Degradation of ribosomal RNA precursors by the exosome

Abstract

The yeast exosome is a complex of 3'-->5' exonucleases involved in RNA processing and degradation. All 11 known components of the exosome are required during 3' end processing of the 5.8S rRNA. Here we report that depletion of each of the individual components inhibits the early pre-rRNA cleavages at sites A(0), A(1), A(2)and A(3), reducing the levels of the 32S, 20S, 27SA(2)and 27SA(3)pre-rRNAs. The levels of the 27SB pre-rRNAs were also reduced. Consequently, both the 18S and 25S rRNAs were depleted. Since none of these processing steps involves 3'-->5' exonuclease activities, the requirement for the exosome is probably indirect. Correct assembly of trans -acting factors with the pre-ribosomes may be monitored by a quality control system that inhibits pre-rRNA processing. The exosome itself degrades aberrant pre-rRNAs that arise from such inhibition. Exosome mutants stabilize truncated versions of the 23S, 21S and A(2)-C(2)RNAs, none of which are observed in wild-type cells. The putative helicase Dob1p, which functions as a cofactor for the exosome in pre-rRNA processing, also functions in these pre-rRNA degradation activities.

Figure 1

Structure and processing of the pre-rRNA in S.cerevisiae. (A) Structure of the 35S pre-rRNA with the location of oligonucleotide probes used for hybridization and primer extension. (B) Major pre-rRNA processing pathway. The primary transcript is processed by a series of sequential cleavages into the mature 18S, 5.8S and 25S rRNA. Initial cleavage in the 3′-ETS by Rnt1p yields the 35S pre-rRNA. The snoRNP-dependent cleavage at site A₀ in the 5′-ETS then generates 33S pre-rRNA, which is rapidly cleaved at site A₁, producing the 32S pre-rRNA. Cleavage at site A₂ in ITS1 then splits the 32S pre-rRNA into the 20S and 27SA₂ pre-rRNAs, destined to form the RNAs of the small and large ribosomal subunit, respectively. The 5′ part of the molecule, 20S pre-rRNA, is exported to the cytoplasm and endonucleolytically cleaved at site D to generate mature 18S rRNA. The 27SA₂ pre-rRNA is processed by two alternative pathways, giving rise to two forms of 5.8S rRNA, the major short form 5.8S_S and a minor long form 5.8S_L. For simplicity, only the major pathway to 5.8S_S is shown. In this pathway, 27SA₂ is cleaved by RNase MRP at site A₃ to generate 27SA₃, which is processed by the 5′→3′ exonucleases Rat1p and Xrn1p to site B_1S, the 5′ end of the 27SB_S pre-rRNA and mature 5.8S_S rRNA. In the alternative pathway, processing occurs at site B_1L, the 5′ end of 27SB_L and 5.8S_L rRNA. The subsequent processing of both 27SB species is identical. Processing at sites C₁ and C₂ releases the mature 25S rRNA and the 7S pre-rRNA. The 7S pre-rRNA is 3′ processed by the exosome complex, generating the 6S pre-rRNA, which is then trimmed to the mature 5.8S. The exosome also degrades the excised spacer region from the 5′ end of the primary transcript to site A₀.

Figure 2

Pre-rRNA processing and degradation in exosome mutants. The inactivation of any of the exosome components results in the inhibition of the early pre-rRNA cleavages. The major intermediates observed in exosome mutants result from (A) inhibition of cleavage at sites A₀–A₂ or (B) inhibition of cleavage at site A₃ in ITS1. (A) The 23S RNA extends from the 5′ end of the primary transcript to site A₃ and is detected in strains mutant for several snoRNAs and many other processing components. The exosome mutants are unusual in accumulating the 23S* RNA (a slightly shortened form of 23S) and the 21S*, the product of cleavage of this RNA at site A₁. (B) The A₂–C₂ RNA extends from site A₂ in ITS1 to site C₂ in ITS2. Mutations in RNase MRP components also inhibit A₃ cleavage and lead to the synthesis of forms of the 5.8S rRNA that are 5′ extended to site A₂ but 3′ processed by the exosome to site D (the mature 3′ end of the 5.8S rRNA). The exosome mutants are unusual in accumulating the A₂–C₂* species that extend to heterogeneous sites in ITS2, between C₂ and the 3′ end of the 5.8S rRNA. The processing pathways shown in (A) and (B) are mutually exclusive, showing that the block in processing at A₀–A₂ is not complete in exosome mutants.

Figure 3

Northern analysis of pre-rRNA processing in exosome mutants. RNA was extracted from GAL::rrp40 and GAL::csl4 strains following transfer from permissive, RSG medium to repressive, glucose medium at 30°C for the times indicated. (A) and (B) Hybridization with probe 001, complementary to ITS1 downstream of site A₃. (C) Hybridization with probe 003, complementary to ITS1 upstream of site A₃. (D) Hybridization with probe 005, complementary to ITS1 downstream of site A₂. (E) Hybridization with probe 006, complementary to ITS2. (F) Hybridization with probe 007, complementary to 25S rRNA. (G) Hybridization with probe 033, complementary to 5′ETS. (H) Hybridization with probe 002, complementary to ITS1 upstream of site A₂. (I) Hybridization with probe 008, complementary to 18S rRNA. Probe names are indicated in parentheses on the left. Lane 1, wild-type, 0 h; lanes 2–5, GAL::rrp40, 0, 2, 6 and 12 h; lanes 6–9, GAL::csl4, 0, 2, 6 and 12 h. The pre-rRNA and rRNA species are schematically represented on the right; rectangles represent the mature rRNA and thin lines the transcribed spacers. The hybridization sites of the probes are indicated on the diagram. The bands labeled 23S* and 21S* are a mixture of the full-length 21S and 23S and the truncated * species, with the truncated forms predominating.

Figure 4

Northern analysis of pre-rRNA processing in single and double mutants. RNA was extracted from the rrp6-Δ strain grown in YPD medium after shift from permissive temperature (30°C; 0 h) to non-permissive temperature (37°C) for the times indicated. GAL::rrp41 strains were grown as described in Figure 2. Probe names are indicated in parentheses. (A) and (B) Hybridization with probe 001. (C) Hybridization with probe 003. (D) Hybridization with probe 005. (E) Hybridization with probe 006. (F) Hybridization with probe 007. (G) Hybridization with probe 033. (H) Hybridization with probe 002. (I) Hybridization with probe 008. Lanes 1 and 6, wild-type; lanes 2–5, rrp6-Δ, 0, 8, 16 and 24 h; lanes 7 and 8, GAL::rrp41, 0 and 6 h; lanes 8–13, GAL::rrp41/rrp6-Δ, 0, 2, 8, 16 and 24 h; lanes 14–16, GAL::U3, 0, 8 and 24 h.

Figure 5

Depletion of Dob1p or exosome components has similar effects on pre-rRNA processing. Growth of GAL-regulated and ts mutants was as described in Figures 2 and 3. Probes are located as indicated in Figure 2. (A) Hybridization with probe 001. (B) Hybridization with probe 003. (C) Hybridization with probe 005. (D) Hybridization with probe 006. (E) Hybridization with probe 007. (F) Hybridization with probe 033. (G) Hybridization with probe 004. (H) Hybridization with probe 008. Lane 1, wild-type; lanes 2–5, GAL::dob1, 0, 2, 6 and 24 h; lanes 6–8, GAL::rrp4, 0, 6 and 24 h; lanes 9–11, mtr3-1, 0, 2 and 6 h at 37°C.

Figure 6

Primer extension analysis through ITS1 in exosome mutants. Primer extension was performed using an oligonucleotide (033) which hybridizes within ITS2. (A) Primer extension stops at sites A₂, A₃, B_1S and B_1L. (B) Stronger exposure of primer extension stop at site A₃. RNA extracted from GAL-regulated constructs or ts mutants, were collected after transfer to repressive glucose medium or 37°C, respectively, for the following lengths of time: lane 1, wild-type, 0 h; lane 2, GAL::rrp40, 12 h; lane 3, GAL::csl4, 12 h; lane 4, mtr3-1, 6 h at 37°C; lane 5, rrp6-Δ at 25°C; lane 6, GAL::dob1, 24 h; lane 7, GAL::U3, 24 h.

Figure 7

Aberrant A₂–C₂ pre-rRNAs accumulate in exosome mutants. RNA was extracted from the GAL::csl4 strain grown on RSG medium (0 h) and after transfer to repressive glucose medium for various lengths of time, and run on a 6% polyacrylamide gel for analysis of low molecular weight RNA. Lane 1, wild-type, 0 h; lanes 2–5, GAL::csl4 for 0, 2, 6 and 12 h. (A) Hybridization with probe 020. (B) Hybridization with probe 017. (C) Hybridization with probe 041. (D) Hybridization with probe 005. (E) Hybridization with probe 002. The weak band visible in all lanes in (D) probably represents cross-hybridization to the mature 5.8S rRNA.