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GENE EXPRESSION

Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing

Sarah J. Lee, Susan J. Baserga
Sarah J. Lee
Department of Therapeutic Radiology and Genetics 1 and
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Susan J. Baserga
Department of Therapeutic Radiology and Genetics 1 and Yale Cancer Center, 2 Yale School of Medicine, New Haven, Connecticut 06520-8040
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DOI: 10.1128/MCB.19.8.5441
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    Fig. 1.

    (A) Pre-rRNA processing in the yeast S. cerevisiae. The nascent 35S transcript is cleaved in three U3 snoRNP-dependent reactions at A0, A1, and A2 to separate the precursors to the SSU (18S) rRNA from those to the LSU (5.8S and 25S) rRNAs. (B) Pre-rRNA processing pathway in yeast depleted of the U3 snoRNA. Genetic depletion of the U3 snoRNA causes an apparent increase in the levels of the nascent 35S precursor and accumulation of the 23S precursor resulting from lack of cleavage at A0, A1, and A2. As a result, decreased levels of the 20S and 18S rRNAs occur. Processing of the LSU pre-rRNA occurs via the 27S precursor (18).

  • Fig. 2.
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    Fig. 2.

    Imp3p and Imp4p interact with Mpp10p in a two-hybrid screen. Interactions were assayed by activation of the beta-galactosidase gene, which, if positive, yields a dark blue color. Two different Mpp10p bait constructs, mpp10-5 andmpp10-6, were tested for interaction with the Imp3p and Imp4p prey.

  • Fig. 3.
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    Fig. 3.

    Imp3p has a high similarity with ribosomal protein S9 (Rps9; E. coli S4). Shown is the GAP (Genetics Computer Group) sequence comparison between Imp3p and Rsp9Ap, one of the two ribosomal protein S9s in yeast. The lines indicate identity, and the dots indicate similarity. The predicted S4 RNA binding domain of Rps9 occurs from aa 107 to aa 170 (1).

  • Fig. 4.
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    Fig. 4.

    Imp3p and Imp4p are essential for cleavage at A0, A1, and A2 in the pre-rRNA. Strains bearing Imp3p or Imp4p expressed from a galactose-inducible, glucose-repressible promoter were constructed and designated pGAL1::IMP3 and pGAL1::IMP4. RNA was extracted from these strains either prior to transfer from galactose to glucose (time zero) or 24 h after transfer to glucose. As a control, RNA was also extracted from an otherwise isogenic strain, YPH259. RNA was analyzed by formaldeyde gel electrophoresis followed by Northern blotting with oligonucleotides complementary to the mature 18S and 25S rRNAs (a and y), to ITS1 (b and c), and to ITS2 (e). The asterisk indicates a precursor that we observe in the nondepleted strains and in the control strain but that we have not observed previously (10). Blotting with oligonucleotide z, complementary to sequences between A0 and A1, suggests that it probably extends from site A1 to A3 (data not shown).

  • Fig. 5.
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    Fig. 5.

    Imp3p and Imp4p are required for pre-rRNA processing. pGAL1::IMP3 and pGAL1::IMP4 were grown in glucose for 48 h and then labelled with [methyl-3H]methionine for 2.5 min. A chase was performed with unlabelled methionine, and samples were removed for RNA analysis immediately after addition of the unlabelled methionine (time zero) or 3 or 12 min postaddition. An otherwise isogenic strain, YPH259, was similarly treated. The RNA was analyzed by agarose-formaldehyde gel electrophoresis followed by transfer to a Zeta-Probe membrane which was subsequently treated with En3Hance (NEN).

  • Fig. 6.
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    Fig. 6.

    (A) Epitope-tagged Imp3 and Imp4 proteins complement their respective null alleles. Either a triple-MYC or a triple-HA tag was placed at the 3′ end of an IMP3 or an IMP4gene and cloned into the expression plasmid p415GPD. Strains were constructed so that the only expressed Imp3 or Imp4 protein is that with the indicated epitope tag. Growth on YPD of the epitope-tagged Imp protein strains was compared to that of a similarly constructed strain that expresses Mpp10p from p415GPD (24). (B) Western blotting with anti-HA epitope antibodies. Total protein was isolated from strains expressing HA-tagged Imp proteins and analyzed by SDS-PAGE and Western blotting. As a control, protein was isolated from strains expressing MYC-tagged protein. (C) Western blotting with anti-MYC epitope antibodies. Total protein was isolated from strains expressing MYC-tagged Imp proteins and analyzed by SDS-PAGE and Western blotting. As a control, protein was isolated from strains expressing HA-tagged protein.

  • Fig. 7.
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    Fig. 7.

    The Imp3 and Imp4 proteins are complexed with Mpp10p in vivo. (A) Immunoprecipitation (IP) followed by Western blotting with anti-MYC epitope antibodies. Immunoprecipitations were performed with lysates and the indicated antibody, and the resulting bound proteins were analyzed by SDS-PAGE and Western blotting with anti-MYC epitope antibodies. (B) Immunoprecipitation followed by Western blotting with anti-Mpp10p antibodies. Immunoprecipitations were performed with lysates and the indicated antibody, and the resulting bound proteins were analyzed by SDS-PAGE and Western blotting with anti-Mpp10p antibodies.

  • Fig. 8.
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    Fig. 8.

    (A) Imp3p and Imp4p are protein components of the U3 snoRNP. Immunoprecipitations were performed with lysates from the epitope-tagged strains and the indicated antibodies. The RNA from bound RNPs was extracted and analyzed by denaturing gel electrophoresis followed by Northern blotting. Probes were used to detect the U3 and U14 box C/D snoRNAs and snR10, a box H/ACA snoRNA. Lanes 1 to 4 represent total RNA extracted from the same amount of lysate used for immunoprecipitation. (B) Imp3p and Imp4p are specific protein components of the U3 snoRNP. Immunoprecipitations were performed with the indicated antibodies and lysates from the indicated strains. RNAs from the resulting bound RNPs was isolated and analyzed by labelling with [5′-32P]cytidine 3′,5′-bis phosphate and T4 RNA ligase, followed by denaturing gel electrophoresis.

  • Fig. 9.
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    Fig. 9.

    Depletion of the Imp proteins does not affect U3 snoRNA levels. RNA was isolated from pGAL1::IMP3 and pGAL1::IMP4 at the switch from galactose to glucose (time zero) and 24 h after the switch to glucose. As a control, RNA was also isolated from an otherwise isogenic strain, YPH259. Equal amounts of RNA were run on a denaturing polyacrylamide gel, blotted, and hybridized with probes complementary to the U3, snR10, snR190, and U14 snoRNAs.

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Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing
Sarah J. Lee, Susan J. Baserga
Molecular and Cellular Biology Aug 1999, 19 (8) 5441-5452; DOI: 10.1128/MCB.19.8.5441

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Imp3p and Imp4p, Two Specific Components of the U3 Small Nucleolar Ribonucleoprotein That Are Essential for Pre-18S rRNA Processing
Sarah J. Lee, Susan J. Baserga
Molecular and Cellular Biology Aug 1999, 19 (8) 5441-5452; DOI: 10.1128/MCB.19.8.5441
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