Development of an in vitro mRNA decay system for Escherichia coli: poly(A) polymerase I is necessary to trigger degradation

Abstract

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from s strain carrying the wild-type gene for PAP I (pcnB+). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) are detected on the 3' end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I-polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.

Figure 1

Incorporation of [³²P]AMP into polysomal RNA. Polysomes were isolated from SK5667 (PAP I⁺) and SK8964 (PAP I⁻) and incubated in the presence of [α-³²P]ATP. Samples were removed at the following time intervals. Lanes 1, 4, 8, and 12, 0.3 min; lanes 5 and 13, 2 min; lanes 2, 6, 9, and 14, 5 min; lanes 3, 7, 10, and 15, 10 min; and lane 11, 15 min. After phenol extraction and ethanol precipitation, the RNA was resolved by 6% PAGE in the presence of 7 M urea. Exogenous PAP I was added at the rate of 1 unit/reaction to lanes 1–3. Polysomes isolated from SK8964(ΔpcnB) transformed with pJL89, a plasmid carrying the pcnB⁺ gene, were used in lanes 12–15. RNA size markers in nucleotides are indicated on the right.

Figure 2

Incorporation of [³²P]AMP into polysomal RNA in the presence or absence of postribosomal S100 supernatants. The experimental procedures are described in the text. Postribosomal extracts were made by centrifuging the lysed cells at 100,000 rpm for 2 hr. The supernatants (S100) were stored on ice overnight. PAP I⁺ is SK5667 (rna-19 thyA715) and PAP I⁻ is SK8964 (ΔpcnB rna-19 thyA715). Lanes 1 and 6 were sampled at 0 min, and all remaining lanes were sampled at 10 min. Lanes 1–5, polysomes from PAP I⁺ to which the postribosomal supernatant from PAP I⁻ was added at the indicated protein concentrations. Lanes 6–10, polysomes from PAP I⁻ with postribosomal supernatant from PAP I⁺ added at the indicated protein concentrations. Lanes 11 and 12, postribosomal supernatant from PAP I⁺ without (lane 11) and with (lane 12) 2 μg of added tRNA. Lanes 13 and 14, postribosomal supernatant from PAP I⁻ without (lane 13) and with (lane 14) 2 μg of added tRNA. Lane 15, polysomes from PAP I⁻ incubated with 1 unit of exogenous PAP I enzyme. RNA size markers in nucleotides are shown on the left.

Figure 3

Northern blot analysis of lpp and trxA mRNA decay in vitro. Polysomes were isolated and incubated with or without ATP as described. Samples were removed at 0, 2, 4, 8 12, and 16 min (lanes 1–6, respectively), resolved using denaturing 6% PAGE, and probed with a full-length fragment for each respective gene. The top three bands represent trxA transcripts, the largest of which is 496 nt. lpp is the single 322-nt species. (A) Polysomes from SK5667 (PAP I⁺) incubated with ATP. (B) Polysomes from SK5667 (PAP I⁺) incubated without ATP. (C) Polysomes from SK8964 (PAP I⁻) incubated with ATP.

Figure 4

Decay of the lpp mRNA in vitro and in vivo. mRNA decay is plotted as the logarithm of the percent signal remaining versus time (2 = 100% while 1 = 10%). A linear regression analysis curve is drawn. (A) In vivo. Data was derived by analyzing Northern blots, such as those presented in Fig. 3, using a Molecular Dynamics model 400A PhosphoImager. □, SK5667 (PAP I⁺); and ◊, SK8964 (PAP I⁻). (B) In vitro. Polysomes were incubated with or without ATP. Samples were removed at the times indicated and analyzed using 6% denaturing PAGE. The Northern blots were analyzed as in A. □, Polysomes from SK5667 (PAP I⁺) plus ATP; ◊, polysomes from SK5667 (PAP I⁺) minus ATP; •, polysomes from SK8964 (PAP I⁻) plus ATP; and ▴, polysomes from SK8964 (PAP I⁻) minus ATP.

Figure 6

Poly(A) tails formed during in vitro mRNA decay. Polysomes were incubated in the presence of [α-³²P]ATP. Samples were removed at various times, deproteinated, and then digested exhaustively with RNase A and RNase T1 as described. An autoradiogram of a 12% polyacrylamide gel is shown. Lanes 1–3, SK5667 (PAP I⁺) at 2, 5, and 10 min, respectively; lane 4, SK8964 (PAP I⁻) at 10 min; lanes 5–7, SK8964 (PAP I⁻) with 0.4 units of exogenous PAP I per reaction at 2, 5, and 10 min, respectively; and lane 8, 10-, 20-, and 30-mers of oligo(dA) end-labeled with [γ-³²P]ATP.

Figure 5

Polyadenylylation in polysomes occurs on mRNA and not rRNA. RNA was isolated and treated as described. Following electrophoresis on 6% denaturing PAGE, the Northern blots were probed with a full-length lpp probe (A) or an oligonucleotide complementary to the 3′ end of the mature 23S rRNA (B). Exogenous PAP was added as indicated at 0.4 units/reaction. All samples were digested with RNase H.

Figure 7

Measurement of endogenous poly(A) tails in polysomal RNA. Samples were taken from an in vitro reaction in which polysomes were incubated in the presence of ATP. Poly(A) tails were visualized by [³²P]pCp end-labeling in the presence of T4 RNA ligase, followed by digestion with RNase A and RNase T and resolution by 12% PAGE, as described. Lane 1, 10-, 20-, and 30-mer end-labeled oligomers of oligo(dA); lanes 2–5, SK5667 (PAP I⁺) sampled at 0, 2, 5, and 10 min, respectively; lanes 6–8; SK8964 (PAP I⁻) plus 0.4 units of exogenous PAP at 2, 5, and 10 min, respectively; and lane 9, postribosomal supernatant from SK5667 (PAP I⁺). Lanes 2 and 9 contain 20 μg of RNA, all others lanes have 10 μg of RNA.