Scaffold function of long noncoding RNA HOTAIR in protein ubiquitination

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1 Yoon et al, page Scaffold function of long noncoding RNA HOTAIR in protein ubiquitination Je-Hyun Yoon,, Kotb Abdelmohsen, Jiyoung Kim, Xiaoling Yang, Jennifer L. Martindale, Kumiko Tominaga-Yamanaka, Elizabeth J. White, Arturo V. Ojalo, John L. Rinn, Stefan G. Kreft, Gerald M. Wilson, and Myriam Gorospe Laboratory of Genetics, National Institute on Aging-Intramural Research Program, NIH, Baltimore, MD, USA; Department of Biochemistry and Molecular Biology and Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA; Biosearch Technologies, Inc., Novato, CA 999, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 8, USA Department of Biology, University of Konstanz, 787 Konstanz, Germany Correspondence: LG, NIA-IRP, NIH Bayview Blvd. Baltimore, MD, USA Tel: (JHY); Fax:

2 PABP p MDM Mexb Mexc Roquin (mouse ) Roquin (mouse ) Snurportin- -catenin Dzip Ataxin- (mouse) Ataxin- (rabbit) AUF TIA- TIAR HuR Ago FMRP Rck TTP KSRP Dicer Drosha Lin8 (Ago IP / IP) Yoon et al, page SUPPLEMENTARY FIGURES a From Fig. d From Fig. 6b Yoon et al, Supplementary Figure S 9 6 Ctrl HuR U6 let7b let7i Ataxin- WT Ataxin- 8Q 7 From Fig. c 7 From Fig. d 6 6 MexbDzip Mexb Dzip Ctrl HuR Ctrl HuR b IP: 6 8 IP: c IP: Dzip IP: Ataxin- IP: Mexb IP: Dzip (8 kda) IP: Ataxin- ( kda) IP: Mexb (9 kda) WB: Dzip WB: Ataxin- H.C. WB: Mexb H.C. H.C. WB: Dzip WB: Ataxin- WB: Mexb IP: Snurportin- IP: Mexc IP: HuR IP: Snurportin- ( kda) WB: Snurportin- L.C. IP: Mexc ( kda) WB: Mexc L.C. IP: HuR (7 kda) WB: HuR L.C. WB: Snurportin- WB: Mexc WB: HuR Supplementary Figure S. Survey of RNA-binding proteins associated with HOTAI and IP control assays. (a) Alternative manner of representing RIP assays used in the main article, where the RNA enrichments in the samples are not set as. but are set relative to the in the respective control group. The graphs represent the means and S.D. from independent experiments. (b) RIP analysis of the interaction of HOTAIR with a panel of RBPs in HeLa cell lysates. Antibodies recognizing the RBPs shown were used for IP in each case; control IP reactions were carried out using a corresponding. HOTAIR levels were measured by RT-qPCR and normalized to the levels of GAPDH mrna levels in the same IP samples measured by RT-qPCR analysis. Data were quantified as enrichment of HOTAIR in the RBP IP relative to the IP. The graphs represent the means and S.D. from independent experiments., P <. using Student s t-test. (c) Using HeLa cell lysates, the quality of the IP reactions was assayed by Western blot (WB) analysis of Dzip, Ataxin-, Mexb, Mexc, HuR, and Snurportin-. Lysates were tested by WB analysis before (lower images for each protein tested) and after IP (top panels for each protein tested). H.C., immunoglobulin heavy chain, L.C., immunoglobulin light chain. Larger fields of blots are shown in Supplementary Figure S7.

3 Relative levels (HOTAIR / 8S) Yoon et al, page a Human HOTAIR (7 nt) Yoon et al, Supplementary Figure S b CLIP analysis HuR IP / IP c HOTAIR(FL) d HOTAIR(-) e HOTAIR (-) HOTAIR (FL) Empty vector WB: Ataxin- ( kda) WB: Dzip (8 kda) (IP) Empty vector HOTAIR FL HOTAIR (-) WB: Actin ( kda) f HOTAIR(-) HOTAIR(-) HOTAIR(FL) GAPDH( UTR) HOTAIR(FL) GAPDH( UTR) His-Dzip 8-76 (8 kda) MBP-HuR (79 kda) Myc-Ataxin- ( kda) Input Pulldown Input Pulldown Supplementary Figure S. Additional characterization of the interaction of HOTAIR with HuR, Dzip, and Ataxin-. (a,b) Schematic of HOTAIR (a) and the segments tested by CLIP analysis (b) following the procedure described in the Methods section, using μg antibody and mg of crosslinked lysate. (c) A plasmid expressing only segments and of HOTAIR (spanning nucleotide positions ~8-7, short blue line) was constructed and expressed in HeLa cells. (d,e) Forty-eight hours after transfecting HeLa cells with plasmids to express full-length [HOTAIR(FL)] or partial HOTAIR [HOTAIR(-)], as assessed by RT-qPCR (d), the levels of endogenous Ataxin-, Dzip, and Actin were determined by Western blot analysis (e). (f) Biotin pulldown analysis to determine the interaction of MBP-HuR (left) or His-Dzip (RNA binding domain) or Myc-Ataxin- (right) with biotinylated HOTAIR(FL), HOTAIR(-) or control GAPDH( UTR). In (b,d), the graphs reflect the means and S.D. from independent experiments. Larger fields of blots are shown in Supplementary Figure S7.

4 HOTAIR / 8S rrna ATXN / 8S rrna SNUPN / 8S rrna Yoon et al, page a HOTAIR # HOTAIR # HuR # Control HuR # Control Yoon et al, Supplementary Figure S IP: Ataxin- IP: Snurportin- WB: Ub - 6 WB: Ub WB: HuR (7 kda) WB: HuR (7 kda) WB: Ataxin- ( kda) WB: Snurportin- ( kda) WB: Actin ( kda) WB: Actin ( kda) b..7.7 Ctrl... HuR # Ctrl HOTAIR # Ctrl HOTAIR # Ctrl HOTAIR # c Dzip # HuR Control Mexb # HuR Control IP: Ataxin- WB: Ub - 6 IP: Snurportin- WB: Ub WB: Dzip (8 kda) WB: Mexb (9 kda) WB: HuR (7 kda) WB: HuR (7 kda) WB: Ataxin- ( kda) WB: Snurportin- ( kda) WB: Tubulin ( kda) WB: Tubulin ( kda) d Ago # - + Control + - Ago # - + Control + - IP: Ataxin- WB: Ub IP: Snurportin- WB: Ub WB: Ago (97 kda) WB: Ago (97 kda) WB: Ataxin- ( kda) WB: Snurportin- ( kda) WB: Dzip (8 kda) WB: Mexb (9 kda) WB: Actin ( kda) WB: Actin ( kda) Supplementary Figure S. Effects of additional HOTAIR- or HuR-directed s. (a) Forty-eight h after silencing HuR with # (Methods), the levels of HuR, Ataxin-, Snurportin-, and loading control Actin were assessed by Western blot analysis; the levels of ubiquitinated Ataxin-, and ubiquitinated Snurportin-, were assessed by IP of Ataxin- or Snurportin followed by ubiquitin Western blot analysis. (b) Forty-eight h after silencing HOTAIR with # (Methods) in the presence of either Ctrl or HuR #, the levels of HOTAIR, ATXN mrna, and SNUPN mrna relative to 8S rrna were assessed by RT-qPCR analysis. Data are the means and S.D. from independent experiments. (c) Fortyeight h after silencing Dzip or Mexb with # (Methods), the levels of HuR, Dzip, Mexb, Ataxin-, Snurportin-, and loading control Tubulin were assessed by Western blot analysis; the levels of ubiquitinated Ataxin-, and ubiquitinated Snurportin-, were assessed by IP of Ataxin- or Snurportin- followed by ubiquitin Western blot analysis. (d) Forty-eight h after silencing Ago with #, the levels of Ago, Dzip, Mexb, Ataxin-, Snurportin-, and loading control Actin were assessed by Western blot analysis; the levels of ubiquitinated Ataxin-, and ubiquitinated Snurportin-, were assessed by IP of Ataxin- or Snurportin followed by ubiquitin Western blot analysis. Larger fields of blots are shown in Supplementary Figure S7.

5 Relative levels (ATXN mrna / /GAPDH mrna) Relative levels (SNUPN mrna / /GAPDH mrna) Relative level Relative level HOTAIR / GAPDH mrna % remaining RNA hotair / gapdh mrna Yoon et al, page Yoon et al, Supplementary Figure S a mire (-6) mirb (8-79) mir (9-8) mirb Let 7i (-6) (-) Human HOTAIR (6 nt) b Ago (97 kda) wt Ago-/- MEFs MEFs.. c 8 HeLa 9 HeLa HSP9 (9 kda) (IP) Ago wt Ago-/- MEFs MEFs Prelet7i (no biot) Prelet7i biotin d Ago (97 kda) Ctrl Ago e HOTAIR Ctrl Ago Pre-let7i GAPDH mrna HSP9 (9 kda) P=. Ctrl Ago Prelet7i Ctrl ~.7 h Pre-let7i ~. h Ago >.9 h Ctrl >. h Pre-let7i >. h Ago >. h Time (h) in actinomycin D f IP g Flag h Ctrl Ago IP Flag-HuR HuR..... Ctrl HuR Ctrl Prelet7i ASlet7i Ctrl i. j Dzip HOTAIR HuR Control Mexb HOTAIR HuR Control Supplementary Figure S. HuR and let7/ago cooperate in promoting HOTAIR decay. (a) HOTAIR sequence with predicted mirna target sites. (b) In mouse embryonic fibroblasts (MEFs) isolated from Ago knockout mice (Ago-/-) or wild type mice (wt), Ago and control HSP9 levels were assessed by Western blot analysis (left), and HOTAIR levels associated with Ago were assessed by RIP analysis followed by RTqPCR (right). (c) Left, RIP analysis of Ago-bound HOTAIR in HeLa cells. Right, 8 h after transfection of HeLa cells with let7i or biotin let7i, the relative enrichment of endogenous HOTAIR in biotin pulldown samples (isolated using streptavidin beads) was assessed by RT-qPCR quantitation of HOTAIR. (d,e) Fortyeight hours after silencing Ago or overexpressing pre-let7i in HeLa cells (d), the steady state levels and half-life of HOTAIR and control GAPDH mrna were assessed as explained in Figure f (e). (f) Forty-eight hours after transfecting HeLa cells with HuR, the association of HOTAIR with Ago was assessed by RIP and RT-qPCR analysis. (g,h) Forty-eight hours after overexpressing Flag-HuR, silencing HuR, overexpressing let7i or expressing a let7i antagomir (AS-let7i), HOTAIR abundance was assessed by RTqPCR. (i,j) The relative abundance of HOTAIR, ATXN, SNUPN mrna, and GAPDH mrna in cells processed as shown in Fig. e,f was assessed by RT-qPCR analysis. In (b-j), the graphs reflect the means and S.D. from independent experiments., P <. using Student s t-test. Larger fields of blots are shown in Supplementary Figure S7.

6 Yoon et al, page 6 a E (Mexb) + Ubiquitin/E + + Snurportin- Yoon et al, Supplementary Figure S E enzymes (Ubc): H H Ha Hb Hc H6 H7 H8 H H WB: Ub Coomassie Blue: Snurportin- b Dzip Mexb HOTAIR HOTAIR HuR HuR Control Dzip (8 kda) Control Mexb (9 kda) HuR (7 kda) HuR (7 kda) Ataxin- ( kda) Snurportin- ( kda) Tubulin ( kda) Tubulin ( kda) c d HOTAIR HuR IP HuR IP Control WB: Ub Empty vector pflag- Ataxin- WT pgfp- Snurportin WB: Actin ( kda) Supplementary Figure S. Survey of Ubiquitin conjugating (Ubc) E enzymes for Snurportin-, and analysis of silencing results. (a) Various E Ubc enzymes were assayed for their effect on ubiquitination of Snurportin- in vitro following the same procedure as that described in the main Fig. b. (b) Western blot analysis of the efficiency of silencing HuR, Dzip, and Mexb in HeLa cells used to measure the half-lives of Ataxin- and Snurportin-. (c) of HOTAIR compared to GAPDH mrna from HuR IP after overexpression of empty vector, Flag-Ataxin- WT, or GFP-Snurportin. Data are the means and S.D. from independent experiments. (d) Western blot analysis to detect ubiquitinated proteins in cell lysates after silencing HuR and/or HOTAIR. Larger fields of blots are shown in Supplementary Figure S7.

7 Relative levels (ATXN mrna / /8S rrna) Relative levels (SNUPN mrna / /8S rrna) HOTAIR / 8S rrna HOTAIR / 8S rrna Relative levels (ATXN mrna / /8S rrna) Relative levels (SNUPN mrna / /8S rrna) HOTAIR / 8S rrna HOTAIR / 8S rrna Yoon et al, page 7 Yoon et al, Supplementary Figure S6 a Dex - Dex b + Dex - Dex Ctrl HOTAIR Dzip Ctrl HOTAIR Mexb WB: Dzip (8 kda) WB: Mexb (9 kda) WB: HuR (7 kda) WB: HuR (7 kda) WB: Actin ( kda) WB: Actin ( kda) c d Dzip Mexb HOTAIR HOTAIR HuR Control HuR Control WB: Dzip (8 kda) WB: Mexb (9 kda) WB: HuR (7 kda) WB: HuR (7 kda) WB: Actin ( kda) WB: Actin ( kda) Supplementary Figure S6. RNA expression in IDH cells. (a,b) IDH cells [proliferating (+Dex) or senescent (-Dex)] were transfected as described in Fig. 6e; days later, the levels of HOTAIR, 8S rrna, ATXN mrna (a) and SNUPN mrna (b) were quantified and plotted. (c,d) Proliferating IDH cells were transfected as in Fig. 8; 8 h later, the levels of HOTAIR, 8S rrna, ATXN mrna (c) and SNUPN mrna (d). In (a-d) the graphs reflect the means and S.D. from independent experiments.., P <. using Student s t-test. Larger fields of blots are shown in Supplementary Figure S7.

8 Yoon et al, page 8 c let-7 c U6 d let-7 d U6 d HuR d Ago (nt) (nt) (nt) (nt) - - d Tubulin e let-7 e His-Ago e MBP HuR (nt) - b MBP-HuR e HuR e AUF e HSP9 g Flag - HuR g HuR g Ago g HSP9

9 Yoon et al, page 9 b Ctrl b HuR a Ataxin- a Snurportin- a HuR a HSP9 c Ataxin- c Ataxin- c Dzip c Snurportin- c Snurportin- c Mexb e UB e Dzip e HuR e Ataxin- - 6 e tubulin f UB f mexb f Snurportin-

10 Yoon et al, page i UB j UB i and j Ago i and j Actin & sd Actin - 6 i Dzip i Ataxin- j Mexb j Snurportin- a UB a Dzip a UBC H6 a Ataxin b UB b UBC H b Snurportin- b mexb -

11 Yoon et al, page c Ataxin- c Ataxin- c Snurportin- 6 c Snurportin- c Tubulin a UB 6a Dzip 6a HuR 6a Ataxin- 6a Tubulin - 6

12 Yoon et al, page 6c Ataxin- 6c Ataxin- 6c Tubulin 6 6 6d Dzip 6d HuR 6d Ataxin- 6d Tubulin 7a HuR 7a HuR 7a p 7a p 7a p 7a p a Ago 7a TERT 7a Actin

13 Yoon et al, page 7c Ctrl +Dex 7c HuR +Dex 7c Ctrl -Dex 7c HuR -Dex 7d UB 7d UB 7d Ataxin- 7d Ataxin- 7d Dzip 7d Dzip 7d and 7d Actin e UB 7e UB 7e Snurportin- 7e Snurportin- 7e Mexb 7e Mexb

14 Yoon et al, page 7f p 7f p 7f p6 7f actin g Ataxin- 7f Snurportin- 7f Actin 8a Ataxin- 8a Snurportin- 8a Actin 8a Ataxin- 8a Snurportin- 8a Actin a Snurportin- 8a Actin 7 8

15 Yoon et al, page 8b Ataxin- 8b Snurportin- 8b Actin 6 8b Ataxin- 8b Snurportin- 8b Actin 6 6 8b Snurportin- 8b Actin 7 8 Sc Dzip Sc Dzip Sc Ataxin- Sc Ataxin- Sc mexb Sc mexb Sc Snurportin- Sc Snurportin- Sc mexc Sc mexc Sc HuR

16 Yoon et al, page 6 Se Ataxin- Se Dzip Se Actin Sf HuR Dzip Ataxin- Sa UB Sa UB Sa HuR - 6 Sa Ataxin- Sa Snurportin- Sa Actin Supplementary Figure S7. Larger fields of blots shown in the Figures -9 and Supplementary Figures S-S6. Larger fields of the blots depited in the main figures are indiated. The corresponding figure and panel, as well as the molecules (proteins or nucleic acids) detected are indicated above each blot., molecular weight markers [kda (kilodaltons) or nt (nucleotides)] are indicated. In blots in which only select lanes were used, red rectangles highlight such lanes.

17 Yoon et al, page 7 Name Species Sequence Notes HOTAIR F human GGGGCTTCCTTGCTCTTCTTATC HOTAIR R human GGTAGAAAAAGCAACCACGAAGC GAPDH F human AGCCACATCGCTCAGACAC GAPDH R human GCCCAATACGACCAAATCC mouse hotair F mouse TGATATGGCTGCACTGAACA mouse hotair R mouse TCCTGTTCTTGGCATCTCTG mouse gapdh F mouse GGGAAATTCAACGGCACAGT mouse gapdh R mouse AGATGGTGATGGGCTTCCC let7b human TGAGGTAGTAGGTTGTGTGGTT let7i human TGAGGTAGTAGTTTGTGCTGTT U6 F human CGCTTCGGCAGCACATATAC U6 R human AAAATATGGAACGCTTCACGA ATXN F human TCGGTGGAGCTTGGTTTACAA ATXN R human GGGAGGACCCAATGAACTGG SNUPN F human CAAGCGGCTGGATTATGTGAA SNUPN R human AGGAACGTCAATTAACCACTCAG 8S rrna F human CGAACGTCTGCCCTATCAACTT 8S rrna R human ACCCGTGGTCACCATGGTA let7 Northern blot human AACCACACAACCTACTACCTCA U6 Northern blot human AAAATATGGAACGCTTCACGA GAPDH 'UTR F human CCAAGCTTCTAATACGACTCACTATAGGGAGA CCTCAACGACCACTTTGTCA GAPDH 'UTR R human GGTTGAGCACAGGGTACTTTATT HOTAIR F human AAAAGGATCC TGAGGCTTGTTAACAAGACCAG HOTAIR R human AAAAGCGGCCGCTTGAGAGACAGTGCACTCACGC HOTAIR fragments amplified HOTAIR segment F human GACAGGGTCTGGGACAGAAG 7- HOTAIR segment R human GAGTCAGAGTTCCCCACTGC HOTAIR segment F human GCAGTGGGGAACTCTGACTC - HOTAIR segment R human GGGTGTTGGTCTGTGGAACT HOTAIR segment F human AGAGAGCACCAGGCACTGAG -8 HOTAIR segment R human TCCCCTACTGCAGGCTTCTA HOTAIR segment F human CAGTGGAATGGAACGGATTT -7 HOTAIR segment R human TCAGACTCTTTGGGGCCTTA HOTAIR segment F human AAGGCCCCAAAGAGTCTGAT -8 HOTAIR segment R human CAGGTCGGTACTGGCTTAGG HOTAIR segment 6F human CTGGCAGAGAAAAGGCTGAA -68 HOTAIR segment 6R human CTTCCCTCCTCTGGCTCTCT HOTAIR segment 7F human AGCCAGAGGAGGGAAGAGAG 6-8 HOTAIR segment 7R human TTTTCCCTTTTCCTCATGGA HOTAIR segment 8F human GGGCACTCACAGACAGAGGT 7-88 HOTAIR segment 8R human TCAGGTTTTTCCAGCGTTCT HOTAIR segment 9F human AGAACGCTGGAAAAACCTGA HOTAIR segment 9R human TGGAGATGATAAGAAGAGCAAGG HOTAIR segment F human GTCAGCCACTGCCCCACAC 9- HOTAIR segment R human GCCAGCTCTCTGGTCTTGTT HOTAIR segment F human TGGCCAAGCACCTCTATCTC 8- HOTAIR segment R human GTGTAGACGCCGCCATATTT HOTAIR segment F human ACGGAACCCATGGACTCATA -7 HOTAIR segment R human TGGTCCCATTTGGATCTTTC HOTAIR segment F human CAAATGTCAGAGGGTTCTGGA 7- HOTAIR segment R human TTGGGGAAGCATTTTCTGAC HOTAIR segment F human TGGGAGTGTGTTTTGTTGGA - HOTAIR segment R human CTACACAACCCCTTCGCTTC HOTAIR segment F human GAAGCGAAGGGGTTGTGTAG -96 HOTAIR segment R human AGGCTAGGGCTGGTTTCACT HOTAIR segment 6F human CCCTAGCCTTTGGAAGCTCT 88-7 HOTAIR segment 6R human TGCTCTGTGCTGCCAGTTAG HOTAIR segment 7F human AGGAATCCACCTGCCTGTTA 68-7 HOTAIR segment 7R human ACCCATGTGTCTCAAGATGC HOTAIR segment 8F human AATGCATCTTGAGACACATGG 7-8 HOTAIR segment 8R human AGAGTGCAAAGTCCCGTTTG HOTAIR segment 9F human CAAACGGGACTTTGCACTCT 8-9 HOTAIR segment 9R human CCCCTTCTGTGTCTACATGC HOTAIR segment F human GCATGTAGACACAGAAGGGGTA 96- HOTAIR segment R human CAGGCATTGGGAATGGTAAT HOTAIR segment F human GCCTGAACTTCCTCCTGCTA -7 HOTAIR segment R human TGCATACCTACCCAATGTATGG HOTAIR segment F human TTCCATACATTGGGTAGGTATGC -6 HOTAIR segment R human GCACAGAAAATGCATCCAGA HOTAIR segment F human TCTGGATGCATTTTCTGTGC 7- HOTAIR segment R human ACCACCACACACACACAACC Supplementary Table S. DNA primers used in this study.

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