Four factors are required for 3'-end cleavage of pre-mRNAs.

Takagaki, Yoshio; Ryner, Lisa; Manley, James L.

doi:10.1101/gad.3.11.1711

Cited by 209 publications

(178 citation statements)

References 52 publications

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“…We used cells expressing different levels of p53, such as MCF7 cells (normal levels) and HeLa cells (low levels). HeLa cells were included in this study because most of the functional properties of the 3 0 processing machinery have been described in this biological system (Takagaki et al, 1989;Hirose and Manley, 1998;Manley, 1999, 2001). As described before, p53 accumulation was detected in samples from MCF7 cells exposed to UV treatment ( Figure 1d, compare lanes 1 and 2).…”

Section: Resultsmentioning

confidence: 99%

p53 inhibits mRNA 3′ processing through its interaction with the CstF/BARD1 complex

et al. 2011

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The mechanisms involved in the p53-dependent control of gene expression following DNA damage have not been completely elucidated. Here, we show that the p53 C terminus associates with factors that are required for the ultraviolet (UV)-induced inhibition of the mRNA 3 0 cleavage step of the polyadenylation reaction, such as the tumor suppressor BARD1 and the 3 0 processing factor cleavage-stimulation factor 1 (CstF1). We found that p53 can coexist in complexes with CstF and BARD1 in extracts of UV-treated cells, suggesting a role for p53 in mRNA 3 0 cleavage following DNA damage. Consistent with this, we found that p53 inhibits 3 0 cleavage in vitro and that there is a reverse correlation between the levels of p53 expression and the levels of mRNA 3 0 cleavage under different cellular conditions. Supporting these results, a tumor-associated mutation in p53 not only decreases the interaction with BARD1 and CstF, but also decreases the UV-induced inhibition of 3 0 processing, all of which is restored by wild-type-p53 expression. We also found that p53 expression levels affect the polyadenylation levels of housekeeping genes, but not of p21 and c-fos genes, which are involved in the DNA damage response (DDR). Here, we identify a novel 3 0 RNA processing inhibitory function of p53, adding a new level of complexity to the DDR by linking RNA processing to the p53 network.

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Section: Resultsmentioning

confidence: 99%

p53 inhibits mRNA 3′ processing through its interaction with the CstF/BARD1 complex

et al. 2011

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“…CF I m is required for cleavage in vitro [162], and appears to be unique to higher eukaryotes. Three polypeptides (25 kD, 59 kD and 68 kD) as well as a less abundant one (72 kD) generally copurify with CF I m activity from HeLa cell nuclear extract.…”

Section: Mammalian Cleavage Factor I (Cf I M )mentioning

confidence: 99%

Protein factors in pre-mRNA 3′-end processing

Mandel

Bai

Tong

2007

Cell. Mol. Life Sci.

364

482

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Most eukaryotic mRNA precursors (pre-mRNAs) must undergo extensive processing, including cleavage and polyadenylation at the 3′-end. Processing at the 3′-end is controlled by sequence elements in the pre-mRNA (cis elements) as well as protein factors. Despite the seeming biochemical simplicity of the processing reactions, more than 14 proteins have been identified for the mammalian complex, and more than 20 proteins have been identified for the yeast complex. The 3′-end processing machinery also has important roles in transcription and splicing. The mammalian machinery contains several sub-complexes, including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factor I (CF I m ), and cleavage factor II (CF II m ). Additional protein factors include poly(A) polymerase (PAP), poly(A) binding protein (PABP), symplekin, and the C-terminal domain (CTD) of RNA polymerase II largest subunit. The yeast machinery includes cleavage factor IA (CF IA), cleavage factor IB (CF IB), and cleavage and polyadenylation factor (CPF).

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“…In this assay, a uniformly 32 P-labeled RNA containing the SV40L poly(A) signals is mixed with DEAEfractionated CPSF, CstF, and CFm (unseparated CFIm and CFIIm) activities (Gilmartin et al 1988;Takagaki et al 1989;Ryan 2007). In the absence of CP, no RNA cleavage takes place (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Nuclear extract preparation was performed as previously described (Ryan 2007). The extract was fractionated on DEAE-Sepharose also as previously described (Ryan 2007), based on established methods (Gilmartin et al 1988;Takagaki et al 1989;Ryan et al 2002). Cleavage factor fractions were concentrated by ammonium sulfate precipitation and dialyzed thoroughly at 4°C in Buffer D50AS.…”

Section: Hela Cleavage Factor Preparationmentioning

confidence: 99%

“…However, it has long been known that the 39 cleavage reaction can be reconstituted in the absence of transcription using cleavage factors supplied by HeLa nuclear extract (Moore and Sharp 1985) or fractionated from it (Gilmartin et al 1988;Takagaki et al 1989). Creatine phosphate (CP), a naturally occurring phosphagen, was initially included in the reconstituted reaction for the intended purpose of maintaining the ATP pool (Moore and Sharp 1985;Takagaki et al 1988), but it was later found that ATP is not required for cleavage by mammalian cleavage factors (Hirose and Manley 1997).…”

Section: Introductionmentioning

confidence: 99%

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Small molecule activators of pre-mRNA 3′ cleavage

Ryan

Khleborodova²,

Pan³

et al. 2009

RNA

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39 Cleavage and polyadenylation are obligatory steps in the biogenesis of most mammalian pre-mRNAs. In vitro reconstitution of the 39 cleavage reaction from human cleavage factors requires high concentrations of creatine phosphate (CP), though how CP activates cleavage is not known. Previously, we proposed that CP might work by competitively inhibiting a cleavagesuppressing serine/threonine (S/T) phosphatase. Here we show that fluoride/EDTA, a general S/T phosphatase inhibitor, activates in vitro cleavage in place of CP. Subsequent testing of inhibitors specific for different S/T phosphatases showed that inhibitors of the PPM family of S/T phosphatases, which includes PP2C, but not the PPP family, which includes PP1, PP2A, and PP2B, activated 39 cleavage in vitro. In particular, NCI 83633, an inhibitor of PP2C, activated extensive 39 cleavage at a concentration 50-fold below that required by fluoride or CP. The testing of structural analogs led to the identification of a more potent compound that activated 39 cleavage at 200 mM. While testing CP analogs to understand the origin of its cleavage activation effect, we found phosphocholine to be a more effective activator than CP. The minimal structural determinants of 39 cleavage activation by phosphocholine were identified. Our results describe a much improved small molecule activator of in vitro pre-mRNA cleavage, identify the molecular determinants of cleavage activation by phosphoamines such as phosphocholine, and suggest that a PPM family phosphatase is involved in the negative regulation of mammalian pre-mRNA 39 cleavage.

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Four factors are required for 3'-end cleavage of pre-mRNAs.

Cited by 209 publications

References 52 publications

p53 inhibits mRNA 3′ processing through its interaction with the CstF/BARD1 complex

p53 inhibits mRNA 3′ processing through its interaction with the CstF/BARD1 complex

Protein factors in pre-mRNA 3′-end processing

Small molecule activators of pre-mRNA 3′ cleavage

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