Qiu L, Moreira A, Kaplan G, Levitz R, Wang J Y, Xu C, Drlica K
Public Health Research Institute, New York, NY 10016, USA.
Mol Gen Genet. 1998 May;258(4):352-62. doi: 10.1007/s004380050741.
Hammerhead ribozymes were used as substrates to examine endoribonucleolytic activities in cell extracts and cultured human cells. Primer-extension analyses showed that ribozymes directed against tumor necrosis factor-alpha mRNA and human immunodeficiency virus type 1 tat mRNA were cleaved at UA and CA dinucleotides by extracts. Preferred cleavage sites were similar to those observed following digestion with RNase A, and cleavage was blocked by RNasin, an inhibitor of pyrimidine-specific ribonucleases. Removal of UA and CA dinucleotides rendered ribozymes more stable when incubated in cell extracts that were not significantly contaminated by extracellular nucleases. Placement of UA dinucleotides adjacent to a ribozyme in mRNA led to excision of the ribozyme from long transcripts during incubation in extracts. UA dinucleotides also made mRNA more labile than a control RNA when expressed from an endogenous plasmid gene in the human myeloid cell line U937. Similarly, UA and CA dinucleotides caused ribozymes to have a shorter half-life when delivered to U937 cells by lipofectin-mediated transformation. Taken together, these data indicate that one or more members of the pyrimidine-specific ribonuclease family is involved in the intracellular degradation of RNA, and they explain the paucity of UA dinucleotides in eukaryotic mRNA. Judicious manipulation of preferred target sequences of pyrimidine-specific ribonucleases may be useful in designing effective hammerhead ribozymes.
锤头状核酶被用作底物来检测细胞提取物和培养的人细胞中的核糖核酸内切酶活性。引物延伸分析表明，针对肿瘤坏死因子-α mRNA和人类免疫缺陷病毒1型tat mRNA的核酶在提取物作用下于UA和CA二核苷酸处被切割。优先切割位点与用核糖核酸酶A消化后观察到的位点相似，并且切割被核糖核酸酶抑制剂核糖核酸酶抑制剂（RNasin）阻断。在未被细胞外核酸酶显著污染的细胞提取物中孵育时，去除UA和CA二核苷酸可使核酶更稳定。在提取物中孵育期间，mRNA中与核酶相邻的UA二核苷酸的存在导致核酶从长转录本中切除。当从人髓样细胞系U937中的内源性质粒基因表达时，UA二核苷酸也使mRNA比对照RNA更不稳定。同样，当通过脂质体介导的转化递送至U937细胞时，UA和CA二核苷酸导致核酶的半衰期缩短。综上所述，这些数据表明嘧啶特异性核糖核酸酶家族的一个或多个成员参与RNA的细胞内降解，并且它们解释了真核mRNA中UA二核苷酸的稀少。对嘧啶特异性核糖核酸酶的优选靶序列进行明智的操作可能有助于设计有效的锤头状核酶。
