Long-Distance RNA-RNA Interactions in the Coronavirus Genome Form High-Order Structures Promoting Discontinuous RNA Synthesis during Transcription
Long-Distance RNA-RNA Interactions in the Coronavirus Genome Form High-Order Structures Promoting Discontinuous RNA Synthesis during Transcription
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DOI：
10.1128/jvi.01782-12
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发表时间：
2013-01-01
影响因子：
5.4
通讯作者：
Sola, Isabel
中科院分区：
文献类型：
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作者：
Mateos-Gomez, Pedro A.;Morales, Lucia;Sola, Isabel
冠状病毒（CoV）转录需要一个高频重组过程，将新合成的负链亚基因组RNA拷贝连接到仅存在一次的先导区域，即基因组的5'端。这个不连续的RNA合成步骤是基于先导区转录调节序列（TRSs）和新生负链RNA中每个基因之前的转录调节序列（TRSs）之间的互补性。此外，模板切换需要RNA基因组结构域的物理接近，它们相距2万到3万个核苷酸。在本报告中，研究表明，这种重组步骤的有效性是通过位于控制每个基因表达的trs附近的RNA基序之间的新的长距离RNA-RNA相互作用以及它们靠近基因组5'端的互补序列来促进的。这些相互作用将把重组过程中涉及的基序聚集在一起。这一发现表明，在冠状病毒基因组中形成高阶RNA结构对于控制至少病毒N基因的表达是必要的。冠状病毒复制子的工程表明，这些长距离相互作用对转录的要求，其中新鉴定的序列之间的互补性被破坏。此外，突变病毒中互补性的破坏导致突变恢复互补性、野生型转录水平和细胞培养中传代的病毒滴度。冠状病毒中所涉及的RNA基序的系统发育保守性加强了这些高阶结构与病毒转录的相关性。
Coronavirus (CoV) transcription requires a high-frequency recombination process that links newly synthesized minus-strand subgenomic RNA copies to the leader region, which is present only once, at the 5' end of the genome. This discontinuous RNA synthesis step is based on the complementarity between the transcription-regulating sequences (TRSs) at the leader region and those preceding each gene in the nascent minus-strand RNA. Furthermore, the template switch requires the physical proximity of RNA genome domains located between 20,000 and 30,000 nucleotides apart. In this report, it is shown that the efficacy of this recombination step is promoted by novel additional long-distance RNA-RNA interactions between RNA motifs located close to the TRSs controlling the expression of each gene and their complementary sequences mapping close to the 5' end of the genome. These interactions would bring together the motifs involved in the recombination process. This finding indicates that the formation of high-order RNA structures in the CoV genome is necessary to control the expression of at least the viral N gene. The requirement of these long-distance interactions for transcription was shown by the engineering of CoV replicons in which the complementarity between the newly identified sequences was disrupted. Furthermore, disruption of complementarity in mutant viruses led to mutations that restored complementarity, wild-type transcription levels, and viral titers by passage in cell cultures. The relevance of these high-order structures for virus transcription is reinforced by the phylogenetic conservation of the involved RNA motifs in CoVs.