Fig 1.
7S-preLSU and 25S-preLSU rRNA tertiary structure with binding sites of ribosomal proteins used for tethered structure probing.
Structures of rRNA, rpL5, rpL27 and rpL35 in 25S-preLSUs (A, C, D) or 7S-preLSUS (B, D, F) are shown in three orientations with either the subunit interface side (A and B), the central protuberance (C and D) or the region formed by LSU rRNA domains I and III (E and F) in the center. In (A-F) the transparent surface of RNA filtered to 20 Angstrom resolution is superimposed on the RNA backbone. The surface representation of 5S rRNA is in blue, of LSU rRNA domain I and terminal ITS2 spacer regions in green and dark green, of LSU rRNA domain III in red and of other rRNA in grey. Spherical atomic models of rpL5, rpL27 and rpL35 are visualized in blue, red and green with the amino-terminal regions in light blue, orange and light green. Pdb files 5apo and 3jct used to create this figure were published in [12,16].
Fig 2.
Tethered MNase cleavages in 27SA-preLSUs, 27SB-preLSUS and 25S-preLSUS as analyzed by northern blotting.
27SA-preLSUs (A), 27SB-preLSUS (B, D-F) and 25S-preLSUS (C) were affinity purified using the indicated TAP tagged biogenesis factors from yeast strains expressing no MNase (lane 2) or MNase in fusion with rpL5 (lane 1), rpL35 (lane 3) or rpL27 (lane 4). After activation of MNase by addition of calcium for 10 minutes at 16°C the resulting rRNA fragments were analyzed by Northern Blotting using the indicated probes (# as prefix, see S1 Fig for rRNA regions recognized by the respective probes). In the upper panel in (A—C) RNA was separated by size by agarose gel electrophoresis, in (D-F) and in the lower panels in (A-C) by acrylamide gel electrophoresis. A ~110 nucleotide long fragment covering the central ITS2 region detected by probes #442 (B) and #3840 (E) is highlighted by an asterisk. Running behavior of RNA fragments of defined length are indicated on the left.
Fig 3.
Tethered MNase cleavage positions in the rRNA sequence as analyzed by primer extension assays.
27SB-preLSUs and 25S-preLSUs were affinity purified via Noc3-TAP or Lsg1-TAP from yeast strains expressing no MNase (No) or MNase fused to rpL5 (L5), rpL35 (L35) or rpL27 (L27). rRNA 5’ ends generated after activation of MNase were analyzed by random primer extension assays and high throughput sequencing or by targeted primer extension assays (see Materials and Methods). In (A) and (B) percent of cleavage (TCR) at the indicated rRNA positions (+/- one nucleotide) was estimated based on high throughput sequencing data. Relative cleavage efficiency was estimated in (C) by inspection of targeted primer extension data shown in S4, S10 and S12 Figs as very strong (++++), strong (+++), weak (++), very weak (+) or not detectable (-).
Fig 4.
Location of rpL27-MNase and rpL35-MNase cleavages in the 25S-preLSU rRNA tertiary structure.
Structures of rRNA, rpL5, rpL27 and rpL35 in 25S-preLSUs (pdb file 5apo, [12]) are visualized using the same color scheme as described in Fig 1, with rRNA cleavage sites highlighted in yellow. In the orientation shown in (A) LSU rRNA domains I and III are in the center, in the orientation shown in (B) the subunit interface side.
Fig 5.
Location of rpL5-MNase cleavages in 25S-preLSU and 7S-preLSU rRNA tertiary structure models.
Structures of rRNA, rpL5, rpL27 and rpL35 in 25S-preLSUs (A) or 7S-preLSUs (B) are shown using the color scheme as in Fig 1, with rRNA cleavage sites at the surface highlighted in yellow. The orientation shown is centered on the central protuberance. The pdb files 5apo and 3jct used to create the Figure were published in [12,16].
Fig 6.
Location of tethered MNase cleavages in secondary and tertiary structure models of the ITS2 rRNA region.
Structures of rRNA, rpL5, rpL27 and rpL35 in 7S-preLSUs are shown using the same color scheme as described in Fig 1, centering on LSU rRNA domains I and III in (A) and on the subunit interface side in (B). The last nucleotides of the terminal ITS2 region resolved in the 7S-preLSU structure model are colored in yellow. The pdb file 3jct used to create the figures (A) and (B) was published in ([16]). In (C-E) three secondary structure models of the ITS2 region are shown which were taken from [29](C), [30](D) and [31](E). In (D) and (E) the terminal parts of the ITS2 region with a secondary structure matching the one observed in cryo electron microscopy analyses of 7S-preLSUs shown in (A) and (B) are highlighted in blue. Position of the endonucleolytic C2 processing site (C2) according to [28,49] and tethered MNase cleavage sites (black arrows) are indicated.
Fig 7.
Release of the central ITS2 fragment from 27SB-preLSUs after site specific ITS2 cleavage by MNAse tethered to rpL27.
In (A) calcium dependent production of the central ~110nt ITS2 fragment was analyzed in cellular extracts of the indicated strains expressing MNase fused to rpL27 with a linker of 2, 24 and 41 amino acids. Extracts were incubated at 16°C for the indicated times in the presence of calcium and RNA was analyzed by Northern blotting using probe #O442 (see Materials and Methods). The central ~110nt ITS2 fragment is marked by an asterisk. In (B) the cellular extract of a strain expressing MNase fused to rpL27 was incubated for the indicated times at 16°C in the presence of calcium (lanes 1–3) and after ten minutes of incubation LSU precursors were affinity purified via Noc3-TAP (lane 4, see Materials and Methods). RNA was analyzed by Northern blotting using probe #O210 in the upper panel and probe #O442 in the lower panel. The central ~110nt ITS2 fragment is marked by a star in the lower panel and a neighboring fragment spanning the 5.8S rRNA region and the 5’ region of ITS2 is marked by two stars in the upper panel. Running behavior of RNA fragments of defined length are indicated on the left.