1. The σ70-like Motif Molecular Cell
Karen A. Wehner, Susan J. Baserga 
Molecular Cell 
Volume 9, Issue 2, Pages (February 2002)
DOI: /S (02)

2. Figure 1 Pre-rRNA Processing Pathway in S. cerevisiae
The nascent rRNA molecules are transcribed by either RNA polymerase I or by RNA polymerase III. Processing of the RNA polymerase III transcript produces the 5S rRNA. The primary RNA polymerase I transcript undergoes extensive modifications and processing to result in the production of the 18S, 5.8S, and 25S rRNAs. The processing sites of the initial transcript are designated (reviewed in Kressler et al., 1999; Venema and Tollervey, 1999). This figure has been adapted from Kressler et al. (1999). The relative positions of the oligonucleotide probes (a, b, c, e, and y) used in Northern blot analysis are indicated in lowercase letters.
Molecular Cell 2002 9, DOI: ( /S (02) )

3. Figure 2 The Rpf Proteins Localize to the Nucleolus
(A) The Rpf1 protein is localized to the nucleolus in living cells. Yeast expressing GFP-Rpf1p (green) were examined both by direct fluorescence and DIC microscopy.
(B) Rpf2p localizes to the nucleolus in fixed cells. Haploid yeast, in which the chromosomal copy of RPF2 was tagged with a triple HA epitope, were used in indirect immunofluorescence. Rpf2p-3×HA is shown in red, the nucleolar marker, Mpp10p, is shown in green, and the DNA (DAPI) is shown in blue. Yellow indicates a complete overlap of the Rpf2 protein with the nucleolar marker.
Molecular Cell 2002 9, DOI: ( /S (02) )

4. Figure 3 The Imp4 Superfamily Is Conserved among Eukaryotes
(A) Phylogenetic tree of the Imp4 superfamily members. Members of individual families are colored accordingly: red (Rpf1), blue (Imp4), green (Rpf2), black (Brx1), and purple (Ssf). GenBank accession numbers are: S. cerevisiae Rpf1p, AAB68926; H. sapiens Rpf1, AF322053; M. musculus Rpf1, TC148051; C. albicans Rpf1, CAB77655; Y. lipolytica Rpf1, CAB55338; D. melanogaster Rpf1, AAF53162; A. thalania Rpf1, CAB77726; C. elegans Rpf1, CAA93858; S. pombe Rpf1, CAB11051; S. cerevisiae Imp4p, 95949; M. musculus Imp4, AF334609; C. elegans Imp4, CAB05841; D. melanogaster Imp4, AAF56395; A. thalania Imp4, AAF09145; S. pombe Imp4, CAB11643; S. cerevisiae Rpf2p, CAA82160; H. sapiens Rpf2, BAB14983; M. musculus Rpf2, BAA95117; D. melanogaster Rpf2, AAF55514; C. elegans Rpf2, AAF60765; S. pombe Rpf2, CAB54156; S. cerevisiae Ssf1p, AAB68379; S. cerevisiae Ssf2p, AAB64748; D. melanogaster Peter Pan, CAB99252; S. cerevisiae Brx1p, CAA99087; M. musculus Brx1, BAB22497; D. melanogaster Brx1, AAF47877; A. thalania Brx1, AC024081; S. pombe Brx1, CAC01521; S. pombe Ssf, CAB11063; C. elegans Peter Pan, AAB97575; A. thalania Peter Pan, BAB10077; L. major Rbp1, AC012381; P. aeruginosa Sigma-70, AAG03965; E. coli rpoD, P00579; M. xanthus Sigma-C, AAA25408; S. aurantiaca Sigma-B, CAA78693; S. coelicolor WHIG, AAA26828.
(B) Western blot of protein extracts prepared from strains in which endogenously expressed Imp4 superfamily members were tagged with a 3×HA epitope. Bands were quantitated to determine relative protein concentrations. Numbers in the matrix indicate the ratio of the proteins on the x axis to the proteins on the y axis.
(C) Sequence comparison of the Imp4 superfamily σ70-like domains and canonical σ70 domains. The two positions that are absolutely conserved are shaded black. Positions at which greater than 46% of the residues are similar to the consensus are shaded in red. Similarity is based upon a functional grouping of amino acids.
(D) The σ70-like motif is essential for viability. 1.6 × 105 cells of pGAL1::RPF1 containing (p415GPD-RPF1), (p415GPD-rpf1ΔN-term), or (p415GPD-rpf1ΔC-term); or pGAL1::RPF2 containing (p415GPD-RPF2) or (p415GPD-rpf2Δσ70-like) were spotted onto 5-FOA media. Absence of growth on 5-FOA indicates that the mutant alleles were not able to complement the genomic null and that the deleted regions are essential for function.
Molecular Cell 2002 9, DOI: ( /S (02) )

5. Figure 4 Imp4 Superfamily Members Are RNA Binding Proteins
(A) Imp4p, Rpf1p, and Rpf2p bind ribohomopolymers. [35S]methionine-labeled luciferase, Imp4p, Rpf1p, and Rpf2p were prepared by in vitro transcription and translation and were assayed for the ability to bind the indicated homoribopolymers, ssDNA anchored to agarose beads, and beads alone. Binding activities of Imp4p (red), Rpf1p (blue), Rpf2p (green), and luciferase (white) represent relative amounts of protein bound, based on the percent of input that was bound.
(B) The 17 amino acids that comprise the σ70-like domains of Rpf1p and Rpf2p were produced as luciferase fusion proteins (Luc-σ70-Rpf1p and Luc-σ70-Rpf2, respectively) and used in RNA binding assays as described in (A). Binding activities were calculated as percent of input bound and are represented as white (luciferase), blue (Luc-σ70-Rpf1p), and green (Luc-σ70-Rpf2) bars.
Molecular Cell 2002 9, DOI: ( /S (02) )

6. Figure 5
The Imp4 Superfamily Proteins Associate with Mature and Precursor rRNA
(A) Triple HA-tagged Imp4 superfamily proteins were examined for their ability to coimmunoprecipitate large subunit pre-rRNA precursors. Following immunoprecipitation (IP), RNA was extracted, separated by electrophoresis, and analyzed by Northern blot analysis using oligonucleotide probes b, c, and e (see Figure 1). The parent (untagged) strain was used as a control. All experiments were also done in parallel without antibody (BA). Total RNA from each extract prior to immunoprecipitation was prepared in the same way and is shown in the panel on the right. The immunoprecipitation supernatants are shown in Supplemental Figure S1 at
(B) RNA coimmunoprecipitated with triple HA-tagged Imp4 superfamily members, with or without anti-HA antibodies (H or B, respectively), was directly labeled with [P32]pCp and separated on an 8% polyacrylamide sequencing gel. The parent (untagged) and Nop1p-3×HA strains were used as controls.
(C) Small RNA molecules coimmunoprecipitated with Rpf2p-3×HA were analyzed by Northern blot analysis. Immunoprecipitations were carried out using anti-HA antibodies (αHA), and mock immunoprecipitations were carried out using beads alone. The parent (untagged) strain was used as a control. All cell extracts were prepared at 150 mM NaCl. The anti-HA immunoprecipitations from Rpf2p-3×HA were done in duplicate and washed in either 150 mM or 300 mM NaCl. RNA molecules were detected using oligos specific for the 7S pre-rRNA (oligo e, Figure 1), 5.8S rRNA, 5S rRNA, and U6 snRNA.
(D) Small RNA molecules coimmunoprecipitating with the other Imp4 superfamily members were also analyzed by Northern blot analysis as described above, except that immunoprecipitations were washed only in 150 mM NaCl. B is beads alone, and H is anti-HA immunoprecipitation.
Molecular Cell 2002 9, DOI: ( /S (02) )

7. Figure 6
Rpf1p, Rpf2p, and Ssf1/2p Are Required for Production of the Large Ribosomal Subunit
(A) Rpf1p depletion results in 27SA precursor accumulation and reduction in 25S rRNA levels. The yeast strain pGAL1::RPF1 was grown in media containing galactose and then switched to media containing glucose for an additional 3 or 9 hr of growth. At 0, 3, or 9 hr after the switch to glucose, 40 OD units of cells were harvested, pulsed for 2.5 min with [methyl-3H]methionine, and chased with cold methionine for 0, 3, 12, or 20 min.
(B) Rpf2p depletion results in slowed processing of 27S precursors and reduction in 25S rRNA levels. pGAL1::RPF2 was prepared for pulse-chase labeling essentially as described for (A), except that cells were grown in glucose for 0, 3, or 6 hr and chased for 0, 3, 6, 12, and 60 min.
(C) Ssfp depletion results in accumulation of 27SA precursors and loss of mature 25S rRNA. pGAL::SSF was grown in media containing galactose/raffinose (GR) and then switched to media containing glucose for an additional 12 hr. RNA was prepared from cultures at 0, 6, and 12 hr after the switch to glucose (Depl.). Cultures continuously grown in GR were used as controls (Undepleted). Equal amounts of RNA were separated on formaldehyde-1.2% agarose gels, transferred to membranes, and hybridized with specific oligonucleotide probes (see Figure 1).
Molecular Cell 2002 9, DOI: ( /S (02) )

8. Figure 7
A Model for the Function of the Imp4 Superfamily in Ribosome Biogenesis
Positions of the Imp4 superfamily proteins: Imp4 (blue), Rpf1 (red), Rpf2 (green), Ssf (purple), and Brx1 (black) proteins are based on our genetic and biochemical evidence. The Imp4 protein is a component of the U3 processome (yellow). Orange Pac-Men facing right represent the 5′ to 3′ exonucleases Rat1p and Xrn1p. Larger orange striped Pac-Men facing left represent the exosome. Yellow scissors represent the endonuclease predicted to function at site C2. Thick black lines represent the rRNA molecules, and thin black lines represent both the internal and external transcribed spacers. Preribosomal subunit complexes and mature ribosomal subunits are represented as light gray or dark gray ovals, respectively.
Molecular Cell 2002 9, DOI: ( /S (02) )