Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates &lt;em&gt;in vitro&lt;/em&gt;

Jablonski, Joseph; Clementz, Mark A.; Ryan, Kevin; Valente, Susana T.

doi:10.3791/51309

Cited by 1 publication

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“…In vitro pre-mRNA 3′ cleavage reactions were carried out as previously described [22,33,35]. The total reaction volume was 12.5 μl and in place of nuclear extract the following DEAE-fractionated cleavage factors (~2–4 mg/ml total protein each) were included: 0.9 μl of CPSF, 0.45 μl of CstF, 1.8 μl of CF m .…”

Section: Methodsmentioning

confidence: 99%

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An investigation into the role of ATP in the mammalian pre-mRNA 3′ cleavage reaction

et al. 2016

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RNA Polymerase II transcribes beyond what later becomes the 3′ end of a mature messenger RNA (mRNA). The formation of most mRNA 3′ ends results from pre-mRNA cleavage followed by polyadenylation. In vitro studies have shown that low concentrations of ATP stimulate the 3′ cleavage reaction while high concentrations inhibit it, but the origin of these ATP effects is unknown. ATP might enable a cleavage factor kinase or activate a cleavage factor directly. To distinguish between these possibilities, we tested several ATP structural analogs in a pre-mRNA 3′ cleavage reaction reconstituted from DEAE-fractionated cleavage factors. We found that adenosine 5′-(β,γ-methylene)triphosphate (AMP-PCP) is an effective in vitro 3′ cleavage inhibitor with an IC50 of ~300 μM, but that most other ATP analogs, including adenosine 5′-(β,γ-imido)triphosphate, which cannot serve as a protein kinase substrate, promoted 3′ cleavage but less efficiently than ATP. In combination with previous literature data, our results do not support ATP stimulation of 3′ cleavage through cleavage factor phosphorylation in vitro. Instead, the more likely mechanism is that ATP stimulates cleavage factor activity through direct cleavage factor binding. The mammalian 3′ cleavage factors known to bind ATP include the cleavage factor II (CF IIm) Clp1 subunit, the CF Im25 subunit and poly(A) polymerase alpha (PAP). The yeast homolog of the CF IIm complex also binds ATP through yClp1. To investigate the mammalian complex, we used a cell-line expressing FLAG-tagged Clp1 to co-immunoprecipitate Pcf11 as a function of ATP concentration. FLAG-Clp1 co-precipitated Pcf11 with or without ATP and the complex was not affected by AMP-PCP. Diadenosine tetraphosphate (Ap4A), an ATP analog that binds the Nudix domain of the CF Im25 subunit with higher affinity than ATP, neither stimulated 3′ cleavage in place of ATP nor antagonized ATP-stimulated 3′ cleavage. The ATP-binding site of PAP was disrupted by site directed mutagenesis but a reconstituted 3′ cleavage reaction containing a mutant PAP unable to bind ATP nevertheless underwent ATP-stimulated 3′ cleavage. Fluctuating ATP levels might contribute to the regulation of pre-mRNA 3′ cleavage, but the three subunits investigated here do not appear to be responsible for the ATP-stimulation of pre-mRNA cleavage.

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Section: Methodsmentioning

confidence: 99%

“…In vitro cleavage was carried out in 12.5 μl at 30 °C for 2 h as described [22,33,35]. The final reaction component concentrations were: 1.25 μg of tRNA, 1.6 mM DTT, 50 mM CP, 6 u of recombinant RNase Inhibitor (Promega), 2.5% PVA, 2 mM EDTA, 3 nM 32 P-labeled L3 substrate, 5 μl of CSF, and PAP and ATP as indicated in Fig.…”

Section: Methodsmentioning

confidence: 99%