Processing and stability of inducibly expressed rpsO mRNA derivatives in Bacillus subtilis

Abstract

The Bacillus subtilis rpsO gene specifies a small (388-nucleotide), monocistronic mRNA that encodes ribosomal protein S15. We showed earlier that rpsO mRNA decay intermediates accumulated to a high level in a strain lacking polynucleotide phosphorylase. Here, we used inducibly expressed derivatives of rpsO, encoding smaller RNAs that had the complex 5' region deleted, to study aspects of mRNA processing in B. subtilis. An IPTG (isopropyl-beta-d-thiogalactopyranoside)-inducible rpsO transcript that contained lac sequences at the 5' end, called lac-rpsO RNA, was shown to undergo processing to result in an RNA that was 24 nucleotides shorter than full length. Such processing was dependent on the presence of an accessible 5' terminus; a lac-rpsO RNA that contained a strong stem-loop at the 5' end was not processed and was extremely stable. Interestingly, this stability depended also on ribosome binding to a nearby Shine-Dalgarno sequence but was independent of downstream translation. Either RNase J1 or RNase J2 was capable of processing lac-rpsO RNA, demonstrating for the first time a particular in vivo processing event that could be catalyzed by both enzymes. Decay intermediates were detected in the pnpA strain only for a lac-rpsO RNA that was untranslated. Analysis of processing of an untranslated lac-rpsO RNA in the pnpA strain shortly after induction of transcription suggested that endonuclease cleavage at 3'-proximal sites was an early step in turnover of mRNA.

FIG. 1.

rpsO transcript. (A) Schematic diagram of the rpsO transcript, showing the extents of the leader region, the CDS, and the 5′-proximal pseudoknot. The vertical filled rectangle represents the rpsO SD sequence. Hatched boxes represent rpsO sequences that are predicted to form relatively stable stem-loop structures. T, transcription terminator sequence. Complementarity of oligonucleotides (oligo) used for Northern blot probing and for RT experiments is shown by leftward-facing arrows. The 5′ portion of rpsO RNA that was replaced with lac sequences in the lac-rpsO construct is indicated. (B) Nucleotide sequence of the 5′ portion of lac-rpsO RNA, indicating the location of the lac operator (in the DNA), the SD sequence, and the XbaI site used for cloning. The location of the mapped 5′ end of the processed lac-rpsO RNA is indicated by the arrowheads, with the large arrowhead indicating the major 5′ end and the small arrowheads indicating the minor 5′ ends. The extent of complementarity of the upstream and downstream lac probes is shown by the leftward-facing arrows. The start codon of the lac-rpsO construct is underlined. The A→C change in the lac operator that makes lac-rpsO transcription IPTG independent is also indicated. The predicted secondary structure and stability of the RNA sequence which gives rise to the abundant “180-nt” decay intermediate in strains lacking PNPase are shown at the right. Complementarity of the ribosome binding site sequence to the 3′ end of 16S rRNA is indicated below the lac sequence.

FIG. 2.

lac-rpsO constructs. Schematic diagrams of native rpsO mRNA (wt) and lac-rpsO constructs (A to J) containing the 42-nt lac sequence (gray rectangle). The designation and length of each RNA are indicated to the right. In the diagrams, two stem-loop structures are drawn in the body of the RNA, one that gives rise to a prominent decay intermediate in the pnpA strain and one that comprises the transcription terminator. The expected sizes of the prominent decay intermediate in the pnpA strain for native rpsO (180 nt), for the lac-rpsO and lac-rpsO(SDX) constructs (102 nt), and for the lac-rpsO(5′-SS,SDX) and lac-rpsO(5′-US,SDX) constructs (128 nt) are indicated. A large X indicates a mutated SD site. The premature stop codon is indicated in constructs H through J.

FIG. 3.

Northern blot analysis of lac-rpsO RNA. (A) rpsO RNAs in the wild-type (wt) and pnpA mutant strains were probed with the 5′ rpsO probe (left) or the downstream lac probe (right). The marker lane (M) contained 5′-end-labeled fragments of a TaqI digest of plasmid pSE420 (4). Values to the left indicate molecular sizes in nucleotides. Migration of full-length (FL) rpsO mRNA, full-length lac-rpsO RNA, and the prominent 180-nt decay intermediate is shown. (For these blots, the marker fragments were run in a lane that was farther away from lanes containing RNA from wild-type and pnpA strains than is shown. The images have been manipulated to have the marker lanes located next to the relevant experimental lanes.) (B) Northern blot analysis of lac-rpsO RNA using the upstream and downstream lac probes. (C) IPTG-induced transcription of lac-rpsO RNA, probed with the downstream lac probe. Above each lane is the time (min) after IPTG addition. (D) Half-lives (t_1/2) of lac-rpsO and +24lac-rpsO RNAs. Above each lane is the time (min) after rifampin (rifampicin) addition. Migration of full-length lac-rpsO RNA and +24lac-rpsO RNA (+24) is indicated. The downstream lac probe was used. (E) Northern blot analysis of steady-state lac-rpsO(SDX) RNA probed with the downstream lac probe. In the lane with RNA from the pnpA strain, the asterisk indicates the RNA decay intermediate with the predicted size, as shown in Fig. 2.

FIG. 4.

RT analysis of 5′ ends of rpsO RNAs in strains containing integrated lac-rpsO, grown in the absence or presence of IPTG. The identities of the primer extension products are indicated at right, with black boxes marking migration of RT products that are likely the result of premature termination of RT transcription (txn). The sequencing reaction on the left was performed with single-stranded M13mp18 DNA, and the results serve as a nucleotide size marker.

FIG. 5.

Northern blot analysis of RNA encoded by lac-rpsO with a 5′-terminal addition. (A) Predicted secondary structure and free energy (kcal mol⁻¹) of the 5′-terminal sequences in lac-rpsO(5′-SS) and lac-rpsO(5′-MS) RNAs. The arrowhead points to the single nucleotide difference between the strong and moderate structures. (B) Patterns of steady-state RNA, showing increased concentrations of lac-rpsO(5′-SS) and lac-rpsO(5′-MS) RNAs. (C) Half-lives (t_1/2) of lac-rpsO RNAs with or without the 5′-terminal secondary structure. Above each lane is the time (min) after rifampin (rifampicin) addition. The half-lives of the full-length RNAs are indicated. Note that the exposure time for the lac-rpsO and lac-rpsO(5′-US) blots was 4 h, while the exposure time for the lac-rpsO(5′-SS) and lac-rpsO(5′-MS) blot was 40 min. (D) Northern blot analyses of half-lives of lac-rpsO constructs with the mutated SD sequence and with or without the 5′-terminal secondary structure. (E) Northern blot analyses of half-lives of lac-rpsO constructs with a stop codon at codon 3 and with or without the 5′-terminal secondary structure. Values to the left indicate molecular sizes in nucleotides. M, marker lane.

FIG. 6.

Northern blot analysis of induction patterns of lac-rpsO(SDX), showing the time course of accumulation of decay intermediates in the pnpA strain. The upstream lac probe was used. RNA was isolated before the addition of IPTG (lane B), immediately after addition of IPTG (lane 0), and at increasing times (min) thereafter. At right are schematic diagrams of lac-rpsO(SDX) decay intermediates, showing the approximate sites of the 3′ ends. Values to the left indicate molecular sizes in nucleotides. M, marker lane; FL, full length.

FIG. 7.

Endonuclease activities in lac-rpsO RNA processing. (A) Processing patterns of lac-rpsO RNA in endonuclease mutants. Names of deleted RNase genes (III, M5, J2, EndoA [A], and Mini-III [m3]) or IPTG-inducible RNase genes (J1, Z, and P) are indicated above the lanes. The control wild-type strain for the RNase J1, Z, and P mutants contained pMAP65. (B) Low-resolution Northern blot analysis of lac-rpsO RNA processing with the RNase J double mutant. The arrow indicates migration of +24-lac-rpsO RNA. (C) High-resolution Northern blot analysis of lac-rpsO RNA processing with the RNase J double mutant. Values to the left indicate molecular sizes in nucleotides. M, marker lane; wt, wild type; FL, full length; +, presence of IPTG; −, absence of IPTG.