Table 1.
Small fragments generated from snoRNAs.
Figure 1.
Conservation of the box H/ACA snoRNAs that encode miRNAs.
Screenshots of the UCSC Genome Browser [48] displaying RefSeq genes (blue lines with hatch marks), miRNA hairpins (red blocks with hatch marks indicating the mature portion) and snoRNAs (green blocks with hatch marks) are displayed above the mammalian conservation track [50] for the genomic regions surrounding ACA36B (A), ACA34 (B), HBI-61 (C) and ACA45 (D).
Figure 2.
Number of snoGPS hits above given scores.
The number of snoGPS hits above scores ranging from 36 to 54 is shown for 676 extended miRNA regions (red), sets of randomly-generated sequences (blue) and sets of randomly-generated hairpins (yellow). For the randomly-generated sequences and hairpins, 100 sets of 676 molecules were run and the average values are shown here. The error bars represent standard deviation.
Table 2.
Best snoGPS hits.
Figure 3.
Secondary structure predictions of H/ACA snoRNAs.
The secondary structure predictions of known H/ACA snoRNAs encoding experimentally detected miRNAs (A) as well as predicted H/ACA snoRNAs encoding known miRNAs (B) were drawn using RNAstructure [34] and RNAviz [51]. Mature miRNAs are drawn in pink. H and ACA boxes are shown respectively in orange and cyan. Guide regions are outlined using dark blue lines.
Figure 4.
Retrogene-like structures encoding miRNAs.
Screenshots of the UCSC Genome Browser [48] displaying RefSeq genes (blue lines with hatch marks), miRNA hairpins (red blocks), snoRNAs (green blocks with hatch marks), repeat-elements (blue blocks with hatch marks) and TSDs (black blocks) are shown for the genomic regions surrounding mir-215 (A), mir-549 (B), mir-1266 (C) and mir-605 (D). The thick regions in the red blocks represent the mature regions of the miRNAs. The 5′ TSD is annotated as TSD5 and the 3′ TSD is annotated as TSD3. Shown below the genomic structure illustrations are the sequences corresponding to these regions. In the sequences, the miRNA hairpins are underlined, the predicted snoRNAs are shown in uppercase italics, the boxes ACA and H are respectively shown in a box and a shaded box and putative poly(A) tails are underlined using a wavy line.
Figure 5.
snoRNA-like miRNA precursors that bind dyskerin.
Nuclear extracts were prepared from HeLa cells stably expressing either free GFP or YFP-dyskerin (YFP-DKC) and immunoprecipitated using an anti-GFP antibody. A Western blot confirming specificity of the immunoprecipitation using an anti GFP antibody. The same membrane was reprobed with an antibody against lamin as a loading control. B Position of the primers used to detect the specified miRNA extended regions. C RT-PCR used to detect co-precipitated hsa-mir-664, hsa-mir-151, hsa-mir-605, has-mir-215 and has-mir-140 miRNA precursors, with E2 box H/ACA snoRNA as positive control and hsa-pri-let-7g miRNA, U3 box C/D snoRNA, U1 snRNA, 5S rRNA and GAPDH pre-mRNA as negative controls for dyskerin-associated RNAs. The lane numbering in panel C refers to the lanes shown in panel A.
Figure 6.
Subcellular localization of H/ACA snoRNA-like miRNA precursors.
Northern blots of HeLa cell extracts fractionated into cytoplasmic, nucleoplasmic and nucleolar fractions were probed for the presence of mir-151 (A) and mir-664 (B) encoding molecules using probes against the respective mature miRNA region. In both panels A and B, bands labeled with ‘a’ represent the expected size of the predicted snoRNAs, those labeled with ‘b’ represent the expected size for the miRNA hairpins and ‘c’ represents the expected size of the mature miRNA. C As controls of the fractionation, northern blots of the same RNA preparations were probed against isoleucine tRNA, U3 and U11.