A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis

Abstract

Although the U3 small nucleolar RNA (snoRNA), a member of the box C/D class of snoRNAs, was identified with the spliceosomal small nuclear RNAs (snRNAs) over 30 years ago, its function and its associated protein components have remained more elusive. The U3 snoRNA is ubiquitous in eukaryotes and is required for nucleolar processing of pre-18S ribosomal RNA in all organisms where it has been tested. Biochemical and genetic analyses suggest that U3 pre-rRNA base-pairing interactions mediate endonucleolytic pre-rRNA cleavages. Here we have purified a large ribonucleoprotein (RNP) complex from Saccharomyces cerevisiae that contains the U3 snoRNA and 28 proteins. Seventeen new proteins (Utp1 17) and Rrp5 were present, as were ten known components. The Utp proteins are nucleolar and specifically associated with the U3 snoRNA. Depletion of the Utp proteins impedes production of the 18S rRNA, indicating that they are part of the active pre-rRNA processing complex. On the basis of its large size (80S; calculated relative molecular mass of at least 2,200,000) and function, this complex may correspond to the terminal knobs present at the 5' ends of nascent pre-rRNAs. We have termed this large RNP the small subunit (SSU) processome.

Figure 1

RNA composition of fractions from the purification of the U3 snoRNP. RNA was extracted from the starting material (extract, lane 2), from the first purification step that enriches for the box C/D snoRNAs (TEV elution, lane 3), and from the final eluate (Flag elution, lane 4). The RNA was directly labelled by ³²p-labelled pCp and T4 RNA ligase, and analysed on an 8% denaturing polyacrylamide gel.

Figure 2

Function of the new components of the SSU processome. a, The Utp proteins are nucleolar. Anti-HA or anti-Myc antibodies were used to detect the tagged Utp proteins (red), whereas anti-Mpp10 antibodies were used to decorate the nucleolus (green). DAPI was used to stain the nucleus (blue). Utp proteins were triply tagged by HA or Myc. b, The Utp proteins and Rrp5 are complexed with Mpp10. Anti-HA immunoprecipitations performed on cell extracts were analysed for the presence of Mpp10 by western blotting with anti-Mpp10 antibodies. T, total (5% of the input for immunoprecipitation); IP, immunoprecipitate. c, The Utp proteins and Rrp5 are associated with U3 snoRNA. Anti-HA immunoprecipitations were performed on cell extracts and were washed at 150 mM (150) or 400 mM (400) NaCl. RNA was extracted and analysed for the presence of the U3 snoRNA by northern blotting. T, total (20% of the input); S, supernatant (20%). d, The Utp proteins are required for 18S rRNA biogenesis. Yeast strains conditionally expressing Utp proteins were grown in medium containing galactose/raffinose (0 h), but switched to glucose for protein depletion (24 or 48 h). RNA was extracted at the indicated time points and analysed for the presence of 25S and 18S rRNAs by northern blot.

Figure 3

The SSU processome sediments at 80S on sucrose gradients. Yeast extracts were analysed on 10–47% sucrose gradients. Anti-Mpp10, anti-Imp3, anti-Imp4 and anti-HA antibodies were used to detect the indicated proteins in each fraction by western blotting. Northern blotting was used to detect the U3 snoRNA. The migration of the 40S, 60S and 80S ribosomal subunits is specified. Utp16 and Nop1 proteins were triply tagged by HA.

Figure 4

The U3 snoRNA and Utp7 are required for terminal knob formation on nascent pre-rRNAs. Yeast strains conditionally expressing either the U3 snoRNA or Utp7 from a galactose promoter were used to make the chromatin spreads. Strains were undepleted (a) or depleted for U3 snoRNA (b) or Utp7 (c). For depletion, the strains were switched from growth in galactose to growth in glucose. Chromatin spreads were made before (0 h) and after (3 h) the switch to glucose and analysed by electron microscopy. Scale, the width of panel a is 0.85 μm.