A role for the exosome in the in vivo degradation of unstable mRNAs

Abstract

In mammals, the mRNAs encoding many proteins involved in inflammation bear destabilizing AU-rich elements (AREs) in the 3'-untranslated region. The exosome, a complex of 3' --> 5' exonucleases, is rate limiting in the destruction of such mRNAs in a mammalian in vitro system, but a role in vivo has not been demonstrated. The phenomenon of ARE-mediated degradation also occurs in the protist parasite Trypanosoma brucei. Messenger RNAs with 3'-untranslated region U-rich elements, which strongly resemble AREs, are extremely unstable in the trypanosome form that parasitizes mammals. The first step in degradation of these mRNAs in vivo is rapid destruction of the 3'-untranslated region; subsequently the mRNA is destroyed by exonucleases acting in both 5' --> 3' and 3' --> 5' directions. We here investigated the roles of three subunits of the trypanosome exosome complex, RRP45, RRP4, and CSL4, in this process, depleting the individual subunits in vivo by inducible RNA interference. RRP45 depletion, which probably disrupts exosome integrity, caused a delay in the onset of degradation of the very unstable RNAs, but did not affect degradation of more stable species. Depletion of RRP4 or CSL4 does not affect the stability of the residual exosome and did not change mRNA degradation kinetics. We conclude that the exosome is required for the initiation of rapid degradation of unstable mRNAs in trypanosomes.

FIGURE 1.

(A) Effect of exosome subunit depletion on trypanosome growth. Cumulative growth curves are shown for depletion of the subunits shown. Below each curve is shown the effect of the depletion of RRP45, RRP4, or CSL4 on the abundances of RRP45, RRP44, and RRP4. The plus sign (+) indicates that normal levels of exosome are present. The vertical, downward-pointing arrow indicates that the level of the exosome subunit indicated above the growth curve is reduced. All panels are Western blots except that for CSL4, which is a Northern blot. CSM is an unrelated protein and SRP is the signal recognition particle RNA, both used as controls. RRP44 is an exonuclease that is not associated with the exosome. (B) The effects of RRP45 depletion on metabolic incorporation of ³⁵S-labeled methionine. The upper panel shows autoradiography and the lower panel the corresponding Coommassie-stained gel. (C) The effects of RRP45, RRP4, or CSL4 depletion on 5.8 S rRNA maturation, analyzed as in Estévez et al. (2001).

FIGURE 2.

Plasmid constructs used to study the effects of exosome subunit depletion on mRNA degradation. The elements indicated are carried in a pGEM backbone. Constructs were linearized within the rRNA spacer region before transfection. The mRNAs produced from the CAT-EP1 plasmid are shown beneath the construct. The CAT mRNAs from the constructs with alternative 3′-UTRs are similar; for ep1ΔURE, only one major polyadenylation site was found whereas for the PGKB mRNAs, several were used (Quijada et al. 2002). Probes used for Figures 3 ▶ and 4 ▶ are shown as dotted lines with circled numbers: (1) CAT probe; (2) EP1 3′-UTR probe; (3) probe specific for longer EP1 3′-UTR.

FIGURE 3.

Effect of RRP45 depletion on mRNA abundance and stability. Trypanosomes expressing different reporter RNAs (indicated above the graphs) were treated with actinomycin D, and RNA was isolated at the various times thereafter. RNAs were detected by Northern blotting and hybridization with [³²P]-labeled probes, and quantitated by phosphorImager using the SRP RNA as an internal control. For each cell line, we show a control Western blot showing typical RRP45 depletion for the cell line. Beneath the graphs are typical Northern blots. Point 0 is before actinomycin D addition; the first time point corresponds to cells that were centrifuged immediately after actinomycin D addition. (Processing takes 5–10 min.) As for Figure 1 ▶, the plus sign (+) indicates that normal levels of RRP45 are present and the vertical, downward-pointing arrow indicates that the level of RRP45 is reduced. Results from three or more experiments are displayed as mean and standard deviation.

FIGURE 4.

(A) The effects of RRP45 depletion on degradation of an RNA containing a strong secondary structure, G₃₀C₃₀, at the 3′-boundary of the CAT cassette. The experiment was performed as for Figure 3 ▶, and RNAs were identified using a probe hybridizing with the CAT gene (probe 1 in Fig. 2 ▶) and the EP1 3′-UTR (probe 2 in Fig. 2 ▶). The identities of the bands were determined using individual CAT and EP1 3′-UTR probes (not shown). As before, the plus sign (+) indicates that RRP45 was present at approximately normal levels (no tetracycline addition, no RNAi induction). The downward arrow indicates that the level of RRP45 was reduced (tetracycline present). (B) The effects of RRP45 depletion on degradation of the full-length CAT-EP1 RNA (analagous mRNA “a” in panel A, but without G₃₀C₃₀). The experiment was performed as for Figure 3 ▶, except that samples were run on a 2% agarose gel. Samples that were predigested with RNase H in the presence of oligo dT serve as markers for fully deadenylated transcripts (A⁻). The bands were detected with an RNA probe to the portion of the 3′-UTR that is absent in the shorter CAT-EP1 transcript (probe 3 in Fig. 2 ▶). The RNA probe cross-hybridized with ribosomal RNA, so we used this signal (lower panel) as a standard for phosphoImager quantitation, which is shown as percentage values on the lanes.