Khleborodova Asya, Pan Xiaozhou, Nagre Nagaraja N, Ryan Kevin
Department of Chemistry and Biochemistry, The City College of New York, The City University, New York, NY 10031, USA; Biochemistry Ph.D. Program, The City University of New York Graduate Center, New York, NY 10016, USA.
Department of Chemistry and Biochemistry, The City College of New York, The City University, New York, NY 10031, USA.
Biochimie. 2016 Jun;125:213-22. doi: 10.1016/j.biochi.2016.04.004. Epub 2016 Apr 6.
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.
RNA聚合酶II的转录范围超出了后来成为成熟信使RNA（mRNA）3'端的区域。大多数mRNA 3'端的形成是由前体mRNA切割后再进行聚腺苷酸化所致。体外研究表明，低浓度的ATP刺激3'切割反应，而高浓度则抑制该反应，但这些ATP效应的起源尚不清楚。ATP可能使切割因子激酶发挥作用或直接激活切割因子。为了区分这些可能性，我们在由DEAE分级分离的切割因子重构的前体mRNA 3'切割反应中测试了几种ATP结构类似物。我们发现腺苷5'-（β，γ-亚甲基）三磷酸（AMP-PCP）是一种有效的体外3'切割抑制剂，IC50约为300μM，但大多数其他ATP类似物，包括不能作为蛋白激酶底物的腺苷5'-（β，γ-亚氨基）三磷酸，促进了3'切割，但效率低于ATP。结合先前的文献数据，我们的结果不支持ATP通过体外切割因子磷酸化刺激3'切割。相反，更可能的机制是ATP通过直接结合切割因子来刺激切割因子活性。已知结合ATP的哺乳动物3'切割因子包括切割因子II（CF IIm）的Clp1亚基、CF Im25亚基和聚（A）聚合酶α（PAP）。CF IIm复合物的酵母同源物也通过yClp1结合ATP。为了研究哺乳动物复合物，我们使用表达FLAG标签的Clp1的细胞系，根据ATP浓度共免疫沉淀Pcf11。FLAG-Clp1在有或没有ATP的情况下都能共沉淀Pcf11，并且该复合物不受AMP-PCP的影响。四磷酸二腺苷（Ap4A）是一种ATP类似物，它比ATP以更高的亲和力结合CF Im25亚基的Nudix结构域，它既不能代替ATP刺激3'切割，也不能拮抗ATP刺激的3'切割。通过定点诱变破坏了PAP的ATP结合位点，但含有不能结合ATP的突变型PAP的重构3'切割反应仍然经历了ATP刺激的3'切割。波动的ATP水平可能有助于前体mRNA 3'切割的调节，但这里研究的三个亚基似乎不负责ATP对前体mRNA切割的刺激作用。
