1. Volume 16, Issue 6, Pages 943-954 (December 2004)
Pre-18S Ribosomal RNA Is Structurally Compacted into the SSU Processome Prior to Being Cleaved from Nascent Transcripts in Saccharomyces cerevisiae 
Yvonne N. Osheim, Sarah L. French, Kristin M. Keck, Erica A. Champion, Krasimir Spasov, François Dragon, Susan J. Baserga, Ann L. Beyer 
Molecular Cell 
Volume 16, Issue 6, Pages (December 2004)
DOI: /j.molcel

2. Figure 1
Cleavage of Nascent rRNA Transcripts in Yeast Cells, but Not in Stress Conditions or in Xenopus Oocytes
(A) Simplified rRNA processing scheme, focusing on the A0, A1, A2 early cleavages that generate the 20S precursor to 18S rRNA. The 27SA2 precursor undergoes six additional cleavages (small arrows) to generate 5.8S and 25S rRNAs.
(B) A yeast rRNA gene (NOY886) from classic Miller spread conditions, showing small terminal knobs (small arrows) and a few larger knobs (large arrow) on transcripts in the middle third of the gene, and mostly cleaved transcripts in the last ∼third of the gene (bracket). Bars in (B)–(E) = 0.5 μm.
(C) A yeast rRNA gene (NOY886; classic Miller spread conditions) after a 4 min heat shock at 37° showing no obvious cleavage of nascent transcripts.
(D) A yeast rRNA gene (YPH499) obtained using more physiological conditions, showing larger terminal knobs on transcripts in the middle third of the gene (large arrows) and mostly cleaved transcripts in the last third of the gene (bracket).
(E) A Xenopus oocyte rRNA gene (classic Miller spreading conditions) showing no obvious nascent transcript cleavage and small 5′ terminal knobs on transcripts starting in the middle third of the gene and remaining to the end.
(B′, D′, and E′) Portions of genes (boxed in [B], [D], and [E]) showing a direct size comparison of small and large 5′ terminal knobs on nascent rRNA transcripts.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Transcript Mapping Shows Length Compaction of Pre-18S RNA to Form the Large SSU Knob Followed by Cleavage in the ITS1 Region
(A–C) Left panels show a yeast rRNA gene from cells grown to OD600 ≅ 0.5 from strains YPH499 (A) and NOY1051 (B and C). Middle panels show interpretive tracing of the genes. DNA is color-coded: the 5′ end to the A2 cleavage site is red, A2 to 3′ end is blue, and the intergene spacer is green. Particles that appear on the transcripts are shown on the tracing: gray particles for small 5′ terminal knobs, pink for newly formed (loose) large SSU knobs, red for mature (tight) SSU knobs, and blue for pre-LSU knobs that form at the 5′ end of cleaved transcripts. Right panels show transcripts linearized and displayed at the appropriate position on the gene map. It can be seen that transcripts increase in length as they transcribe the 5′ ETS and 18S sequence but then are significantly compacted at a point near ITS1, resulting in the start of a second length gradient. Note the very short transcripts in this region, such as #29 on gene A, #15 on B, and #19 on C. At a point further along the gene, the compacted 18S sequence in the large (red) SSU knob is cleaved from the transcript. The downward arrows marked “compaction” or “cleavage” indicate that most transcripts after that point have undergone compaction or cleavage, respectively.
(D) RNP fibril maps for the genes in (A)–(C), drawn with the DNA on a slope so that 5′ RNA sequences are aligned vertically to the left in each map. Note that the 5′ ends of the earliest cleaved transcripts in each gene extrapolate approximately to the ITS1 region (green dashed lines), which contains the A2 and A3 separating cleavage sites. Some of the cleaved transcripts acquire new terminal knobs (blue), which compact the transcript length.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Verification of Cleavage and Decrease in Frequency as Cells Are Grown to Higher Densities
(A) Genes were exposed to detergent to partially unfold the nascent transcripts, thus showing the length difference between cleaved and uncleaved transcripts. Two genes are shown together with higher magnification views of their 3′ halves. Cleaved transcripts are indicated by a bracket. The inset in the right panel is a tracing of the gene in that panel. The gene on the left was prepared in 0.1% Sarkosyl; the gene on the right in 0.025% Triton.
(B) Three representative genes from NOY886 cell culture grown to OD600 readings of 0.6 (top), 1.8 (middle), or 2.7 (bottom) showing that the frequency of cotranscriptional cleavage drops as cells are grown to higher density. Note the efficient cleavage of the large SSU knobs (arrows) in the top gene as shown by the absence of knobs at the 3′ end (bracket), while the bottom gene (from the same culture at a later time point) shows large SSU knobs persisting on most transcripts all the way to the end of the gene. The middle gene displays an intermediate mix of transcripts plus and minus the large SSU knob in the 3′ region. Quantitative analysis of these results is shown in Table 1.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Progressive Formation of a Pre-60S Particle after Cleavage to Remove the Pre-40S particle
(A) Shown are three genes with efficient cleavage of nascent transcripts followed by formation of new 5′ terminal knobs on the LSU portion of the transcripts. Each panel shows the full-length gene as a small insert and a larger view of the 3′ half of the gene. Thin arrows: small particle or structured element that is present immediately after cleavage at the 5′ end, and sometimes can be seen in the same region of the transcript before cleavage. Medium arrows: new LSU 5′ terminal knobs that form after cleavage of the large SSU knobs. Large arrows: large SSU knobs.
(B) A gene with very little cleavage of nascent transcripts is shown; large SSU knobs persist to the end of the gene (large arrows). Note that there is a small particle kinked to one side at the site that will form the LSU knob (thin arrows), but that these structures do not progressively enlarge as they do after removal of the SSU knob, as seen in genes in (A).
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Representative Genes Showing Effects of Depletion of Specific Components on SSU Knob Formation and Nascent Transcript Cleavage
For all panels, cells were grown to OD600 of 0.4–0.6. For (B)–(I), time of depletion refers to time after the cells were switched from growth in galactose medium to growth in glucose medium.
(A) Control rRNA gene from YPH499 strain, which is the parental strain for most of the depletion strains. Large SSU knobs (large arrows) and transcript cleavage are evident.
(B) Depletion of U3 snoRNA for 3 hr; strain JH84. Transcripts have very little particulate structure and no obvious cleavage.
(C–E) Depletion of SSU processome components Utp7, Imp3, and Mpp10, respectively, all for 6 hr. Small terminal knobs are seen on some transcripts (arrows), but no significant cleavage.
(F) Depletion of U14 snoRNA for 6 hr. Transcripts are similar to those seen in SSU processome-component depletions in (C)–(E), with occasional small particles, and are not as naked as when U3 RNA is depleted (B).
(G) Depletion of RRP2, the RNA component of RNase MRP, for 6 hr. Transcripts do not show formation of large SSU knobs and do not show cleavage, though smaller knobs are seen (arrows).
(H) Depletion of Rpf2, which is required for LSU synthesis, for 6 hr. Depletion of Rpf2 allows cotranscriptional formation of large SSU knobs (arrows), though cleavage is somewhat delayed.
(I) The gene is from a cold-sensitive strain, grown at 23° for 6 hr, carrying a mutant mpp10 with the last 95 amino acids deleted (mpp10-5). Large SSU knobs form (arrows), but they are not cleaved while nascent, consistent with slowed cleavage at A2. The knobs appear somewhat looser than typical SSU knobs (compare [A] or [H]).
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Model for Cotranscriptional Processing of S. cerevisiae rRNA Transcripts
At the top is shown an rRNA gene schematic, with intergene spacer regions as thin black lines and transcribed gene sequences color-coded red (before A2 site) or blue (after A2 site) as in Figure 2. The arrow indicates the transcribed segment and the time required for transcription, 112 s, as determined previously (French et al., 2003).
The middle panel shows our interpretation of events visualized on nascent yeast rRNA transcripts. Particles are color-coded as in Figure 2. Transcripts increase in length until Pol I reaches about the end of the 18S sequence. The 5′ ETS and most of the 18S rRNA sequences are then compacted into a large loose (pink) SSU knob, followed shortly by tightening of this particle into a smaller, more electron-dense knob (red) and incorporation of the entire 18S sequence. This occurs ∼55 s after the start of transcription in early log phase cells (though later in late log phase cells). About 30 s later on average, the large SSU knob is no longer seen on nascent transcripts, which we interpret to represent cleavage at the A2 site (Figure 2). Although the released pre-40S ribosomes are not seen in chromatin spreads since they are no longer chromatin-associated, other results suggest that most processome components are released from the cleaved 20S RNA, which is exported to the cytoplasm for maturation to 18S RNA. The downstream cleavage product, nascent 27SA2 RNA, acquires a new 5′ terminal particle that enlarges and encompasses more RNA as the transcripts mature (blue particles). Cleavage is a stochastic process, and not all transcripts near the 3′ end are cleaved, as shown by two transcripts that retain the SSU knob. Release of the transcript at the 3′ end of the gene is by cotranscriptional cleavage by Rnt-1 in the 3′ ETS (Kufel et al., 1999).
The schematic at the bottom shows how nascent eukaryotic rRNA transcripts have appeared in most previous EM studies, with small 5′ terminal knobs and no obvious cleavage.
Molecular Cell  , DOI: ( /j.molcel )