be affected through transcription regulation. For the second aim, the fact that the general splicing factor U2AF65 is able to interact with several histone marks could lead to a better understanding of the mechanism of splicing regulation by chromatin structure, which is still unclear.