Detection of 5'- and 3'-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli

Abstract

Evidence is accumulating that small, noncoding RNAs are important regulatory molecules. Computational and experimental searches have led to the identification of approximately 60 small RNA genes in Escherichia coli. However, most of these studies focused on the intergenic regions and assumed that small RNAs were >50 nt. Thus, the previous screens missed small RNAs encoded on the antisense strand of protein-coding genes and small RNAs of <50 nt. To identify additional small RNAs, we carried out a cloning-based screen focused on RNAs of 30-65 nt. In this screen, we identified RNA species corresponding to fragments of rRNAs, tRNAs and known small RNAs. Several of the small RNAs also corresponded to 5'- and 3'-untranslated regions (UTRs) and internal fragments of mRNAs. Four of the 3'-UTR-derived RNAs were highly abundant and two showed expression patterns that differed from the corresponding mRNAs, suggesting independent functions for the 3'-UTR-derived small RNAs. We also detected three previously unidentified RNAs encoded in intergenic regions and RNAs from the long direct repeat and hok/sok elements. In addition, we identified a few small RNAs that are expressed opposite protein-coding genes and could base pair with 5' or 3' ends of the mRNAs with perfect complementarity.

Figure 1

Northern analysis of RNAs derived from 5′-UTR sequences. Samples were isolated from MG1655 wild type (wt) and hfq-1 mutant cells from exponential (E) and stationary (S) phase cells. Arrows indicate the predominant bands. RNA molecular weight markers were run with each set of samples for direct estimation of RNA transcript length. The RNA marker lane for one of the panels is shown, but only represents the approximate sizes for the other panels because there was slight variation in the running of the gels.

Figure 2

Northern analysis of RNAs derived from 3′-UTR sequences. The samples were the same as in Figure 1, and the RNA marker lane similarly corresponds to one of the panels. Small arrows indicate the full-length mRNA and large arrows indicate processed transcripts. The arrangement of the adjacent genes is shown below each panel. Genes present on the clockwise or Watson strand are denoted above the central line (>) and the genes on the counterclockwise or Crick strand are denoted below the central line (<).

Figure 3

Northern analysis of the (A) RyfB, RyfC and (B) RyfD RNAs derived from intergenic sequences. The samples were the same as in Figure 1, and the RNA marker lane similarly corresponds to one of the panels. Arrows indicate predominant bands. The arrangement of adjacent genes is shown below each panel as in Figure 2. Sequences capable of base pairing and diagrams of possible pairing arrangements are given below the gene arrangements. Bases that differ between E.coli 0157 and E.coli K12 are indicated in red. The 5′ ends of the RyfB, RyfC and RyfD RNAs (indicated by balls) all correspond to the actual transcription start.

Figure 4

Northern analysis of RNAs derived from hok/sok repetitive sequences. The samples were the same as in Figure 1, and the RNA marker lane similarly corresponds to one of the panels.

Figure 5

Northern analysis of antisense RNAs expressed near the (A) 5′ end or (B) 3′ end of the mRNA encoded on the opposing strand. The samples were the same as in Figure 1, and the RNA marker lane similarly corresponds to one of the panels. Small arrows indicate the full-length mRNA and large arrows indicate processed transcripts. The arrangement of adjacent genes is shown below each panel as in Figure 2. The 5′ ends of the RyjB and RyjC (indicated by balls) correspond to the actual transcription start.