Department of Biochemistry, Medical University of Vienna and University of Veterinary Medicine, Vienna, Austria.
Nucleic Acids Res. 2010 Jun;38(11):3794-808. doi: 10.1093/nar/gkq032. Epub 2010 Mar 26.
An unexpectedly high number of regulatory RNAs have been recently discovered that fine-tune the function of genes at all levels of expression. We employed Genomic SELEX, a method to identify protein-binding RNAs encoded in the genome, to search for further regulatory RNAs in Escherichia coli. We used the global regulator protein Hfq as bait, because it can interact with a large number of RNAs, promoting their interaction. The enriched SELEX pool was subjected to deep sequencing, and 8865 sequences were mapped to the E. coli genome. These short sequences represent genomic Hfq-aptamers and are part of potential regulatory elements within RNA molecules. The motif 5'-AAYAAYAA-3' was enriched in the selected RNAs and confers low-nanomolar affinity to Hfq. The motif was confirmed to bind Hfq by DMS footprinting. The Hfq aptamers are 4-fold more frequent on the antisense strand of protein coding genes than on the sense strand. They were enriched opposite to translation start sites or opposite to intervening sequences between ORFs in operons. These results expand the repertoire of Hfq targets and also suggest that Hfq might regulate the expression of a large number of genes via interaction with cis-antisense RNAs.
最近发现了大量的调控 RNA，它们可以在基因表达的各个层面上精细地调节基因的功能。我们采用了基因组 SELEX 方法，该方法用于鉴定基因组中编码的蛋白结合 RNA，以在大肠杆菌中寻找更多的调控 RNA。我们使用全局调控蛋白 Hfq 作为诱饵，因为它可以与大量的 RNA 相互作用，促进它们的相互作用。富集的 SELEX 池被进行深度测序，8865 个序列被映射到大肠杆菌基因组上。这些短序列代表基因组 Hfq-适体，是 RNA 分子内潜在调控元件的一部分。富含 5'-AAYAAYAA-3'的基序在所选 RNA 中富集，并赋予 Hfq 低纳摩尔亲和力。通过 DMS 足迹法证实了该基序与 Hfq 的结合。Hfq 适体在编码蛋白基因的反义链上的出现频率比在有义链上高出 4 倍。它们在翻译起始位点的对面或操纵子中 ORF 之间的间隔序列的对面富集。这些结果扩展了 Hfq 靶标的范围，也表明 Hfq 可能通过与顺式反义 RNA 的相互作用来调节大量基因的表达。
