Monoallelically expressed noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression
- Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, United States
- Department of BioHealth Informatics, School of Informatics and Computing, IUPUI, United States
- Department of Computer Science, University of Illinois at Urbana-Champaign, United States
- Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Ireland
- Pacific Biosciences, United States
- Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, United States
- Ionis Pharmaceuticals Inc, United States
- Department of Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, United States
- Department of Biochemistry, University of Illinois at Urbana-Champaign, United States
- Department of Cell and Molecular Biology, Northwestern University, United States
- Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, United States
- Department of Biomedical Engineering, Georgia Tech, United States
- Cancer Center at Illinois, University of Illinois at Urbana-Champaign, United States
Figures
Figure 1 with 3 supplements
SNUL-1 forms RNA clouds in human cell lines.
(A) RNA-FISH of SNUL-1 (green) in WI-38 cells. Nucleoli are visualized by rRNA (red). (B) RNA-FISH of SNUL-1 (green) in hTERT-RPE1 and U2OS cell lines. Nucleoli are visualized by rRNA (red). (C) …
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Figure 1—source data 1
Quantification of nucleoli and SNUL-1 cloud numbers in different cell lines.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig1-data1-v2.zip
Figure 1—figure supplement 1
SNUL-1 forms RNA clouds in human cell lines.
A RNA-FISH of SNUL-1 (green) in different human cell lines. For all images, nucleoli are visualized by rRNA (red). Scale bars, 5 µm.( B) RNA-FISH of SNUL-1 after nuclease treatments in WI-38 cells. …
Figure 1—figure supplement 2
SNUL-1 forms RNA clouds in human cell lines.
A Local alignment between SNUL-1 Probe 4 and SNUL-2 probe. Note the imperfect [CT] repeat in SNUL-2 probe and the poor alignment between the two probes beyond the [CT]-rich region. (B) Schematic …
Figure 1—figure supplement 3
SNUL-1 forms RNA clouds in human cell lines.
A Schematic, showing the positions of the rRNA and ITS1 probes, ASO-SNUL and SNUL-1 CS candidates relative to pre-rRNA sequence. Please note that the dashed lines represent imperfect and/or gapped …
Figure 2 with 1 supplement
SNUL-1 is an RNA Pol I transcript and forms constrained nucleolar territory.
(A) Representative SIM image of the SNUL-1 (red) distribution relative to DFC/FC units in WI-38 cells. FC is marked by RPA194 (blue) and DFC is marked by mNeonGreen (NG)-FBL (green). Scale bars, …
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Figure 2—source data 1
Quantification in Figure 2B, F and G.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig2-data1-v2.zip
Figure 2—figure supplement 1
SNUL-1 is an RNA Pol I transcript and forms constrained nucleolar territory.
(A) Visualization of the tripartite structure within a single HeLa nucleolus by SIM. FC is marked by UBF (red), DFC is marked by mNeonGreen (mNG)-FBL (green), and GC is marked by mTagBFP2-B23 …
Figure 3 with 1 supplement
SNUL-1 is associated with the NOR of one Chr. 15 allele.
(A) DNA-RNA-FISH of SNUL-1 RNA (green) and distal junction (DJ) DNA (red) in WI-38 cells. (B) DNA-RNA-FISH of SNUL-1 RNA and Chr. 15, Chr. 13, and Chr. 22 marked by probes painting the q-arms of the …
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Figure 3—source data 1
Quantification of association rates between SNULs and different chromosomes in Figure 3C.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig3-data1-v2.zip
Figure 3—figure supplement 1
SNUL-1 is associated with the NOR of one Chr. 15 allele.
(A) RNA-FISH showing the distribution of SNUL-1 in WI-38 cells during mitosis. Arrows point at the prominent SNUL-1 cloud in early G1 daughter nuclei. Arrow heads point at the relatively weak SNUL-1 …
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Figure 3—figure supplement 1—source data 1
Quantification of association rates between SNULs and different chromosomes in Figure 3—figure supplement 1F.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig3-figsupp1-data1-v2.zip
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Figure 3—figure supplement 1—source data 2
Quantification of relative integrated density of the rDNA on the two Chr.15 alleles in Figure 3—figure supplement 1H.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig3-figsupp1-data2-v2.zip
Figure 4 with 1 supplement
The SNUL-1 cloud displays mitotically-inherited random monoallelic association.
(A) Representative RNA-FISH images showing the association of SNUL-1 with the 15Sat III or 15CEN in WI-38 nuclei. (B) Plot showing the relative integrated density of the 15Sat III signal in WI-38 …
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Figure 4—source data 1
Quantification in Figure 4D, F and H.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig4-data1-v2.zip
Figure 4—figure supplement 1
The SNUL-1 cloud displays mitotically-inherited random monoallelic association.
(A) DNA-RNA-FISH showing the localization SNUL-1 RNA cloud and 15Sat III in hTERT-RPE1 cell nucleus. (B) Plot showing the relative integrated density of the 15Sat III signals. Relative integrated …
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Figure 4—figure supplement 1—source data 1
Quantification in Figure 4—figure supplement 1B,D,H and J.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig4-figsupp1-data1-v2.zip
Figure 5 with 7 supplements
SNUL-1 influences rRNA biogenesis.
(A) RNA-FISH of SNUL-1 (green) and SNUL-2 (red) in WI-38 cells transfected with ctr-ASO or ASO-SNUL oligonucleotides. (B) 5-FU immunostaining in control and SNUL-depleted WI-38 cells. Scale bars, 5 …
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Figure 5—source data 1
Quantification in Figure 5C, E, F and H.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig5-data1-v2.zip
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Figure 5—source data 2
Uncropped image of Northern Blot in Figure 5J.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig5-data2-v2.zip
Figure 5—figure supplement 1
SNUL-1 influences rRNA biogenesis.
(A) Representative SNUL-1 RNA FISH in Ctr-ASO, ASO-SNUL, and ASOs targeting individual SNUL-1 CS candidates-treated cells (ASO112 and 113). Scale bars, 10 µm. (B) Representative RNA-FISH images …
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Figure 5—figure supplement 1—source data 1
Quantification of pre-rRNA signal intensities in different cell lines, related to Figure 5—figure supplement 1C,E,G.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig5-figsupp1-data1-v2.zip
Figure 5—figure supplement 2
SNUL-1 influences rRNA biogenesis.
(A) SIM image of a single nucleolus showing the nascent pre-rRNA detected by 5’ETS-2 probe (red) in Ctr and SNUL-depleted cells. DFC is marked by mNG-FBL (green) and FC is marked by RPA194 (blue). …
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Figure 5—figure supplement 2—source data 1
Uncropped image of Northern Blot in 1511 Figure 5—figure supplement 2C and D.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig5-figsupp2-data1-v2.zip
Figure 5—figure supplement 3
SNULs influence Chr.15 replication timing.
(A-B) Representative images of replication timing-specific hybridization (ReTiSH) showing the replication timing of rDNA regions in HTD114 control and SNUL-depleted cells. HTD114 cells are labeled …
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Figure 5—figure supplement 3—source data 1
Quantification of ReTiSH assay showing the relative integrated density of the rDNA signal on the two Chr.15 alleles, related to Figure 5—figure supplement 3C.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig5-figsupp3-data1-v2.zip
Figure 5—figure supplement 4
SNULs influence Chr.15 replication timing.
(A-B) Representative images of ReTiSH showing the replication timing of rDNA regions in WI38 control and SNUL-depleted cells. WI38 cells are labeled with BrdU for 5 or 14 hr prior to harvest and …
Figure 5—figure supplement 5
SNULs influence Chr.15 replication timing.
(A-B) Representative images of EdU staining followed by rDNA and 15CEN DNA FISH in HTD114 control and SNUL-depleted cells. Cells are synchronized by double thymidine block and released at different …
Figure 5—figure supplement 6
rDNA clusters do not show allele-specific histone modifications.
(A-C) Representative images of active histone marks (H3K4me3, H4Ac AND H3Ac) distribution in HTD114 Ctrl and SNUL-depleted metaphase spreads. DNA-FISH is performed to detect rDNA (magenta) and 15CEN …
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Figure 5—figure supplement 6—source data 1
Quantification of histone mark signal intensities on Chr.15 alleles in HTD114 cell line, related to Figure 5—figure supplement 6D.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig5-figsupp6-data1-v2.zip
Figure 5—figure supplement 7
rDNA clusters do not show allele-specific histone modifications.
(A-C) Representative images of active histone marks (H3K4me3, H3Ac AND H4Ac) distribution in RPE1 Ctrl and SNUL-depleted metaphase spreads. DNA-FISH is performed to detect rDNA (magenta) and 15Sat …
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Figure 5—figure supplement 7—source data 1
Quantification of histone mark signal intensities on Chr.15 alleles in RPE1 cell line, related to Figure 5—figure supplement 7D.
- https://cdn.elifesciences.org/articles/80684/elife-80684-fig5-figsupp7-data1-v2.zip
Author response image 1
RNA-FISH (green) using unique probes designed from the chr.
17 RNA (without including the repeat) in WI-38 cells showed the absence of SNUL-1 cloud in the nucleolus.
Tables
Author response table 1
| QUERY | nucDNA1_PK_combo__HQ_transcript/2305 |
|---|---|
| SCORE | 1743 |
| START | 2 |
| END | 2523 |
| QSIZE | 3003 |
| IDENTITY | 88.4% |
| CHROM | chr13 |
| STRAND | + |
| Ref_START | 9250197 |
| Ref_END | 9252367 |
| SPAN | 2171 |
Additional files
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Supplementary file 1
Sequence of the SNUL-1 probe.
Please see the Excel file
- https://cdn.elifesciences.org/articles/80684/elife-80684-supp1-v2.xlsx
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Supplementary file 2
The CS candidate sequences.
Please see the Excel file
- https://cdn.elifesciences.org/articles/80684/elife-80684-supp2-v2.xlsx
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Supplementary file 3
The dissimilarity score between each isoform and its best match from human transcript database.
For each isoform the empirical p-value is the empirical probability of observing a dissimilarity score greater than or equal to its dissimilarity score. The product of the 5 p-values being in the order of rejects the hypothesis of all isoforms being the transcripts of the same known gene.
- https://cdn.elifesciences.org/articles/80684/elife-80684-supp3-v2.xlsx
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Supplementary file 4
The pairwise dissimilarity for the isoforms.
The pairwise dissimilarity for the isoforms. Global pairwise alignment was used to compute the dissimilarity score for each pair of isoforms. The dissimilarity score was computed by taking the ratio of mismatching to matching sites where both isoforms do not contain gaps. For each pair of isoforms, the p-value shows the probability of observing a dissimilarity score greater than or equal to their dissimilarity by approximating the empirical distribution of pairwise dissimilarity with erlang distribution.
- https://cdn.elifesciences.org/articles/80684/elife-80684-supp4-v2.xlsx
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Supplementary file 5
Probes used in this study.
Please see the Excel file.
- https://cdn.elifesciences.org/articles/80684/elife-80684-supp5-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/80684/elife-80684-mdarchecklist1-v2.docx
