Periodic Reporting for period 1 - POL2-TERM (Structural basis of co-transcriptional pre-mRNA 3’-end processing)
Reporting period: 2020-04-01 to 2022-03-31
In eukaryotes, mRNAs encoding for proteins are transcribed by RNA polymerase II (Pol II). However, the RNA emerging from Pol II, pre-mRNA, as the name suggests requires numerous enzymatic modifications prior to becoming mature, export-competent mRNA that can be handed over to the translation machinery. A critical step prior to the nuclear export of mRNA is the addition of a poly-adenine (poly-A) tail. The poly-A tail is a crucial factor governing the stability of the mRNA molecule and protects it from exosomal degradation, making it an important regulatory element for gene expression. Furthermore, numerous human pathologies have been associated with aberrations in 3’-end processing. In previous studies, the presence of Pol II has been shown to stimulate the cleavage of pre-mRNA and many studies suggest that the 3’-end processing machinery associates with the flexible C-terminal domain (CTD) of the largest subunit of Pol II. Taken together, it is widely accepted that 3’ end processing is a co-transcriptional event and remains to be one of the final frontiers into understanding transcription at a molecular level, but to date we lack the understanding of this at a structural level.
As Pol II transcribes past the last exon, motifs required for the recruitment of the 3’-end processing machinery are transcribed. Past studies have shown that these specific elements recruit specific sub-complexes to the nascent pre-mRNA. A U-rich upstream element is thought to recruit the CFIm complex, followed by the “AAUAAA” consensus hexamer that recruits the CPSF-core complex, the cleavage site that is actioned by the CPSF-cleavage module, and a downstream U/GU-rich element that recruits the CstF complex. These sub-complexes are thought to assemble onto scaffolding proteins that interact with the CTD of Pol II. Exactly how the complex assembles on the surface of Pol II and which factors govern co-transcriptional 3’-end processing remains to be established. Understanding the molecular and structural basis of co-transcriptional 3’-end processing will pave the way to elucidating the mechanisms of downstream processes such alternative-polyadenylation.
In order to tackle this fundamental biological problem, we set out to achieve the following objectives:
• Produce recombinant protein factors that govern 3’-end processing and endogenous Pol II
• Produce nucleic acid scaffolds suitable for complex formation between the above two
• Produce stable complexes of the above, and perform structural studies on them
As an outcome of the project, we have established protocols for the expression and purification of the 3’-processing subcomplexes. We have also in-vitro transcribed pre-mRNA substrates and incorporated them into a complex with Pol II and DNA. The comprehensive structural analysis of the co-transcriptional 3’-end processing complex is in progress and is anticipated to not only further the field of mRNA maturation but also provide better understanding of transcription regulation itself.
As Pol II transcribes past the last exon, motifs required for the recruitment of the 3’-end processing machinery are transcribed. Past studies have shown that these specific elements recruit specific sub-complexes to the nascent pre-mRNA. A U-rich upstream element is thought to recruit the CFIm complex, followed by the “AAUAAA” consensus hexamer that recruits the CPSF-core complex, the cleavage site that is actioned by the CPSF-cleavage module, and a downstream U/GU-rich element that recruits the CstF complex. These sub-complexes are thought to assemble onto scaffolding proteins that interact with the CTD of Pol II. Exactly how the complex assembles on the surface of Pol II and which factors govern co-transcriptional 3’-end processing remains to be established. Understanding the molecular and structural basis of co-transcriptional 3’-end processing will pave the way to elucidating the mechanisms of downstream processes such alternative-polyadenylation.
In order to tackle this fundamental biological problem, we set out to achieve the following objectives:
• Produce recombinant protein factors that govern 3’-end processing and endogenous Pol II
• Produce nucleic acid scaffolds suitable for complex formation between the above two
• Produce stable complexes of the above, and perform structural studies on them
As an outcome of the project, we have established protocols for the expression and purification of the 3’-processing subcomplexes. We have also in-vitro transcribed pre-mRNA substrates and incorporated them into a complex with Pol II and DNA. The comprehensive structural analysis of the co-transcriptional 3’-end processing complex is in progress and is anticipated to not only further the field of mRNA maturation but also provide better understanding of transcription regulation itself.
We have cloned and expressed full-length human proteins in insect cells as a strategy to preserve potential contacts that involve otherwise disordered regions. Due to the size and complexity of the target, we explored what tags and gene combinations were feasible for the expression and purification of stable sub-complexes. Initial trials of the assembly of the co-transcriptional 3’-end processing complex appear to be promising and are undergoing screening with cryo-EM analysis.
Due to the effects of COVID-19 pandemic, the research fellow was not able to attend any conferences or meetings during the project funding period. However the research fellow engaged in numerous local collaborative projects as part of the two-way transfer of knowledge and produced results that have been exploited in the form of high-impact journal publications and disseminated accordingly. These results and progress has helped the research fellow towards becoming an independent researcher by giving opportunities to exercise leadership and mentorship for junior researchers.
Due to the effects of COVID-19 pandemic, the research fellow was not able to attend any conferences or meetings during the project funding period. However the research fellow engaged in numerous local collaborative projects as part of the two-way transfer of knowledge and produced results that have been exploited in the form of high-impact journal publications and disseminated accordingly. These results and progress has helped the research fellow towards becoming an independent researcher by giving opportunities to exercise leadership and mentorship for junior researchers.
Our work represents the next step in systematically attempting to structurally characterize the co-transcriptional 3’-end processing complex incorporating the state-of-the-art knowledge. The cloning conducted in this project paves the way for future mutagenesis studies and will have value for the broader research community interested in this field. We have also improved methods involved in larger complex assembly and isolation, which is already being used by others in the department.