Kühn Uwe, Buschmann Juliane, Wahle Elmar
Institute of Biochemistry and Biotechnology, Martin Luther University Halle-Wittenberg, 06099 Halle, Germany.
RNA. 2017 Apr;23(4):473-482. doi: 10.1261/rna.057026.116. Epub 2017 Jan 17.
The nuclear poly(A) binding protein (PABPN1) has been suggested, on the basis of biochemical evidence, to play a role in mRNA polyadenylation by strongly increasing the processivity of poly(A) polymerase. While experiments in metazoans have tended to support such a role, the results were not unequivocal, and genetic data show that the ortholog of PABPN1, Pab2, is not involved in mRNA polyadenylation. The specific model in which PABPN1 increases the rate of poly(A) tail elongation has never been examined in vivo. Here, we have used 4-thiouridine pulse-labeling to examine the lengths of newly synthesized poly(A) tails in human cells. Knockdown of PABPN1 strongly reduced the synthesis of full-length tails of ∼250 nucleotides, as predicted from biochemical data. We have also purified Pab2 and the poly(A) polymerase, Pla1, and examined their in vitro activities. Whereas PABPN1 strongly increases the activity of its cognate poly(A) polymerase in vitro, Pab2 was unable to stimulate Pla1 to any significant extent. Thus, in vitro and in vivo data are consistent in supporting a role of PABPN1 but not Pab2 in the polyadenylation of mRNA precursors.
基于生化证据，有人提出核聚腺苷酸结合蛋白（PABPN1）通过显著提高聚腺苷酸聚合酶的持续合成能力，在mRNA聚腺苷酸化过程中发挥作用。虽然后生动物中的实验倾向于支持这一作用，但结果并不明确，而且遗传数据表明，PABPN1的直系同源物Pab2不参与mRNA聚腺苷酸化。PABPN1提高聚（A）尾延伸速率的具体模型从未在体内进行过研究。在这里，我们使用4-硫尿苷脉冲标记来检测人类细胞中新合成的聚（A）尾的长度。如生化数据所预测的那样，敲低PABPN1会强烈降低约250个核苷酸的全长尾的合成。我们还纯化了Pab2和聚腺苷酸聚合酶Pla1，并检测了它们的体外活性。虽然PABPN1在体外能强烈提高其同源聚腺苷酸聚合酶的活性，但Pab2在很大程度上无法刺激Pla1。因此，体外和体内数据一致支持PABPN1而非Pab2在mRNA前体聚腺苷酸化中的作用。
