RNA-Seq and RNA immunoprecipitation analyses of the transcriptome of Streptomyces coelicolor identify substrates for RNase III

Abstract

RNase III is a key enzyme in the pathways of RNA degradation and processing in bacteria and has been suggested as a global regulator of antibiotic production in Streptomyces coelicolor. Using RNA-Seq, we have examined the transcriptomes of S. coelicolor M145 and an RNase III (rnc)-null mutant of that strain. RNA preparations with reduced levels of structural RNAs were prepared by subtractive hybridization prior to RNA-Seq analysis. We initially identified 7,800 transcripts of known and putative protein-coding genes in M145 and the null mutant, JSE1880, along with transcripts of 21 rRNA genes and 65 tRNA genes. Approximately 3,100 of the protein-coding transcripts were categorized as low-abundance transcripts. For further analysis, we selected those transcripts of known and putative protein-coding genes whose levels changed by ≥ 2-fold between the two S. coelicolor strains and organized those transcripts into 16 functional categories. We refined our analysis by performing RNA immunoprecipitation of the mRNA preparation from JSE1880 using a mutant RNase III protein that binds to transcripts but does not cleave them. This analysis identified ca. 800 transcripts that were enriched in the RNA immunoprecipitates, including 28 transcripts whose levels also changed by ≥ 2-fold in the RNA-Seq analysis. We compare our results with those obtained by microarray analysis of the S. coelicolor transcriptome and with studies describing the characterization of small noncoding RNAs. We have also used the RNA immunoprecipitation results to identify new substrates for RNase III cleavage.

Fig 1

RT-PCR analysis of transcripts from S. coelicolor M145 (parental strain) and JSE1880 (rnc-null mutant). RT-PCR was performed as described previously (8, 16), and products were analyzed after 20 PCR cycles to ensure linearity between band intensity and the number of cycles. Lane 1, size standards (the arrow indicates the 500-bp standard); lane 2, mRNA from M145 as the template for reverse transcription; lane 3, M145 BARD RNA; lane 4, M145 mock BARD RNA; lanes 5 and 8, JSE1880 mRNA; lanes 6 and 9, JSE1880 BARD RNA; lanes 7 and 10, JSE1880, mock BARD RNA. The primers used for the PCRs for lanes 2 to 7 were specific for the readthrough transcript from the S. coelicolor rpsO-pnp operon and produced a 460-bp fragment, while the primers used for lanes 8 to 10 were specific for ramR, producing a 583-bp fragment.

Fig 2

Single-gene resolution plot of the transcriptomes of S. coelicolor M145 and JSE1880. The RNA-Seq analysis was performed as described in Materials and Methods. The transcripts that are noted specifically in the plot are those for which the largest numbers of sequencing reads were observed with either M145 or JSE1880 mRNAs as templates. It should be noted that the S. coelicolor chromosome is linear (4), thus the gene ID numbers indicate the order of genes beginning at the left end of the chromosome.

Fig 3

Outline of the RNA immunoprecipitation (RIP) procedure. As described in detail in Materials and Methods, the procedure involved binding the RNase III mutant protein, RNase III D70A, to S. coelicolor mRNA, binding anti-RNase III D70A antibody to the RNA-RNase III D70A complexes, and isolation of the antibody-RNA-RNase III D70A complexes using magnetic beads.

Fig 4

Scatter plot of the enrichment ratios for BARD transcripts. Enrichment ratios, defined as the percentage of the total reads for each transcript in the BARD divided by the percentage of the total reads for the same transcript in the starting JSE1880 mRNA pool, were calculated and are plotted against gene ID number. The data are plotted using a semilog scale to spread the points over the area of the graph. Open squares represent the enrichment ratios for the 28 genes whose transcript levels increased by ≥2-fold in the JSE1880 mRNA pool compared with those of M145.

Fig 5

Primer extension of products from RNase III cleavage assays. RNase III digestions of nonradioactive transcripts of SCO5737 and SCO3982 to SCO3988 were performed as described previously (10). Primer extension of digestion products was performed as described previously (10), and the same primer used for extension was also used to generate the sequencing ladders shown in the figure. Lanes 1, 2, and 3 show the extension products obtained representing RNase III cleavages in SCO5837, SCO3987, and SCO3988. The results shown in the left panel were taken from a previously published figure (15) and are reproduced here with permission from the Journal of Bacteriology.

Fig 6

Mfold (49) models of the regions of SCO5737, SCO3987, and SCO3988 that contain RNase III cleavage sites. Bases are numbered from the first base in the translation start codon for each gene, and arrows indicate the major RNase III cleavage sites determined by primer extension.