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1.

Figure. From: Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein.

Fig. 4. Cells depleted of Rrp10p accumulate 20S pre-rRNA in the cytoplasm. Electron microscopic detection of the ITS1: (A) in wild-type yeast cells, the labelling is exclusively detected in the nucleolus (35S, 33S, 32S and 20S pre-rRNAs); (B) in Δxrn1 cells, the labelling is found in the nucleolus and in the cytoplasm (fragment D-A2 from the ITS1); (C) in GAL::PROTA-RRP10 cells grown for 16 h in YPD medium (Glu), the labelling is detected in the nucleolus and in the cytoplasm. np, nucleoplasm; no, nucleolus; ct, cytoplasm. Bar, 200 nm.

Emmanuel Vanrobays, et al. EMBO J. 2001 August 1;20(15):4204-4213.
2.

Figure. From: Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein.

Fig. 3. Depletion of Rrp10p results in reduced synthesis of 18S rRNA. (A) YO296 (GAL::PROTA-RRP10) strain grown at 30°C on YNB Gal without methionine; (B) YO296 grown at 30°C in YPG medium (Gal) then shifted for 14 h to YPD medium (Glu), and finally grown for 9 h in YNB Glu without methionine. Cells were labelled for 4 min with (methyl-3H) methionine and chased with a large excess of unlabelled methionine for 1–20 min.

Emmanuel Vanrobays, et al. EMBO J. 2001 August 1;20(15):4204-4213.
3.

Figure. From: Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein.

Fig. 5. Rrp10p is a cytoplasmic protein. Subcellular localization using GFP-tagged proteins (A and B). (A) GFP-Rrp10p: cells transformed by pEV24 (GFP::RRP10) exhibit a cytoplasmic pattern of fluorescence. (B) Gar1p-GFP: in cells transformed with pZUT3 (GAR1::GFP) a punctate pattern characteristic of a nucleolar staining is observed. Positions of nuclei were determined by DAPI staining (blue). Overlay images are shown by superposition of the blue and green stainings. Immunolocalization by electron microscopy (C and D). (C) ProtA-Rrp10p; (D) Gar1p-ProtA. Tagged proteins were detected by treatment with anti-protein A antibodies followed by incubation with colloidal gold-conjugated protein A. no, nucleolus; np, nucleoplasm; ct, cytoplasm. Bars in C and D, 200 nm.

Emmanuel Vanrobays, et al. EMBO J. 2001 August 1;20(15):4204-4213.
4.

Figure. From: Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein.

Fig. 6. Sedimentation profiles of ProtA-Rrp10p and 20S pre-rRNA in a glycerol gradient. A total extract was loaded on a 10–30% glycerol gradient and subjected to centrifugation. Fractions were collected, and proteins and RNA in each fraction were analysed. (A) Sedimentation profiles of ProtA-Rrp10p, rpS4p, rpL3p and rpL32p. Proteins pre cipitated with TCA and separated by SDS–PAGE were revealed by western blotting. (B) Sedimentation pattern of 18S and 25S rRNAs and of 20S pre-rRNA. Fractions containing the peaks of 40S, 60S ribosomal subunits and 80S ribosomes are indicated. (C) 20S pre-RNA selectively co-purifies with Rrp10-ProtAp. 1/400 of the clarified cell lysate (T) or the bulk of the immunoprecipitated RNA (IP) was probed with either a mix of probes 1 and 6 in Figure 1 (18S, 25S) or a D-A2 probe prepared by multiprime labelling (20S). The fraction of each rRNA or pre-rRNA recovered in the immunoprecipitate was determined by phosphoimager quantification (IP/T).

Emmanuel Vanrobays, et al. EMBO J. 2001 August 1;20(15):4204-4213.
5.

Figure. From: Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein.

Fig. 2. Depletion of Rrp10p specifically affects the steady-state levels of mature 18S rRNA species and results in 20S pre-rRNA accumulation. Northern blot analysis of pre-rRNA processing: YO296 (GAL::PROTA-RRP10) cells were grown in YPG medium (Gal), or in YPD medium for up to 24 h (Glu). At the indicated time points, total RNA was extracted and separated in 1% agarose-formaldehyde gels to analyse 35S, 32S, 27SA2, 25S, 20S and 18S species, and in 6% polyacrylamide gels for 7S(L), 7S(S) and 5.8S species analysis. Equal amounts of total RNA (5 µg) were loaded in every lane. (A) Ethidium bromide staining of the gel. Northern blots of the gel were hybridized to: (B) probe 2 complementary to ITS1 upstream of site A2; (C) probe 1 complementary to 18S rRNA, a 7 h exposure (upper panel in C) and a 90 min exposure (lower panel in C) of the same blot are shown; (D) probe 4 complementary to 5.8S, probe 5 complementary to ITS2 and probe 6 complementary to 25S rRNA. Pre-rRNA and rRNA species are schematically represented on the right side; full rectangles represent the mature rRNAs and thin lines the transcribed spacers.

Emmanuel Vanrobays, et al. EMBO J. 2001 August 1;20(15):4204-4213.
6.

Figure. From: Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein.

Fig. 1. Pre-rRNA processing in S.cerevisiae. (A) Structure of the 35S pre-rRNA. In the primary transcript the sequences of the mature 18S, 5.8S and 25S rRNAs are flanked by the external transcribed spacers (5′ and 3′ETS) and separated by the internal transcribed spacers (ITS1 and ITS2). Cleavage sites are indicated by uppercase letters A0–E, and oligonucleotide probes used in northern blot hybridizations by numbers 1–6. (B) Pre-rRNA processing pathway. Sequential cleavages of the 35S pre-rRNA at sites A0 and A1 generate the 33S and 32S pre-rRNAs. Cleavage of the 32S pre-rRNA at site A2 in ITS1 yields the 27SA2 and 20S pre-rRNAs, which are precursors to the RNA components of the large and small ribosomal subunits, respectively. The 27SA2 precursor is either processed at site A3 by RNase MRP, generating the 27SA3 pre-rRNA rapidly digested by the 5′–3′ exonucleases Rat1p and Xrn1p to site B1S yielding the 27SBS pre-rRNA. This constitutes the major pathway. Approximately 15% of the 27SA2 molecules are processed at site B1L leading to the 27SBL intermediate. Processing at site B2, the 3′ end of the 25S rRNA, occurs concomitantly with 27SB 5′-end formation. The 27SBS and 27SBL pre-rRNAs both follow the same processing pathway to 25S and 5.8SS/L through cleavage at site C2 in ITS2, followed by 3′–5′ exonucleolytic digestion of 7SS and 7SL from site C2 to E by the exosome complex, and 5′–3′ exonucleolytic digestion to the 5′ end of the 25S rRNA. The final maturation of the 20S pre-rRNA by an endonucleolytic cleavage at site D occurs in the cytoplasm and produces the mature 18S rRNA and a fragment D-A2 (5′ ITS1). The D-A2 fragment is then degraded by the 5′–3′ exonuclease Xrn1p.

Emmanuel Vanrobays, et al. EMBO J. 2001 August 1;20(15):4204-4213.

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