ASAR lncRNAs control DNA replication timing through interactions with multiple hnRNP/RNA binding proteins

  1. Mathew Thayer  Is a corresponding author
  2. Michael B Heskett
  3. Leslie G Smith
  4. Paul T Spellman
  5. Phillip A Yates
  1. Department of Chemical Physiology and Biochemistry,Oregon Health & Science University, United States
  2. Department of Molecular and Medical Genetics, Oregon Health & Science University, United States
  3. Stanford Cancer Institute, United States
  4. Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Oregon Health & Science University, United States
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
RNA binding proteins interact with an ~7 kb domain within ASAR6-141 RNA.

(A) RNA-DNA FISH images of ASAR6-141 expression in six individual HTD114 cells. ASAR6-141 RNA (green; green arrows) and chromosome 6 DNA (chromosome paint, red), and DNA was stained with DAPI. (B) …

Figure 1—source data 1

The location of significant peaks of eCLIP reads that map within ASAR and XIST genes.

https://cdn.elifesciences.org/articles/95898/elife-95898-fig1-data1-v1.xlsx
Figure 1—source data 2

eCLIP reads for 120 RBPs in 10 kb windows within ASARs.

https://cdn.elifesciences.org/articles/95898/elife-95898-fig1-data2-v1.xlsx
Figure 1—source data 3

Fosmids, BACs, and primers.

https://cdn.elifesciences.org/articles/95898/elife-95898-fig1-data3-v1.xlsx
Figure 1—figure supplement 1
CRISPR/Cas9 deletion of the ~7 kb RBPD.

(A) Integrative Genomics Viewer view showing the genomic location of ASAR6-141. The genomic locations of RNAseq and eCLIP reads from HepG2 and K562 (from ENCODE) are shown. The zoomed in view shows …

Figure 1—figure supplement 1—source data 1

Original image file of PCR products shown in Figure 1—figure supplement 1C.

Figure 1-figure supplement 1C, the original image file of the PCR products shown in panel C. Figure 1-figure supplement 1C (labels), the original image file for panel C with labels and regions of the image used are marked by the black boxes. Figure 1-figure supplement 1D, the original image file of the PCR products shown in panel D. Figure 1-figure supplement 1D (labels), the original image file for panel D with labels and regions of the image used are marked by the black boxes.

https://cdn.elifesciences.org/articles/95898/elife-95898-fig1-figsupp1-data1-v1.zip
Figure 2 with 1 supplement
Replication timing in cells with ASAR6−141~7 kb RBPD deletion or ectopic integration.

(A) Schematic illustration of the BrdU terminal label protocol (Smith and Thayer, 2012). Cells were treated with BrdU (green) for 5 hr and then harvested for mitotic cells. (B) BrdU incorporation in …

Figure 2—figure supplement 1
Identification of mouse chromosomes containing the ~7 kb RBPD transgenes.

(A) DNA FISH on mouse chromosomes from clone ~7 kb(+)A5. Mitotic spread showing DNA FISH using the ~7 kb RBPD (magenta; arrow) and a mouse chromosome 5 BAC (RP24-107D17; red; arrowheads). (B and C) …

Figure 3 with 2 supplements
Depletion of RBPs results in disruption of the chromosome territory localization of ASAR6-141 RNA.

(A–J) shRNA mediated depletion of RBPs. K562 cells were transfected with empty vector (A, C, E, G, and I) or vectors expressing shRNAs directed against HNRNPU (B), HNRNPUL1 (D), HNRNPC (F), HNRNPM (H

Figure 3—figure supplement 1
RBP protein levels in cells expressing shRNAs.

K562 or HTD114 cells were transfected with empty vector (-) or shRNA against RBPs and cell extracts were processed for western blots using antibodies (Key Resource Table) as indicated: (A and B) …

Figure 3—figure supplement 1—source data 1

Original image files for the western blots.

Figure 3-figure supplement 1A, the original image file for the western blot with HNRNPU (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HNRNPU. Figure 3-figure supplement 1A (labels), the same image as above showing labels for the location of HNRNPU and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1B, the original image file for the western blot with HNRNPU (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against HNRNPU. Figure 3-figure supplement 1B (labels), the same image as above showing labels for the location of HNRNPU and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3—figure supplement 1C, the original image file for the western blot with UCHL5 (green) and tubulin (red) antibodies on K562 cells with and without shRNA against UCHL5. Figure 3-figure supplement 1C (labels), the same image as above showing labels for the location of UCHL5 and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1D, the original image file for the western blot with HNRNPC (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HNRNPC. Figure 3-figure supplement 1C (labels), the same image as above showing labels for the location of HNRNPC and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3—figure supplement 1E, the original image file for the western blot with HNRNPC (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against HNRNPC. Figure 3-figure supplement 1E (labels), the same image as above showing labels for the location of HNRNPC and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1F, the original image file for the western blot with HNRNPL (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HNRNPL. Figure 3—figure supplement 1F (labels), the same image as above showing labels for the location of HNRNPL and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1G, the original image file for the western blot with HNRNPM (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HNRNPM. Figure 3-figure supplement 1G (labels), the same image as above showing labels for the location of HNRNPM and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1H, the original image file for the western blot with HNRNPM (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against HNRNPM. Figure 3-figure supplement 1H (labels), the same image as above showing labels for the location of HNRNPM and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1I, the original image file for the western blot with HNRNPL (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against HNRNPL. Figure 3-figure supplement 1I (labels), the same image as above showing labels for the location of HNRNPL and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1J, the original image file for the western blot with KHSRP (green) and tubulin (red) antibodies on K562 cells with and without shRNA against KHSRP. Figure 3-figure supplement 1J (labels), the same image as above showing labels for the location of KHSRP and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1K, the original image file for the western blot with KHSRP (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against KHSRP. Figure 3-figure supplement 1K (labels), the same image as above showing labels for the location of KHSRP and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1L, the original image file for the western blot with HNRNPUL1 (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against HNRNPUL1. Figure 3-figure supplement 1L (labels), the same image as above showing labels for the location of HNRNPUL1 and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1M, the original image file for the western blot with HLTF (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HLTF. Figure 3-figure supplement 1M (labels), the same image as above showing labels for the location of HLTF and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1N, the original image file for the western blot with SAFB2 (green) and tubulin (red) antibodies on K562 cells with and without shRNA against SAFB2. Figure 3-figure supplement 1N (labels), the same image as above showing labels for the location of SAFB2 and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1O, the original image file for the western blot with PTBP1 (red) antibody on K562 cells with and without shRNA against PTBP1. Figure 3-figure supplement 1O (labels), the same image as above showing labels for the location of PTBP1, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1P, the original image file for the western blot with HNRNPA1 (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HNRNPA1. Figure 3-figure supplement 1P (labels), the same image as above showing labels for the location of HNRNPA1 and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red.

https://cdn.elifesciences.org/articles/95898/elife-95898-fig3-figsupp1-data1-v1.zip
Figure 3—figure supplement 2
RNA FISH on cells transfected with shRNAs that did not disrupt ASAR localization.

(A–D) K562 cells were transfected with empty vector (A) or shRNA expression vectors for MATR3 (B), PTBP1 (C), or PTBP2 (D). Cells were processed for RNA FISH using probes for ASAR6-141 (green; …

Ectopic integration of the ASAR6−141~7 kb RBPD transgene into an inactive X chromosome.

(A) Two color RNA FISH assay for expression of the sense ~7 kb RBPD transgene plus Xist RNA. Examples of three different cells showing colocalization of the ASAR RNA and Xist RNA. Individual cells …

Figure 5 with 1 supplement
Depletion of RBPs results in disruption of the chromosome territory localization of ASAR RNAs.

shRNA-mediated depletion of RBPs in K562 cells (A–K) or HTD114 cells (L–Q). Cells were transfected with empty vector (A–C and L–N) or vectors expressing shRNAs directed against HNRNPA1 (D), HNRNPC (E

Figure 5—figure supplement 1
Disruption of the chromosome territory localization of ASAR1-187 and ASAR6 RNAs following shRNA depletion of RBPs.

HTD114 cells were transfected with empty vector (A) or shRNA expression vectors for HNRNPA1 (B), HNRNPC (C), HNRNPL (D), HNRNPM (E), HNRNPUL1 (F), KHSRP (G), HLTF (H), or UCHL5 (I). Cells were …

Figure 6 with 2 supplements
Depletion of RBPs results in asynchronous replication on autosome pairs.

(A) BrdU incorporation in parental HTD114 cells. Cells were exposed to BrdU for five hours and processed for BrdU incorporation using an antibody against BrdU. This panel represents chromosomes from …

Figure 6—figure supplement 1
Replication timing in cells transfected with shRNA expression vectors.

(A and B) HTD114 cells were transfected with PTBP1 shRNA expression vector, exposed to BrdU for 5 hr, harvested for mitotic cells, and processed for BrdU incorporation (green) and DNA FISH using a …

Figure 6—figure supplement 2
Replication timing alterations in shRNA depleted cells.

HTD114 cells were transfected with shRNA expression vectors for HNRNPA1 (A), HNRNPC (B), HNRNPL (C), HNRNPM (D), HLTF (E), KHSRP (F), HNRNPU (G), or UCHL5 (H), exposed to BrdU for 5 hr, harvested …

Human C0T-1 DNA detects ASAR6 RNA.

(A and B) RNA-DNA FISH on mouse cells containing an ASAR6 BAC transgene integrated into mouse chromosome 3 Donley et al., 2013. (A) RNA-DNA FISH using an ASAR6 fosmid probe to detect ASAR6 RNA …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-HLTF
(rabbit polyclonal)
SigmaRRID:AB_18572631:1000
AntibodyAnti-HNRNPA1
(rabbit polyclonal)
SigmaHPA0042761:500
AntibodyAnti-HNRNPC
(rabbit polyclonal)
SigmaRRID:AB_26813351:1000
AntibodyAnti-HNRNPL
(rabbit polyclonal)
SigmaRRID:AB_26815881:500
AntibodyAnti-HNRNPM
(rabbit polyclonal)
SigmaRRID:AB_18508791:2000
AntibodyAnti-HNRNPU
(rabbit polyclonal)
SigmaRRID:AB_26837961:500
AntibodyAnti-HNRNPUL1
(rabbit polyclonal)
SigmaRRID:AB_26796161:1000
AntibodyAnti-KHDRBS1
(rabbit polyclonal)
SigmaRRID:AB_26814171:500
AntibodyAnti-KHSRP
(rabbit polyclonal)
SigmaRRID:AB_106015821:500
AntibodyAnti-PTPB1
(mouse monoclonal)
InvitrogenRRID:AB_25330821:250
AntibodyAnti-SAFB
(rabbit polyclonal)
SigmaRRID:AB_18506221:500
AntibodyAnti-SAFB2
(rabbit polyclonal)
SigmaRRID:AB_26812661:500
AntibodyAnti-UCHL5
(rabbit polyclonal)
SigmaRRID:AB_18585851:2000
AntibodyAnti-alpha-tubulin
(mouse monoclonal)
SigmaRRID:AB_26171161:10,000
AntibodyAnti-BrdU FITC
(mouse monoclonal)
RocheRRID:AB_1104262750 µg/ml
AntibodyAnti-mouse IgG, Dylight 680
(goat polyclonal)
InvitrogenRRID:AB_19659561:10,000
AntibodyAnti-mouse IgG, Dylight 650
(goat polyclonal)
Thermo FisherRRID:AB_109423011:10,000
AntibodyAnti-rabbit IgG, Dylight 800
(goat polyclonal)
Thermo FisherRRID:AB_25567751:10,000
AntibodyAnti-rabbit IgG, Alexa Fluor Pus 405
(goat polyclonal)
InvitrogenRRID:AB_28905481:100
AntibodyAnti-goat IgG, Alexa Fluor 594
(rat monoclonal)
InvitrogenRRID:AB_25360801:100
Recombinant DNA reagentshRNA:HLTF
(plasmid)
SigmaTRCN0000272562
Recombinant DNA reagentshRNA:HNRNPA1
(plasmid)
SigmaTRCN0000006586
Recombinant DNA reagentshRNA:HNRNPC
(plasmid)
SigmaTRCN0000006644
Recombinant DNA reagentshRNA:HNRNPL
(plasmid)
SigmaTRCN0000017246
Recombinant DNA reagentshRNA:HNRNPM
(plasmid)
SigmaTRCN0000314949
Recombinant DNA reagentshRNA:HNRNPU
(plasmid)
SigmaTRCN0000350256
Recombinant DNA reagentshRNA:HNRNPUL1
(plasmid)
SigmaTRCN0000074732
Recombinant DNA reagentshRNA:KHDRBS1
(plasmid)
SigmaTRCN0000428104
Recombinant DNA reagentshRNA:KHDRBS1
(plasmid)
SigmaTRCN0000428752
Recombinant DNA reagentshRNA:KHSRP
(plasmid)
SigmaTRCN0000013256
Recombinant DNA reagentshRNA:PTBP1
(plasmid)
SigmaTRCN0000231420
Recombinant DNA reagentshRNA:SAFB
(plasmid)
SigmaTRCN000022099
Recombinant DNA reagentshRNA:SAFB2
(plasmid)
SigmaTRCN0000075057
Recombinant DNA reagentshRNA:UCHL5
(plasmid)
SigmaTRCN0000234907

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