Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells - PubMed (original) (raw)
Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells
Natalia Shcherbik et al. EMBO Rep. 2010 Feb.
Abstract
Most transcripts in growing cells are ribosomal RNA precursors (pre-rRNA). Here, we show that in mammals, aberrant pre-rRNA transcripts generated by RNA polymerase I (Pol I) are polyadenylated and accumulate markedly after treatment with low concentrations of actinomycin D (ActD), which blocks the synthesis of full-length rRNA. The poly(A) polymerase-associated domain-containing protein 5 is required for polyadenylation, whereas the exosome is partly responsible for the degradation of the short aberrant transcripts. Thus, polyadenylation functions in the quality control of Pol I transcription in metazoan cells. The impact of excessive aberrant RNAs on the degradation machinery is an unrecognized mechanism that might contribute to biological properties of ActD.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Figures
Figure 1
Low doses of ActD induce the accumulation of short rRNA transcripts. (A) Metabolic labelling of RNA in mouse 3T3 cells with [3H]Uridine. Cells were treated with 20 ng/ml of ActD where indicated 10 min before labelling. Incorporation of the label into mature rRNAs (28S and 18S) and their main precursors (45S, 32S) was detected by fluorography (F). Methylene blue (MB) staining of the same membrane shows equal loading. (B) Hybridization analysis of pre-rRNA in untreated cells or cells treated with 20 ng/ml of ActD for 30 or 60 min. The main precursors detected with each probe are indicated. A range of co-migrating precursors is indicated with a ‘/'; for example, 47S/45S indicates 47S, 46S and 45S. The asterisk indicates abortive transcripts that accumulate in ActD-treated cells. (C) Structure of the primary transcript (47S) synthesized by Pol I in mouse cells. The 47S pre-rRNA contains rRNA sequences flanked by ETS and separated by ITS, which are removed during processing into mature rRNAs. The main processing sites and intermediates are shown. Probes specific for spacer regions (indicated at the top) allow detection of pre-RNAs but do not hybridize to mature rRNAs. (D) Rapid decay of pre-synthesized rRNA transcripts after transcription shut-off. Cells were either untreated or treated with 20 ng/ml ActD for 30 min to induce accumulation of abortive 5′ETS transcripts. Cells were then treated with 2 μg/ml of ActD to stop further synthesis. RNA was isolated after the indicated time and analysed by northern hybridization with probe 5′ETS-1. ActD, actinomycin D; ETS, external transcribed spacer; ITS, internal transcribed spacer; pre-rRNA, rRNA precursors; rRNA, ribosomal RNA.
Figure 2
Polyadenylated pre-rRNAs in normal conditions and after ActD treatment. (A) Poly(A)+ RNA was prepared as described in the Methods section and analysed by northern hybridizations with probes, as indicated in Fig 1C. (B) The site of probe hybridization determines the minimal length of molecules detected in a population of RNAs with a fixed 5′ end and heterogeneous 3′ ends. The asterisks denote RNAs that would be detectable with the probe. ActD, actinomycin D; pre-rRNA, precursor rRNA; rRNA, ribosomal RNA.
Figure 3
Polyadenylation of aberrant Pol I transcripts involves Papd5 and the exosome. (A) RT–PCR assay for semiquantitative analysis of polyadenylated pre-rRNA transcripts. (B) Hybridization of RT–PCR products to detect polyadenylated 5′ETS transcripts before and after 30 min treatment with 20 ng/ml of ActD in cells transfected with the indicated siRNAs. Average values of the signal from phosphorimager quantification of two separate PCR assays with primers 5′ETSd2 and 5′ETSd4 is shown relative to the signal in ActD-treated cells transfected with a non-targeting siRNA pool. Amplification using primer 5′ETSd2 (17 PCR cycles) is shown as an example. (C) RNA samples used for the assay shown in (B) were analysed by northern hybridization with probe 5′ETS-1 to detect nascent transcripts; 18S rRNA was used as a loading control. (D) Levels of 47S pre-rRNA in untreated cells were quantified by phosphorimager analysis of hybridizations with probe 5′ETS-1. The hybridization signal in each sample was normalized to 18S rRNA to compensate for loading variations. The data are average values from three independent transfections; one set used for the quantification is shown in (C). (E) Ratio of the 5′ETS fragments accumulating after ActD treatment to the 47S primary transcript level in the same cells before treatment. Analysis was performed as in (D). (F) Polyadenylated pre-rRNA fragments detected under normal conditions. The same RNA samples from untreated cells as in (B) were assayed using 22 PCR cycles. Error bars in all graphs indicate s.e. values. ActD, actinomycin D; Ctrl, control; ETS, external transcribed spacer; Exosc, exosome component; Papd5, poly(A) polymerase-associated domain-containing protein 5; Pols, DNA polymerase sigma; pre-rRNA, precursor rRNA; rRNA, ribosomal RNA; RT–PCR, reverse transcriptase PCR; siRNA, small interfering RNA.
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