A twin UGUA motif directs the balance between gene isoforms through CFIm and the mTORC1 signaling pathway
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eLife assessment
In this valuable report, the authors explore the connection between mTORC signaling and APA regulated by the CFIm complex. Using a combination of genetic and functional genomics approaches, the study reveals that a twin UGUA motif is a potent cis acting regulation of PAS usage that is recognized by CFIm. Overall, the evidence in general is convincing and supports the conclusions and provides the field with additional mechanistic insight of how signaling connects to APA regulatory machinery.
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Abstract
Alternative polyadenylation (APA) generates mRNA isoforms and diversifies gene expression. Here we report the discovery that the mTORC1 signaling pathway balances the expression of two Trim9/TRIM9 isoforms through APA regulation in human and mouse. We showed that CFIm components, CPSF6 and NUDT21, promote the short Trim9/TRIM9 isoform ( Trim9-S/TRIM9-S ) expression. In addition, we identified an evolutionarily conserved twin UGUA motif, UGUAYUGUA, in TRIM9-S polyadenylation site (PAS) that is critical for its regulation by CPSF6. We found additional CPSF6-regulated PASs with similar twin UGUA motifs in human and experimentally validated the twin UGUA motif functionality in BMPR1B , MOB4 , and BRD4-L . Importantly, we showed that inserting a twin UGUA motif into a heterologous PAS was sufficient to confer regulation by CPSF6 and mTORC1. Our study reveals an evolutionarily conserved mechanism to regulate gene isoform expression by mTORC1 and implicates possible gene isoform imbalance in cancer and neurological disorders with mTORC1 pathway dysregulation.
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eLife assessment
In this valuable report, the authors explore the connection between mTORC signaling and APA regulated by the CFIm complex. Using a combination of genetic and functional genomics approaches, the study reveals that a twin UGUA motif is a potent cis acting regulation of PAS usage that is recognized by CFIm. Overall, the evidence in general is convincing and supports the conclusions and provides the field with additional mechanistic insight of how signaling connects to APA regulatory machinery.
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Reviewer #1 (Public Review):
The authors set out to further probe how mTORC signaling can impact metabolism by modulating the function of the APA machinery. The major strength of the paper is the identification of a 'twin UGUA' motif that governs PAS selection as dictated by the CFIm complex. Further, the authors show that the twin UGUA motif is not just necessary but is sufficient to confer sensitivity to mTORC activity and the CFIm complex regulation. The weaknesses of the paper include a tenuous connection between mTORC signaling and CFIm as it was not rigorously established how CFIm gets activated/deactivated when mTORC is modulated.
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Reviewer #2 (Public Review):
In this work, Herron et al. investigated the impact of mTORC1 and CFIm on the expression of the Trim9/TRIM9 isoforms in both mouse and human. They extend upon their cTAG-PAPERCLIP method and demonstrated that systemic AAV injection of cell type-specific Cre recombinases to cTag-PABP mice is a feasible method of APA profiling. From this they show that mTORC1 hyperactivation promotes a shift towards the long Trim9 isoform, Trim9-L. They further provide evidence that the mTORC1 signalling pathway controls Trim9/TRIM9 isoform usage in both human and mouse with high mTORC1 promoting usage of the long isoform and low mTORC1 favouring the short isoform. They also show that the CFIm subunits CPSF6 and NUDT21 play a crucial role in the use of the TRIM9-S/Trim9-S isoform and demonstrate the importance of a twin UGUA …
Reviewer #2 (Public Review):
In this work, Herron et al. investigated the impact of mTORC1 and CFIm on the expression of the Trim9/TRIM9 isoforms in both mouse and human. They extend upon their cTAG-PAPERCLIP method and demonstrated that systemic AAV injection of cell type-specific Cre recombinases to cTag-PABP mice is a feasible method of APA profiling. From this they show that mTORC1 hyperactivation promotes a shift towards the long Trim9 isoform, Trim9-L. They further provide evidence that the mTORC1 signalling pathway controls Trim9/TRIM9 isoform usage in both human and mouse with high mTORC1 promoting usage of the long isoform and low mTORC1 favouring the short isoform. They also show that the CFIm subunits CPSF6 and NUDT21 play a crucial role in the use of the TRIM9-S/Trim9-S isoform and demonstrate the importance of a twin UGUA motif in this PAS for its regulation by CPSF6. Additionally, they find that this twin UGUA motif is functionally present in the human BMPR1B, MOB4 and BRD4 genes and that insertion of the twin UGUA motif into a heterologous PAS is enough to confer regulation by both CPSF6 and mTORC1. Critically, the position of the twin UGUA motif directs preferential cleavage and polyadenylation to generate an isoform, such that it's presence can result in the use of a short isoform (TRIM9) or a long isoform (BMPR1B, MOB4 and BRD4). The work expands upon the known cis-regulatory motifs for CPSF6 and provides further evidence of a connection between the mTORC1 signalling pathway and CPSF6-mediated alternative polyadenylation. The mechanistic connection between TORC1 signalling and CPSF6 function is, however, still opaque. An experiment probing the connection between TORC1 signalling and the nuclear-cytoplasmic shuttling of CPSF6 with its activity (regulating APA) would significantly strengthen the study. Most conclusions are well supported by the presented data.
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Reviewer #3 (Public Review):
Alternative polyadenylation is an important aspect of RNA processing that can alter the type or amount of proteins that are produced from a gene, with consequences for many aspects of biology. Herron et al. set out to identify how the mTORC1 pathway, which regulates cellular metabolism, influences alternative polyadenylation in the mouse brain. They identified a novel mTORC1-regulated gene with alternative polyadenylation - TRIM9 - and convincingly demonstrate that its alternative polyadenylation is controlled by the CFIm complex of the cleavage and polyadenylation machinery. A major strength of these results is that the authors use multiple orthogonal methods - including PAPERCLIP, qPCR and western blotting, to demonstrate that TRIM9 is regulated by mTORC1 and CFIm. They also demonstrate that this …
Reviewer #3 (Public Review):
Alternative polyadenylation is an important aspect of RNA processing that can alter the type or amount of proteins that are produced from a gene, with consequences for many aspects of biology. Herron et al. set out to identify how the mTORC1 pathway, which regulates cellular metabolism, influences alternative polyadenylation in the mouse brain. They identified a novel mTORC1-regulated gene with alternative polyadenylation - TRIM9 - and convincingly demonstrate that its alternative polyadenylation is controlled by the CFIm complex of the cleavage and polyadenylation machinery. A major strength of these results is that the authors use multiple orthogonal methods - including PAPERCLIP, qPCR and western blotting, to demonstrate that TRIM9 is regulated by mTORC1 and CFIm. They also demonstrate that this regulation is conserved between mice and humans by using multiple different model systems, and use synthetic reporter constructs to identify the cis-regulatory elements that are responsible for TRIM9 regulation by CFIm. These results highlight the importance of alternative polyadenylation in controlling gene expression and are important for researchers wishing to understand how the mTORC1 pathway functions.
The authors also identify that a "twin" UGUA motif in the poly(A) site of the short form of TRIM9 is responsible for its regulation by CFIm. They show that this motif is conserved across mammals and suggest that the adjacent UGUA motifs are necessary for regulation by CFIm. The evidence supporting this aspect of the manuscript is incomplete because the authors only ever mutate both UGUA motifs of TRIM9, and so it is not possible to determine whether the full motif or only one of the UGUA motifs is necessary for regulation, nor whether the effect of the two UGUA motifs is simply additive. The only evidence for the necessity of the full twin motif comes from a synthetic JUNB reporter construct, where a single UGUA motif was insufficient to induce proximal polyadenylation. However, given that there is previous evidence that individual UGUA motifs can act as enhancers of polyadenylation, this may be due to context-specific issues with the JUNB reporter, and evidence from different contexts would make the authors conclusions more convincing.
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