RNase E Cleaves at Multiple Sites in Bubble Regions of RNA I Stem Loops Yielding Products That Dissociate Differentially from the Enzyme

Vladimir R Kaberdin,Yen-Huei Chao,Sue Lin-Chao

doi:10.1074/jbc.271.22.13103

Vladimir R Kaberdin, Yen-Huei Chao + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.22.13103

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Abstract

Earlier work has shown that RNase E cleaves RNAI, the antisense repressor of replication of ColE1-type plasmids, producing pRNAI-5, whose further decay is mediated by the poly(A)-dependent activity of polynucleotide phosphorylase and other 3' to 5' exonucleases. Using a poly(A) polymerase-deficient strain to impede exonucleolytic decay, we show that RNAI is additionally cleaved by RNase E at multiple sites, generating a series of decay intermediates that are differentially retained by the RNA binding domain (RBD) of RNase E. Primer extension analysis of RNAI decay intermediates and RNase T1 mapping of the cleavage products of RNAI generated in vitro by affinity-purified RNase E showed that RNase E can cleave internucleotide bonds in the bubble regions of duplex RNA segments and in single-stranded regions. Chemical in situ probing of a complex formed between RNAI and the RBD indicates that binding to the RBD destabilizes RNAI secondary structure. Our results suggest a model in which a series of sequential RNase E-mediated cleavages occurring at multiple sites of RNAI, some of which may be made more accessible to RNase E by the destabilizing effects of its RBD, generate RNA fragments that are further degraded by poly(A)-dependent 3' to 5' exonucleases.

Highlights

Earlier work has shown that the degradation of RNA I (Fig. 1), the antisense repressor of the replication of ColE1-type
Inactivation of Poly(A) Polymerase Results in Accumulation of Several rne-dependent Degradative Intermediates of RNA I—Northern blot hybridization to a 32P-labeled riboprobe complementary to RNA I was used to detect full-length RNA I and its degradative products in preparations of total cellular RNA isolated from wild-type E. coli and from strains mutated in polynucleotide phosphorylase (PNPase), RNase E, or poly(A) polymerase
The preferential accumulation of low molecular weight (LMW) products in a strain deficient for poly(A) polymerase suggests that RNA I decay in wild-type E. coli may involve multiple endonucleolytic cleavages producing short RNA I fragments that are made unstable by polyadenylation and undergo rapid digestion by PNPase, RNase II, or other nucleases

Summary

MATERIALS AND METHODS

Bacterial Strains, Plasmids, and Growth Conditions—The bacterial ase [8, 9]. The latter accelerates mRNA decay by means of the addition of poly(A) tails to the 3Ј-end [8] and has been suggested to play an important role in regulating the stability of messenger RNA (for review, see Ref. 10). Labeled GGGRNA I was purified in an 8% polyacrylamide sequencing gel, and 1–5 pmol of it was incubated with 50 ng of full-length RNase E (affinity purified under non-denaturing conditions) or with 2–3 ␮g of catalytic domain (affinity purified under denaturing conditions and refolded) in 50 ␮l of 20 mM Tris-HCl (pH 8.0), 5 mM magnesium chloride, 100 mM sodium chloride, 5% glycerol, 0.1% Triton X-100, 0.1 mM dithiothreitol. Both protein preparations were kindly provided by McDowall and Cohen (Stanford University). The products of cleavage were eluted from the gel, dissolved in a sequencing buffer, and run on an 8% sequencing gel using RNase T1 digest and partial alkaline hydrolysate of terminally labeled RNA I as a size marker

RESULTS

LMW intermediate

DISCUSSION