The 28 nt leader sequence of scaRNA9 is important for stability and protein interactions. (A) RNA pulldown of WT and mutant scaRNA2 and scaRNA9. Proteins recovered in the pulldown reactions were subjected to SDS-PAGE, western blot transfer, and probing with the indicated antibodies. Background binding of proteins was determined using reactions containing beads alone without RNA bait (lane 2). The input lane (lane 1) accounts for 3% of the lysate used in the pulldown reactions. The location of coilin and coilp1 are indicated, and the relative amount of these proteins recovered by scaRNA9 with the deleted leader sequence (lane 6) compared to WT scaRNA9 (lane 5) is shown in the histogram (n=5 experimental repeats, ***P<0.0005, data are mean±s.e.m.). (B) Northern blotting of RNA isolated from untransfected cells (lane 1) or cells transfected with a DNA construct expressing WT scaRNA9 (lane 2) or scaRNA9 with a deleted leader sequence (lane 3). Probe B, shown in Fig. 1, was used to detect full-length scaRNA9 and the mgU2-30 domain. The blot was also probed for 5S rRNA to verify that equal amounts of RNA were present. (C) RNA FISH/IF was used to localize WT scaRNA9 and scaRNA9ΔLeader. Probe D, shown in Fig. 1, was used to detect full-length scaRNA9 and the mgU2-30 domain by RNA FISH. Arrows indicate Cajal bodies (detected by coilin localization) and arrowheads demarcate nucleoli (determined by DAPI negative regions). The relative intensity of nucleolar to nucleoplasmic RNA FISH signal was quantified for each RNA (histogram, *P<0.05, data are mean±s.e.m., n=20 cells). Scale bars: 2 μm.

Coilp1 contributes to the processing of scaRNA 9 and 17. For coilp1 reduction experiments, cells were treated for 24 h with control or coilp1 siRNA, followed by transfection with DNA constructs expressing scaRNA 9 or 17 and harvest/RNA isolation 24 h later (A,C). For coilp1 overexpression experiments, cells were co-transfected with plasmids expressing scaRNA 9 or 17 with GFP-coilp1 or GFP alone (B,D). The RNA was then subjected to Northern blotting using the probes indicated in Fig. 1. Original, as well as adjusted, images are shown. Adjustments were made to images using the High and Low transformation settings of the QuantityOne software. Gamma levels were not changed and the transformation was applied evenly across the entire image for each individual panel. Histograms were generated from the adjusted images and display the quantification of the processed fragment relative to the full-length scaRNA normalized to the control condition. *P<0.05, ***P<0.0005, data are mean±s.e.m., n=3 experimental repeats; scaRNA9 (A,B), scaRNA17 (C,D). Note that ectopic scaRNA9 is expressed from a construct containing an intron. Consequently, full-length ectopic scaRNA9 is the same size as endogenous scaRNA9, which is encoded within an intron. Full-length endogenous scaRNA17 is difficult to detect using Dig probes.

Regulatory RNPs. Base pairing between the nucleolus-enriched fragments of scaRNA2, 9 and 17 with snoRNA. Also shown is the potential for the 3′ loop of mgU2-30 (top right) to base pair with 28S rRNA, possibly blocking ACA3 binding to this site, as well as serve as a binding site for U20 snoRNA. The 5′ loop of scaRNA17-derived mgU4-8 (bottom right), which contains a sequence found in U6 snRNA, might serve as a binding sink for U94 and HBII-166 (not shown) snoRNAs, thereby altering C60 and C62 methylation of U6 snRNA. We propose that these fragments derived from scaRNA 2, 9 and 17 form regulatory RNPs (regRNPs) that influence the modification of sites within 18S rRNA, 28S rRNA and the nucleolar trafficked U6 snRNA.

Methylation of 18S A484 is increased upon reduction of scaRNA17. RNA from control or scaRNA17 siRNA treated cells was subjected to primer extension using 5 different dNTP amounts (indicated) during the reverse transcription step with a radioactive primer complementary to nucleotides 524-544 of 18S rRNA. Samples were run on a denaturing acrylamide gel followed by detection of the radioactive signals. Quantification demonstrates that the A484 pause signal is 1.7-fold more abundant in the scaRNA17 knockdown condition compared to control knockdown (n=6 experimental repeats, P<0.005).

Disruption of the snoRD16/18S rRNA interaction by a fragment of scaRNA17. (A) Extra base paring (shown in amber) (van Nues et al., 2011) between snoRD16 and 18S rRNA predicted to facilitate the methylation of A484 (shown in purple) is shown. (B) The looped region of snoRD16 (from nt 30-40) base pairs with scaRNA17 (regRNP17) from nt 412-402 (Fig. 4). The binding of the nucleolus-enriched fragment derived from scaRNA17, mgU4-8 (regRNP17), may disrupt the interaction of snoRD16 with 18S rRNA, resulting in decreased A484 methylation. (C) RNase protection assays using fragments of snoRD16, 18S rRNA (31 nt, 3′ Dig labeled) and scaRNA17. Reactions containing the indicated RNA were denatured, followed by annealing and incubation with RNase A/T1. The reactions were then resolved on a 15% TBE-urea polyacrylamide gel, followed by Northern blot transfer and detection with anti-Dig antibodies. Because of the intensity of the signal, only 20% of the reaction with 18S rRNA fragment lacking RNase A/T1 was run on the gel (lanes 1 and 6), and the exposure of these lanes is shorter than that for lanes 2-5. Additionally, the reaction sample in lane 6 was supplemented with Dig-labeled DNA oligonucleotides to serve as size markers (indicated by bands at 22 and 18 nt). A protected fragment is observed in lane 4 but not in lane 5.

The A484 region of 18S rRNA, and snoRD16 associated RNAs. (A) A schematic of 18S rRNA is shown, along with interacting snoRNAs. Note that snoRD16 can form extra base pairing with 18S rRNA (amber nt) by the formation of a loop within snoRD16 (van Nues et al., 2011). Modification of G509, A512 and C517 involves guide snoRNAs that have an overlapping binding site. The underlined sequence in 18S rRNA including C517 is found exactly in snoRD16, which means that snoRD56 can associate with snoRD16. (B) Schematic of snoRD16, which is 101 nt long. The looped region of snoRD16, which interacts with regRNP17, is indicated. The sequence in snoRD16 that is exactly identical to that found in 18S rRNA is indicated by a yellow rectangle. The locations of the snoRD16 C/D and C′/D′ boxes are shown, as well as associations with other snoRNAs.