Koslowsky D J, Yahampath G
Department of Microbiology, Michigan State University, East Lansing 48824, USA.
Mol Biochem Parasitol. 1997 Dec 1;90(1):81-94. doi: 10.1016/s0166-6851(97)00133-3.
The organization of the mitochondrial maxicircle genome of Trypanosoma brucei is unique in the close packing of the mRNA genes. For many of them, the 5' and 3' ends of adjacent transcripts overlap and formation of the proper 3' or 5' end can eliminate a portion of the coding sequence of the adjacent gene. Large, polycistronic transcripts have been detected. suggesting that mechanisms for precise cleavages at both 5' and 3' gene boundaries must exist. However, no common sequences near the ends of the mRNAs that could be candidates for control regions have been detected. In addition, nothing is known about how RNA editing interacts with and affects 5' and 3' processing and/or polyadenylation. Edited precursor transcripts have been detected, indicating that editing complexes can assemble prior to transcript cleavage. Because editing often initiates near the 3' end of the mRNA, the assembly of an editing complex in this region may influence the cleavage selection process. In order to determine the extent that RNA editing and 3' end-processing interact, RNAs were analyzed to determine the extent of editing in precursor RNAs and to determine if unedited transcripts can be cleaved and polyadenylated. Two overlapping RNA junctions were analyzed; the junction between NADH dehydrogenase (ND) subunit 7 and cytochrome oxidase (CO) subunit III, and the junction between CO subunit II and maxicircle unidentified reading frame (MURF) II. For both of these RNAs, editing affects restriction endonuclease recognition sequences, allowing us to analyze editing patterns by differential restriction digests. These analyses suggest that when the gRNA is supplied in trans, RNA editing and cleavage/polyadenylation are independent events and while they may influence one another, one event is not dependent on the other. Conversely, for the COII transcript, where the gRNA is located at the 3' end of the mRNA and appears to be supplied in cis, edited precursors were not detected. This suggests a requirement for a precise intramolecular interaction for COII editing that cannot form prior to 3' end-maturation.
布氏锥虫线粒体最大环基因组的组织方式在mRNA基因的紧密排列方面独具特色。对于其中许多基因而言，相邻转录本的5'端和3'端相互重叠，正确的3'端或5'端的形成会消除相邻基因的部分编码序列。已检测到大型多顺反子转录本，这表明在5'和3'基因边界处必定存在精确切割机制。然而，尚未检测到mRNA末端附近可能作为控制区域候选序列的共同序列。此外，对于RNA编辑如何与5'和3'加工及/或聚腺苷酸化相互作用并产生影响，目前一无所知。已检测到经过编辑的前体转录本，这表明编辑复合体可在转录本切割之前组装。由于编辑通常在mRNA的3'端附近起始，该区域编辑复合体的组装可能会影响切割选择过程。为了确定RNA编辑与3'端加工相互作用的程度，对RNA进行了分析，以确定前体RNA中的编辑程度，并确定未编辑的转录本是否能够被切割和聚腺苷酸化。分析了两个重叠的RNA连接点；烟酰胺腺嘌呤二核苷酸脱氢酶（ND）亚基7与细胞色素氧化酶（CO）亚基III之间的连接点，以及CO亚基II与最大环未鉴定阅读框（MURF）II之间的连接点。对于这两种RNA，编辑都会影响限制性内切酶识别序列，这使我们能够通过差异限制性酶切分析编辑模式。这些分析表明，当引导RNA（gRNA）以反式提供时，RNA编辑与切割/聚腺苷酸化是独立事件，虽然它们可能相互影响，但一个事件并不依赖于另一个事件。相反，对于COII转录本，其gRNA位于mRNA的3'端且似乎以顺式提供，未检测到经过编辑的前体。这表明COII编辑需要精确的分子内相互作用，而这种相互作用在3'端成熟之前无法形成。
