Enhancers regulate 3′ end processing activity to control expression of alternative 3′UTR isoforms

Kwon, Buki; Fansler, Mervin M.; Patel, Neil D.; Lee, Jihye; Ma, Weirui; Mayr, Christine

doi:10.1101/2020.08.17.254193

Cited by 5 publications

(6 citation statements)

References 121 publications

(351 reference statements)

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“…In other cases, endogenous 3′UTR deletions led to the discovery of 3′UTR-dependent mRNA localization phenotypes (Terenzio et al, 2018). Although our data indicate that p53 abundance regulation is 3′UTR-independent, the 3′UTR may still have important functions possibly through control of protein localization or protein activity as has been shown for other proteins (Berkovits and Mayr, 2015;Moretti et al, 2015;Terenzio et al, 2018;Lee and Mayr, 2019;Mayr, 2019;Bae et al, 2020;Fernandes and Buchan, 2020;Kwon, 2020).…”

Section: Discussionmentioning

confidence: 49%

Endogenous p53 expression in human and mouse is not regulated by its 3′UTR

Mitschka

Mayr

2021

eLife

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The TP53 gene encodes the tumor suppressor p53 which is functionally inactivated in many human cancers. Numerous studies suggested that 3′UTR-mediated p53 expression regulation plays a role in tumorigenesis and could be exploited for therapeutic purposes. However, these studies did not investigate post-transcriptional regulation of the native TP53 gene. Here, we used CRISPR/Cas9 to delete the human and mouse TP53/Trp53 3′UTRs while preserving endogenous mRNA processing. This revealed that the endogenous 3′UTR is not involved in regulating p53 mRNA or protein expression neither in steady state nor after genotoxic stress. Using reporter assays, we confirmed the previously observed repressive effects of the isolated 3′UTR. However, addition of the TP53 coding region to the reporter had a dominant negative impact on expression as its repressive effect was stronger and abrogated the contribution of the 3′UTR. Our data highlight the importance of genetic models in the validation of post-transcriptional gene regulatory effects.

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Section: Discussionmentioning

confidence: 49%

Endogenous p53 expression in human and mouse is not regulated by its 3′UTR

Mitschka

Mayr

2021

eLife

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“…Notably, a recent study that deleted 3′UTR sequences in several cytokine genes found similar discrepancies between reporter-based assays and gene expression from native contexts (Zhao et al, 2017). Although our data indicate that p53 abundance regulation is 3′UTR-independent, the 3′UTR may still have important functions possibly through control of protein localization or protein activity as has been shown for other proteins (Berkovits and Mayr, 2015; Moretti et al, 2015; Terenzio et al, 2018; Lee and Mayr, 2019; Fernandes and Buchan, 2020; Bae et al, 2020; Kwon, 2020; Mayr, 2019).…”

Section: Discussionmentioning

confidence: 53%

“…Our observations support the recently established role of the coding region as major regulator of mRNA stability and translation 13,14 . Although our data indicate that p53 abundance regulation is 3’UTR-independent, the human p53 3’UTR may still have important functions possibly through control of protein localization or protein activity as has been shown for other proteins 9,15-17 .…”

Section: Mainmentioning

confidence: 52%

Endogenous p53 expression in human and mouse is not regulated by its 3′UTR

Mitschka

Mayr

2020

Preprint

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Inactivation of p53 occurs in many cancers. microRNAs and RNA-binding proteins are thought to repress p53 expression through its 3’ untranslated region (3’UTR), thus contributing to tumorigenesis. We used CRISPR/Cas9 to delete the human and mouse p53 3’UTRs while preserving endogenous mRNA processing. This revealed that the endogenous 3’UTR is not involved in the regulation of p53 mRNA or protein expression, neither in steady state nor after genotoxic stress.

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“…RBBP6 did not copurify with CPSF and was therefore expressed and purified separately. As a model pre-mRNA substrate, we used a 218-nt fragment of the SV40 pre-mRNA, which has been shown to be cleaved efficiently in vivo ( Ryner and Manley 1987 ; Kwon et al 2021 ). We omitted the PAP enzyme and ATP from the reactions to focus on the cleavage step of pre-mRNA 3′ end processing ( Fig.…”

Section: Resultsmentioning

confidence: 99%

RBBP6 activates the pre-mRNA 3′ end processing machinery in humans

et al. 2022

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3′ end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3′ end of the mature transcript, which is important for mRNA localization, translation, and stability. Cleavage must therefore be tightly regulated. Here, we reconstituted specific and efficient 3′ endonuclease activity of human CPSF with purified proteins. This required the seven-subunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm), and, importantly, the multidomain protein RBBP6. Unlike its yeast homolog Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to humans. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in mRNA processing.

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Enhancers regulate 3′ end processing activity to control expression of alternative 3′UTR isoforms

Cited by 5 publications

References 121 publications

Endogenous p53 expression in human and mouse is not regulated by its 3′UTR

Endogenous p53 expression in human and mouse is not regulated by its 3′UTR

Endogenous p53 expression in human and mouse is not regulated by its 3′UTR

RBBP6 activates the pre-mRNA 3′ end processing machinery in humans

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