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Figure 2. Comparison of the stabilities of exogenous polyadenylated and deadenylated mRNAs in RSW either lacking or containing poly(A)-binding protein (PABP). RSW was first depleted of PABP. It was then incubated as described in Protocol 8 with either of three substrates: polyadenylated p-globin mRNA, p; deadenylated p-globin mRNA, A(-)3; or histone mRNA, H, which normally lacks poly(A). Some reactions were supplemented with purified yeast PABP. Lane 1, DNA size markers (sizes of two fragments are given in base pairs on the left); lanes 2-7, polyadenylated 3-globin, 3/ and deadenylated histone, H; mRNAs incubated for the times indicated with PABP-depleted RSW or with PABP-depleted RSW plus purified yeast PABP; lanes 8-13, deadenylated p-globin mRNA, A( - )p incubated as per lanes 2-7. Reprinted with permission from ref. 24.

Figure 1 shows the decay of 7-globin mRNA, whose intracellular half-life is approximately 17 h, 5-globin mRNA (intracellular half-life 4h) and c-myc mRNA, with an intracellular half-life of 0.5 h. This experiment illustrates an essential property of this in vitro mRNA decay system: it reflects known intracellular mRNA half-lives.

6.1.2 Radiolabeled substrate incubated with ribosomal salt wash

The experiment of Figure 2 was performed for two reasons:

(a) To compare the half-lives of two radiolabeled globin mRNA substrates, one with an 80 nt 3' poly(A) tract, the other lacking poly(A).

(b) To determine whether poly(A)-binding protein (PABP) influences the in vitro decay rate of either substrate. The experiment involved the following:

• Preparation of three 32P-labelled substrates, polyadenylated human (3-globin mRNA, deadenylated human p-globin mRNA, and human H4 histone mRNA, by transcribing appropriate cDNA clones with SP6 RNA polymerase. Histone mRNA is one of the few mammalian mRNAs without a poly(A) tract. It served as a control in some reactions.

• Preparation of RSW from tissue culture cells (human erythroleukaemia) as per Protocol 3 and then depletion of the RSW of endogenous PABP.

• Incubation of substrates with RSW as per Protocol 8 for the times indicated in Figure 2. Some reactions also contained highly purified PABP from yeast.

• Extraction of total RNA from each reaction and electrophoresis of each RNA in a denaturing polyacrylamide/urea gel.

• Autoradiography of the gel.

Figure 2 shows the rapid decay of polyadenylated globin mRNA and of histone mRNA in the PABP-depleted RSW. In contrast, p-globin mRNA is at least 10-fold more stable than histone mRNA in reactions supplemented with purified PABP. Therefore, PABP contributes to the long half-life of polyadenylated (3-globin mRNA in these reactions. However, PABP does not stabilize deadenylated p-globin mRNA. Therefore, PABP probably helps to stabilize mRNA by interacting with the 3' poly(A) tract.

6.1.3 Effect of a fra/is-acting mRNA stability regulatory factor in the post-polysomal supernatant (Si 30)

The experiment of Figure 3 was performed to determine whether the destabiliza-tion of 7-globin and other host cell mRNAs following Herpes simplex virus infection could be reproduced in vitro and, if so, in what subcellular fraction the destabilizing factor resided. The following experiment was performed:

• Two sets of tissue culture cells were used. One was infected with Herpes simplex virus, the other was 'mock infected', meaning that it was treated in the same way but no virus was added.

• At 60, 180, and 360 min following virus inoculation (mixing virus and cells), cells were harvested and post-polysomal supernatant (SI30) was prepared, as per Protocol 2.

• In vitro mRNA decay reactions were performed by mixing polysomes from uninfected cells with SI30 from either mock-infected or virus-infected cells, as per Protocol 6.

• Each reaction was incubated for 0 to 150 min; then total RNA was isolated and annealed with a 32P-labelled DNA probe for human 7-globin mRNA. The amount of undegraded 7-globin mRNA in each reaction was determined by incubation with nuclease SI and electrophoresis of nuclease-resistant DNA in a gel.

Mock

I I I in vitro

Y-Globin-*

Wild-type

Post-Inoculation S 60 180 360

0 15 30 90 150 0 15 30 90 150 0 15 30 90 150 < Incubation

Figure 3. Identification of an mRNA destabilizing factor in the S130 of cells infected with Herpes simplex virus. Tissue culture cells were either mock-infected or infected with Herpes simplex virus for up to 6 h, as described in the text. S130 was prepared from each culture flask and was added to polysomes from uninfected cells. The reactions were incubated as per Protocol 6. The stability of polysome-associated 7-globin mRNA was determined by nuclease S1 mapping. Top panel, polysomes plus S130 from mock-infected cells, i.e. cells not exposed to virus. Bottom panel, polysomes plus S130 from cells infected for the indicated times [wild-type, post-inoculation time (min)] with Herpes simplex virus. The time course in min of each incubation is shown [in vitro incubation time (min)] above each lane. Reprinted with permission from ref. 6.

Figure 3 shows that polysomal, endogenous 7-globin mRNA is very stable in reactions supplemented with SI30 from mock-infected cells (no virus) but that SI30 from cells infected with the virus for 60, 180, or 360 min contains a potent mRNA destabilizer. This destabilizer is now known to be a protein encoded by the virus and an integral part of the virion itself. By an unknown mechanism, this protein accelerates the degradation of almost all cellular mRNAs, even those like 7-globin which are normally very stable in uninfected cells.

6.2 Data interpretation

As discussed in Section 1 and Protocol 6, mRNA decay rates are slower in vitro than in intact cells. Relatively slow reaction rates are also observed in cell-free transcription and translation systems. Therefore, it seems reasonable to exploit cell-free mRNA decay systems to measure relative, as opposed to absolute, decay rates. To assess whether the system is functioning properly, the decay rates of mRNAs which are known to have different intracellular half-lives should be determined. mRNAs that are unstable in cells should also be unstable in vitro, relative to stable mRNAs.

Some caution must be exercised in plotting and interpreting in vitro mRNA decay data. In cells, mRNA is degraded in a logarithmic fashion, according to the formula:

C/C0 = e-V, where C and C0 are mRNA levels at time t and time 0, respectively, and kd is the decay constant (reviewed in ref. 25).

The problem with applying similar parameters to in vitro data is that decay rates may decrease as the reaction time increases. For example, the author has found that the c-myc mRNA decay rate decreases at least two-fold after several hours of incubation, presumably because critical reaction components become rate-limiting (J. Ross and G. Brewer, unpublished observations). Therefore, it seems reasonable to plot the in vitro data as either a linear or logarithmic function, provided that each mRNA is analysed by the same criteria (see Figure I as an example).

6.3 Troubleshooting

Several potential obstacles and trouble spots which might arise during the course of cell-free mRNA decay experiments are listed below, as well as some suggestions for solving them.

6.3.1 Excessive RNA degradation during preparation of polysomes and extracts

Precautions for reducing endogenous, non-specific RNase activity while preparing polysomes and during in vitro incubations are discussed in Protocol 2. If these are inadequate, it might be helpful to avoid using primary animal cells and to use instead a tissue culture cell line synthesizing the mRNA(s) of interest. Some cells lines contain less 'non-specific' nuclease activity than others. Try screening several cell lines to find one which allows intact polysomes to be isolated.

6.3.2 All mRNAs in the mRNA decay reactions are degraded at similar rates

Again, non-specific RNase activity might be overwhelming the system. Several modifications should be tried:

• Add more RNase inhibitor or try various combinations of inhibitors.

• Modify the reaction conditions. For example, non-specific RNase activity might be depressed by changing the monovalent or divalent cation concentrations, the pH, or the reaction temperature.

• Add carrier single-stranded RNA, for example, poly(C) at l-2pg/25pl reaction (see Protocols 6-8). Avoid higher amounts of exogenous RNA, which can slow mRNA decay processes.

6.3.3 No mRNA degradation is observed

The simplest explanation for this result would be inactivation of messenger RNases (and other RNases) during extract preparation. If no RNA is degraded during in vitro reactions, the composition and pH of all extraction buffers should be monitored carefully. An unlikely explanation is that all RNase activity in the extract is blocked by the placental RNase inhibitor. Therefore, reactions should be carried out without inhibitor.

6.3.4 Regulated decay is not observed

For example, a particular mRNA might be known to be degraded five-fold less rapidly in cells treated with a translational inhibitor, as compared with untreated cells. However, the mRNA is found to be degraded in vitro at the same rate with polysomes isolated from treated and untreated cells. There could be many explanations for this result, but the following can be investigated:

• Add S130 {Protocol 2) to the reactions. Perhaps the regulatory factor is in the SI 30 and is not poly some-associated.

• Alter the reaction conditions, as per Section 6.3.2.

• Prepare a lysolecithin extract (Protocols 4 and 9) rather than a detergent-free extract from both sets of cells, to encourage translation of the mRNA, since the regulation of some mRNAs might be linked to their translation. In this case, the mRNA might be unstable under conditions of efficient translation elongation, but stabilized when elongation is retarded by an inhibitor. The in vitro system would then reflect the intracellular situation only by comparing the stability of elongating versus non-elongating mRNA. Some translation does occur in the lysolecithin extract but little is observed in detergent-free extract. To determine whether the act of translational elongation itself influences the stability of the mRNA, compare half-lives in lysolecithin extracts with or without added translational inhibitor.

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