EL-SEWER FEMS Microbiology Letters I33 (1995) 209-2 13

micF RNA is a substrate for RNase E

Matthew Schmidt ‘, Nicholas Delihas *

Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, NY I 1794-5222, USA

Received 17 August 1995; revised I September 1995; accepted 5 September 1995

Abstract

Ribonuclease E (RNase E) is known to play an important role in mRNA decay and RNA processing in Escherichiu co/i. While several substrates for RNase E have been identified, the specificity for the recognition and cleavage sites has not been

completely determined. In this study, micF RNA, an antisense RNA found in E. coli and related bacteria, was found to be a substrate for RNase E in vitro. Two cleavage sites were mapped, and both are found in the sequence context UA/UUU and are located within 10 nucleotides upstream of stem-loop structures. These results help define a generalized RNase E cleavage/recognition pattern.

Keywords: Escherichia coli; RNase E cleavage sites; micF RNA; Antisense RNA

1. Introduction

RNase E was initially identified as an rRNA-

processing nuclease [ll but was subsequently shown to specifically cleave other RNAs [2,3], including RNA I, an antisense RNA found in Escherichia coli

[4]. Recently, RNase E has been shown to be associ-

ated with polynucleotide phosphorylase (PNPase) in a protein complex, providing evidence that it plays a

general role in mRNA degradation [5,6]. The cleavage site specificity of RNase E is not yet

completely defined. A consensus sequence of RAUUW (R = G or A; W = U or A) was proposed

by Ehretsmann et al. [7], but more recent work does

* Corresponding author. Tel.: f 1 (516) 632 8779; Fax: + 1

(516) 632 8891; E-mail: ndelihas@asterix.bio.sunysb.edu.

’ Present address: State University of New York Empire State

College, 250 Veterans’ Memorial Highway, Hauppauge, NY I 1788, USA.

Federation of European Microbiological Societies

SSDl 0378- 1097(95)00352-5

not support the idea of a canonical RNase E cleavage

sequence, and suggests that RNase E exerts its activ- ity at sites whose sequence is AU-rich [4,8]. It has

also been suggested that while RNase E cleavage sites occur in single-stranded regions, the presence of a stable hairpin 3’ to the cleavage site is important in

defining an RNase E-susceptible location [7,9]. How-

ever, McDowall et al. [lo] find that RNase E-suscep- tible constructs which lack stem-loops present in the

natural substrates are efficiently cleaved. Addition- ally, it has been proposed that cleavage by RNase E is facilitated by the presence of five or more un- paired nucleotides at the 5 end of an RNA [l 11. Clearly, more work must be done to determine how RNase E recognizes and cleaves an RNA.

The present study was initiated to determine if

micF RNA, a 93-nucleotide trans-encoded antisense RNA found in E. coli and other bacteria [ 12,131, is a substrate for RNase E. In this study, micF RNA has proven to be very useful in the analysis of RNase E cleavages due to the prevalence of AU-rich regions

210 M. Schmidr, M. Delihas/ FEMS Microbiology Letters 133 (1995) 209-213

in various segments of the molecule; also, its sec- ondary structure is known [ 141.

2. Materials and methods

2. I. Enzymes and reagents

Restriction enzymes, T4 polynucleotide kinase, calf intestinal alkaline phosphatase, and nucleotides were from both Promega and Boehringer Mannheim. T4 RNA ligase was from New England Biolabs. [ y- 32 P]ATP was from either NEN Research Products or ICN. [5’-32P)pCp was obtained from NEN Re- search Products. [ cy- 32 P]UTP was from Amersham.

2.2. In vitro transcription

To synthesize micF RNA, plasmid pUCT7micF DNA [ 153 was digested with DraI restriction en- donuclease; for t95d RNA, plasmid pGM95 DNA [9] was also digested with DraI restriction endonucle- ase. In vitro transcription with T7 RNA polymerase was performed according to published protocols [ 14,151. 32P end labelling of RNA and RNA renatu- ration were as previously described [ 151

2.3. Digestion with ribonuclease E

15 ~1 of renatured RNA solution was added to 6 ~1 40% polyethylene glycol (PEG) A4, 8000, 2 ~1 1% Triton X-100, 1 ~1 sterile bovine serum albumin (BSA) (0.3 mg ml-‘), and 5 ~1 sterile, double-de- ionized water. RNase E was added to a final concen- tration of 1 ng t.~l- ’ , and the mixture was incubated at 30°C for varying amounts of time. The final reaction volumes were 30 ~1. The digestions were terminated by the addition of 12 ~1 of 90% for- mamide in 0.5 X TBE and 0.05% tracking dyes to a 4-~1 aliquot of the digestion sample.

2.4. Preparations of RNase E

RNase E samples were a kind gift from Dr. George Mackie. Two different preparations of RNase E were used depending upon the experiment. The ‘AS-26’ preparation consists of partially purified RNase E, and results from ammonium sulfate precip-

itation of a crude protein fraction obtained from an E. coli strain (GM402) harboring plasmid pGM102. This plasmid contains the me gene and upon induc- tion causes the overexpression of the me gene prod- uct, RNase E. The ‘180~kDa’ preparation contains only the purified, renatured RNase E polypeptide, which migrates as a 180-kDa protein although it is predicted to be smaller (1 14 kDa) based on the sequence of the me gene [31.

3. Results

When incubated with RNase E, 5’- and 3’ end- labelled samples of micF RNA showed a specific pattern of cleavage. Representative autoradiograms of polyacrylamide gel separations of RNase E diges- tion products are shown in Figs. I and 2. The RNase E cleavage sites are graphically displayed on a sec- ondary structural model for micF RNA [14] in Fig. 3.

One major and one minor RNase E cleavage site are observed when micF RNA is exposed to RNase E. The major cleavage occurs at the phosphodiester bond linking micF RNA residues A60 and U61 (Fig. 1). This cleavage is observed in both 5’- and 3’- labelled preparations of micF RNA (Fig. 2). The site of cleavage is found in the sequence context UA/UUU (Fig. 3).

A minor cleavage of micF RNA by RNase E is observed only when 5’-labelled RNA is used. This cleavage is also found in the sequence context UA/UUU (Fig. 2) and occurs between residues Al9 and U20 of micF RNA (Figs. 2 and 3). Since this cleavage is much less prominent than the one occur- ring after position A60, one would not expect to see this cleavage when using 3’-labelled RNA, because under these conditions only additional cleavages oc- curring between positions 60 and 93 could be de- tected. Other than cleavages at Al9 and A60, no additional RNase E sites were observed in micF

RNA.

4. Diiusfkn

Based on in vitro digestion with purified ribonu- clease E, we show that micF RNA is a substrate for

M. Schmidt, N. Delihas / FEMS Microbiolog? Letters 133 (1995) 209-213 211

RNase E and define the sites of cleavage. Both micF

RNA cleavage sequences are in agreement with the current view that RNase E acts at AU-rich sequences located in single-stranded regions of the RNA [lo]. The major cleavage exists in the primary structural context CUA/UUUC, a contiguous stretch of five residues consisting of A and U, while the minor cleavage is found in the context CUUUA/UUUAU- UAC, a continuous region of 11 A or U bases. It is possible that while the sequences of both RNase E cleavage sites are the same, this is not a requirement,

90 80

70

60

0 30 60120 L Tl 40

90

80

70

60 -

50

.60

50

Fig. 1. Denaturing acrylamide gel showing the cleavage pattern of

micF RNA after digestion with RNase E, positions 58-93. 5’-

Labelled sample on 12% polyacrylamide gel: lanes marked 0, 30,

60. and 120 are digestions with purified RNase E at a concentra-

tion of 1 ng ~1~’ for 0, 30, 60, and 120 min, respectively. The

sequencing lane marked TI is a partial digestion with RNase Ti

under denaturing conditions; the lane marked L is an alkaline

hydrolysis ladder.

- - -

5’4abeled 3’4abelsd micf RNA micF RN.4

0 .5 1 2 L Tl U2 0 .5 1 2 L Tl U2

10

20

30

40

60

80

-

Fig. 2. Denaturing acrylamide gels showing the cleavage pattern

of micF RNA after digestion with RNase E. (A) 5’-Labelled

sample on 12% polyacrylamide gel: lanes marked 0, 0.5, 1. and 2

are digestions with purified RNase E at a concentration of 1 ng

~1~’ for 0,0.5, I, and 2 h. respectively. Sequencing lanes marked

TI and U2 are partial digestions with RNases Tl and U2,

respectively, under denaturing conditions: the lane marked L is an

alkaline hydrolysis ladder. (B) 3’-Labclled sample on 12% poly-

acrylamide gel: lanes marked 0, 0.5. I. and 2 are digestions with

purified RNase E at a concentration of 1 ng PI-’ for 0. 0.5, I.

and 2 h, respectively. Sequencing lanes marked Tl and U2 are

partial digestions with RNases Tl and U2, respectively, under

denaturing conditions; the lane marked L is an alkaline hydrolysis

ladder.

212 M. Schmidt. N. Delihas/ FEMS Microbiology Letters 133 (1995) 209-213

I II

u

C C

U U C.G

u c 40 G.C

A." ".A 80

C.G A-U

".A 10 G."

lJ.A G-C

A.U C.G

C.G ”

30” C.G

G." c A-" 1 10 2" 50 60 5,) GC"A"CAUCAUUAAC"""AUU"AU"ACCCCUAU""CA.U""U"U"

t 4 RNase E RNase E

Fig. 3. Cleavages of micF RNA by RNase E. Arrows mark the ribonuclease E cleavage sites. The boldness of the arrow corresponds to the

intensity of the cleavage. micF RNA secondary structure is from [ 141.

and the sites of cleavage are largely reflective of the

AU continuity of the cleavage region. Several other similar regions of four or five continuous A and U

nucleotides occur in micF RNA, at positions lo- 14

(AUUAA), 22-25 (UAUU), 36-39 (AUUU), and 51-54 (UUUA), and no cleavage is observed in these sequences. The AU-rich sequence at positions

36-39 (AUUU) finds itself partly located in a hair- pin region of secondary structure [14]; this likely

explains the lack of cleavage at this location. The

AU-rich sequences close to hairpin I, positions 22-25 and 51-54 may be not be cleaved due to steric hindrance or to an ordered structure in these se-

quences. For example, evidence of base-stacking was found at positions 51 and 52 [ 141. The lack of cleavage at the AU-rich single-stranded positions

lo-14 may be due to the large distance of these sequences from hairpin I or because the sequence context is not suitable for RNase E cleavage. It seems likely that while AU continuity may be an important factor, it is not solely responsible for RNase E cleavage. There may be a complex relation- ship between the AU continuity and the exact se- quence for RNase E recognition. In addition, the higher order structural context of an AU-rich region or the distance of this region from a hairpin may also determine whether RNase E will cleave this site.

Acknowledgements

We thank Dr. George Mackie for his generous

gift of purified and partially purified RNase E, along with his generous sharing of techniques and advice. We additionally thank Craig DeLoughery for purifi- cation of T7 RNA polymerase. This work was sup-

ported by SUNY at Stony Brook Institutional and Departmental funds.

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