Ribozyme Stability, Exon Skipping, and a Potential Role for RNA Helicase in Group I Intron Splicing by Coxiella burnetii
Ribozyme Stability, Exon Skipping, and a Potential Role for RNA Helicase in Group I Intron Splicing by Coxiella burnetii
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DOI：
10.1128/jb.05472-11
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发表时间：
2011-10-01
影响因子：
3.2
通讯作者：
Minnick, Michael F.
中科院分区：
文献类型：
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作者：
Hicks, Linda D.;Warrier, Indu;Minnick, Michael F.
贝氏柯克斯体（Coxiella burnetii）是人类Q热的病原体，其23 S rRNA基因含有异常高数量的保守、自私的遗传元件，包括两个I组内含子，称为Cbu. L1917（L1917）和Cbu. L1951（L1951）。为了更好地理解内含子在柯克斯体生物学中的作用，我们确定了内含子的内在稳定时间段，L1917和L1951的编码核酶的体外半衰期分别为15天和5天，这可能是由于它们的大小不同（分别为551和1，559个碱基）、各自RNA结构的相对紧密度和单链RNA的量。两个内含子的体内半衰期也被确定为类似于11分钟通过使用RNA酶保护试验和大肠杆菌模型。在同步培养的C. Burnetii，并发现与16 S rRNA的相似;即，第0天至第3天，核酶水平显着增加，然后保持稳定，直至感染后8天。16 S rRNA和核酶水平下降，在静止和死亡阶段（第8至14天）。Coxiella内含子RNA的显著稳定性可能是由它们与核糖体的结合所赋予的，这种化学计量关系被确定为每500个核糖体中有一种核酶。在培养的前5天，发现剪接的不准确性（外显子2跳跃）增加，每130万个拷贝中约有一个不正确剪接的23 S rRNA。L1917内含子剪接的体外效率显着增强的重组柯克斯体RNA DEAD盒解旋酶（CBU_0670）的存在下，相对于对照组，这表明这种酶可能作为一个内含子RNA剪接促进剂在体内。
The 23S rRNA gene of Coxiella burnetii, the agent of Q fever in humans, contains an unusually high number of conserved, selfish genetic elements, including two group I introns, termed Cbu.L1917 (L1917) and Cbu.L1951 (L1951). To better understand the role that introns play in Coxiella's biology, we determined the intrinsic stability time periods (in vitro half-lives) of the encoded ribozymes to be similar to 15 days for L1917 and similar to 5 days for L1951, possibly due to differences in their sizes (551 and 1,559 bases, respectively), relative degrees of compactness of the respective RNA structures, and amounts of single-stranded RNA. In vivo half-lives for both introns were also determined to be similar to 11 min by the use of RNase protection assays and an Escherichia coli model. Intron RNAs were quantified in synchronous cultures of C. burnetii and found to closely parallel those of 16S rRNA; i.e., ribozyme levels significantly increased between days 0 and 3 and then remained stable until 8 days postinfection. Both 16S rRNA and ribozyme levels fell during the stationary and death phases (days 8 to 14). The marked stability of the Coxiella intron RNAs is presumably conferred by their association with ribosomes, a stoichiometric relationship that was determined to be one ribozyme, of either type, per 500 ribosomes. Inaccuracies in splicing (exon 2 skipping) were found to increase during the first 5 days in culture, with a rate of approximately one improperly spliced 23S rRNA per 1.3 million copies. The in vitro efficiency of L1917 intron splicing was significantly enhanced in the presence of a recombinant Coxiella RNA DEAD-box helicase (CBU_0670) relative to that of controls, suggesting that this enzyme may serve as an intron RNA splice facilitator in vivo.