RNase III participates in GadY-dependent cleavage of the gadX-gadW mRNA - PubMed (original) (raw)

RNase III participates in GadY-dependent cleavage of the gadX-gadW mRNA

Jason A Opdyke et al. J Mol Biol. 2011.

Abstract

The adjacent gadX and gadW genes encode transcription regulators that are part of a complex regulatory circuit controlling the Escherichia coli response to acid stress. We previously showed that the small RNA GadY positively regulates gadX mRNA levels. The gadY gene is located directly downstream of the gadX coding sequence on the opposite strand of the chromosome. We now report that gadX is transcribed in an operon with gadW, although this full-length mRNA does not accumulate. Base pairing of the GadY small RNA with the intergenic region of the gadX-gadW mRNA results in directed processing events within the region of complementarity. The resulting two halves of the cleaved mRNA accumulate to much higher levels than the unprocessed mRNA. We examined the ribonucleases required for this processing, and found that multiple enzymes are involved in the GadY-directed cleavage including the double-strand RNA-specific endoribonuclease RNase III.

Published by Elsevier Ltd.

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Figures

Fig. 1

Fig. 1

GadY RNA-directed mRNA cleavage. (a) Diagram of the gadXWY region. The gadY promoter mutation is indicated by the X. (b) Levels of the gadX (∼1.0 kb) and gadW (∼1.1 kb) mRNAs in cells without a plasmid, carrying the vector control (pRI), or overexpressing GadY (pRI-GadY). The top band in both panels of (b) is likely to be due to cross hybridization with the 16S rRNA. (c) Diagram of the cat replacement of gadX. (d) Levels of the cat (∼0.8 kb) mRNA in cells carrying the vector control (pRI) or overexpressing GadY (pRI-GadY) in a background (GSO129) in which the gadX promoter and coding sequence were replaced with the cat promoter and coding sequence. (e) Diagram of the cat replacement of gadX and gfp replacement of gadW. (f) Levels of the cat (∼0.8 kb, ∼1.7 kb) an_d gfp_ (∼ 0.9 kb, ∼1.7 kb) mRNAs in cells carrying the vector control (pRI) or overexpressing GadY (pRI-GadY) in a background (GSO403) in which the gadX promoter and coding sequence were replaced with the cat promoter and coding sequence and a promoterless gfp gene was inserted downstream and separated from cat by the sequences complementary to GadY. In all cases, total RNA (5 μg each) isolated from cultures grown in LB media to OD600 = 0.7 was separated in a 1% agarose−0.05 M MOPS−1 mM EDTA gel and then transferred to a nylon membrane. The individual mRNAs were detected by specific oligonucleotides (gadX-R, gadW-A2, cat-A1 and gfp-R).

Fig. 2

Fig. 2

(a) Map of the lacZgadX reporter. (b) Plate with lacZgadX reporter strain (GSO404) harboring pRI, pRI-GadY, pRI-GadY(90), pRI-GadY(59). The strains were streaked on an LB plate containing 100 μg/ml of X-gal. At OD600 = 0.7, the levels of β-galactosidase activity in the strains harboring pRI and pRI-GadY were 320 and 0.3 Miller units, respectively (data not shown).

Fig. 3

Fig. 3

All three forms of GadY direct processing of its complementary sequence. (a) Primer extension analysis to map 5′ ends of the processed gadX-gadW mRNA. (b) Primer extension analysis to map 5′ ends of the processed lacZgadX reporter mRNA. For both (a) and (b), primer extension assays were performed with total RNA isolated from strains GSO109 and GSO404 carrying pRI, pRI-GadY, pRI-GadY(90) and pRI-GadY(59) grown to OD600 = 0.7 in LB media using oligonucleotides GadW-A2 and lacZ-R2, respectively. The same oligonucleotides were used to prime the adjacent sequences from p_lac-gadXgadY_-10 mutant and pACYC-lacZgadX. Black bars indicate the extent of the different GadY transcripts. (c) Positions of cleavage. Sequences of base paired gadX-gadW mRNA and GadY RNA are given. The 5′ end of the three forms of the GadY RNA are labeled and indicated by the small boxes. The arrows and larger box denote the Rho-independent terminator. Red arrows indicate the GadY-dependent 5′ ends, blue arrows indicate the GadY(90)-dependent 5′ ends and green arrows indicated the pRI-GadY(59)-dependent 5′ ends. The larger arrows indicate the most intense bands. The band decreased in rnc mutant strains (see Fig. 4) is indicated by the asterick.

Fig. 4

Fig. 4

GadY-directed processing in RNase mutant strains. Total RNA was isolated from wild type and RNase mutant (rnc, rnc rng, rnc rne, rnc rnpA) derivative of the lacZgadX reporter strain harboring pBAD-RI, pBAD-GadY, pBAD-GadY(90) or pBAD-GadY(59) 20 min following the addition of arabinose to 0.2% to actively growing cultures (OD 600 ≈ 0.7). For the strains carrying the temperature sensitive rne and rnpA alleles, half of the culture was shifted to the non-permissive temperature (43.5°C) for 30 min prior to induction. Primer extension analysis was performed using labeled oligonucleotide lacZ-R2 and the products were separated on a 6% polyacrylamide gel. The band decreased in rnc mutant strains is indicated by the asterick, and the extra band present in rng mutant strains is indicated by the bullet.

Fig. 5

Fig. 5

GadY can direct processing of gadX in vitro. (a) Diagram of the gadX and GadY constructs used for the in vitro assay. Black arrows indicate the in vitro synthesized gadX and GadY RNAs. Gray bars indicate the 140 nt 5′ and 100 nt 3′ fragments detected when the gadX RNA was processing in the presence of RNase III. (b) A 32P-labeled gadX mRNA fragment (mRNA) was incubated at 37°C for 1 hr with the indicated combinations of in vitro synthesized GadY sRNA (GadY), GSO430 crude cell extract (protein) and heat treated GSO430 crude cell extract (HT protein). Treated samples were separated on a 6% polyacrylamide gel alongside a Perfect Marker RNA ladder.

Fig. 6

Fig. 6

RNase III is partially responsible for GadY-directed processing. (a) Processing activity from column fractions from final purification step. The 32P-labeled gadX mRNA fragment was incubated with GadY and 5 μl of Mono S column fractions for 1 hr at 37°C. Treated samples were separated on a 6% polyacrylamide gel alongside Perfect Marker RNA ladder. (b) TCA precipitated protein from column fractions from the final purification step were separated on a 10-20% tris glycine SDS PAGE gel and stained with GelCode Blue. The protein band indicated by the arrow was excised from the gel and analyzed by mass spectrometry.

Fig. 7

Fig. 7

GadY-directed processing in the absence of RNase III. (a) Plate with lacZ reporter strain harboring pRI, pRI-GadY in wild type and rnc mutant backgrounds (GSO404 and GSO405, respectively). (b) Levels of the cat (∼0.8 kb, 1.7 kb) and gfp (∼0.9 kb, ∼1.7 kb) mRNAs in cells carrying the vector control (pRI) or overexpressing GadY in a wild type and rnc mutant backgrounds (GSO403 and GSO432, respectively). (c) Primer extension analysis of gfp transcripts in cells carrying the vector control (pRI) or overexpressing GadY in a wild type and rnc mutant backgrounds (GSO403 and GSO432, respectively). (d) A 32P-labeled gadX mRNA fragment (mRNA) incubated at 37°C for 1 hr with the indicated combinations of in vitro synthesized GadY sRNA (GadY), a crude cell extract from the GSO431 rnc mutant (protein) and heat-treated mutant crude cell extract (HT protein).

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