Characterization of dominant-negative mutants of the DEAH-box splicing factors Prp22 and Prp16
Characterization of dominant-negative mutants of the DEAH-box splicing factors Prp22 and Prp16
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DOI：
10.1074/jbc.m112473200
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发表时间：
2002-05-03
影响因子：
4.8
通讯作者：
Schwer, B
中科院分区：
文献类型：
--
作者：
Schneider, S;Hotz, HR;Schwer, B
酿酒酵母 Prp22 和 Prp16 是前 mRNA 剪接所需的 RNA 依赖性 ATP 酶。这两种蛋白都是核酸依赖性 NTPase 的 DEXH-box 家族的成员。之前对 Prp22 和 Prp16 的突变分析确定了保守基序 I (GYGKT)、II (DEAH) 和 VI (QRXGRXGR) 内的残基，这些残基是其生物活性所必需的。无功能的 Prp22 和 Prp16 突变体对细胞生长产生显着的负面影响。在这里，我们发现致命的 Prp22 突变体的过度表达会导致体内未剪接的前 mRNA 和切除的内含子的积累。通过纯化和表征重组突变蛋白来确定体内剪接的致死性和抑制性的生化基础。基序 II 中的致死性 Prp22 突变体 D603A 和 E604A 以及基序 VI 中的 Q804A 和 R808A 在 ATP 水解和剪接体释放 mRNA 方面存在缺陷，但在促进步骤 2 酯交换反应中具有活性。基序 I 中的致死性 Prp16 突变体 G378A 和 K379A；基序 II 中的 D473A 和 E474A；基序 VI 中的 Q685A、G688A、R689A 和 R692A 在 ATP 水解和步骤 2 酯交换化学方面存在缺陷。 Prp16 和 Prp22 的 ATP 酶缺陷突变体在体外与剪接体结合，并阻断各自野生型蛋白的反式功能。比较 Prp16 和 Prp22 的突变效应突出了前 mRNA 剪接中 ATP 依赖性步骤的共同和独特的结构要求。
Saccharomyces cerevisiae Prp22 and Prp16 are RNA-dependent ATPases required for pre-mRNA splicing. Both proteins are members of the DEXH-box family of nucleic acid-dependent NTPases. Prior mutational analysis of Prp22 and Prp16 identified residues within conserved motifs I (GYGKT), II (DEAH), and VI (QRXGRXGR) that are required for their biological activity. Nonfunctional Prp22 and Prp16 mutants exerted a dominant negative effect on cell growth. Here we show that overexpression of lethal Prp22 mutants leads to accumulation of unspliced pre-mRNAs and excised introns in vivo. The biochemical basis for the lethality and inhibition of splicing in vivo was determined by purifying and characterizing recombinant mutant proteins. The lethal Prp22 mutants D603A and E604A in motif II and Q804A and R808A in motif VI were defective for ATP hydrolysis and mRNA release from the spliceosome, but were active in promoting step 2 transesterification. Lethal Prp16 mutants G378A and K379A in motif I; D473A and E474A in motif II; and Q685A, G688A, R689A, and R692A in motif VI were defective for ATP hydrolysis and step 2 transesterification chemistry. The ATPase-defective mutants of Prp16 and Prp22 bound to spliceosomes in vitro and blocked the function of the respective wild-type proteins in trans. Comparing the mutational effects in Prp16 and Prp22 highlights common as well as distinct structural requirements for the ATP-dependent steps in pre-mRNA splicing.