Analysis of post-transcriptional RNA metabolism in prokaryotes

Abstract

Post-transcriptional RNA metabolic pathways play important roles in permitting prokaryotes to operate under a variety of environmental conditions. Although significant progress has been made during the last decade in deciphering RNA processing pathways in a number of bacteria, a complete understanding of post-transcriptional RNA metabolism in any single microorganism is far from reality. Here we describe multiple experimental approaches that can be used to study mRNA stability, tRNA and rRNA processing, sRNA metabolism, and polyadenylation in prokaryotes. The methods described here can be readily utilized in both Gram-negative and Gram-positive bacteria with simple modifications.

Keywords: Cleavage specificity; Northern analysis; RNA isolation; RNAseq; SMART cDNA; mRNA decay; tRNA processing.

Fig. 1.

Steady-state RNA isolated from a wild-type and RNase III deletion (Δrnc) strains using the RNAsnap™ method. Total RNA (500 ng/lane) was separated on a 1% agarose gel and visualized with ethidium bromide staining. The various rRNA species are indicated to the right of the gel. The presence of the 30S primary rRNA transcript can easily be distinguished in the Δrnc mutant.

Fig. 2.

Analysis of 5' ends of leuX tRNA primary transcripts by primer extension [24]. All E. coli strains used in this study were derived from MG1693 (rph-1), which was considered wild-type. MG1693 has a single base pair deletion in the rph gene resulting in a frame-shift mutation [31]. The genotypes of the strains are noted at the top of the lane. Total RNA isolated from all the strains were reverse transcribed using a ³²P-end labelled leuX tRNA specific primer and the cDNAs were separated on a 6% polyacrylamide sequencing gel. A leuX DNA sequencing ladder (CTAG) generated by same leuX tRNA specific primer was also separated along with the cDNAs and was used to identify the transcription initiation site (I) and the mature 5' end (II). The primer extension analysis also identified several RNase E cleavage sites (indicated as *) in the rnpA49 mutant, which disappeared in the rne-1 rnpA49 strain.

Fig. 3.

Graphical presentation of SMART™ cloning. P1, gene specific RT primer; P2, SMART template switching primer and P3, anchor primer. The procedure is as described in the text (section 6.2)

Fig. 4.

Analysis of proK tRNA 5' and 3' end processing in an RNase P mutant using the self-ligation RT-PCR cloning technique. The cartoon at the top represents a graphical presentation of genomic organization of the monocistronic proK gene. The transcription initiation sites (underlined) upstream of the mature proK tRNA (rectangle) and the downstream immature sequences with the transcription terminator are shown [32]. The downward arrow (↓) represents the positions of either a 5’ or a 3’ end based on sequencing of the cDNAs. The numbers of 5' and 3' ends detected were noted on top of the arrow. The number of transcripts with unprocessed 5' ends increased significantly in the rnpA49 mutant compared to the wild-type control [32]. Both strains also contain rph-1 allele (see legend to Figure 2.)