Competition for XPO5 binding between Dicer mrna, pre-mirna and viral RNA regulates human Dicer levels

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1 CORRECTION NOTICE Nat. Struct. Mol. Biol. 18, (211) Competition for binding between mrna, pre-mirna and viral RNA regulates human levels Yamina Bennasser, Christine Chable-Bessia, Robinson Triboulet, Derrick Gibbings, Carole Gwizdek, Catherine Dargemont, Eric J Kremer, Olivier Voinnet & Monsef Benkirane In the version of this supplementary file originally posted online, an incorrect scan was mistakenly used in Supplementary Figure 2 (the western blot of TAP in the unbound material, bottom left panel). The error has been corrected in this file as of 22 January 216. NATURE STRUCTURAL & MOLECULAR BIOLOGY

2 Competition for binding between DICER mrna, pre-mirna and viral RNA regulates human DICER levels Yamina Bennasser 1,7, Christine Chable-Bessia 1, Robinson Triboulet 1, Derrick Gibbings 2, Carole Gwizdek 3, Catherine Dargemont 3, Eric J Kremer 4, Olivier Voinnet 2 and Monsef Benkirane 1,7. 1 CNRS. Institut de Génétique Humaine UPR1142. Laboratoire de Virologie Moléculaire, Montpellier, France. 2 Institut de Biologie moléculaire des plantes, IBMP CNRS, Université de Strasbourg, France 3 Institut Jacques Monod, Université Paris Diderot, CNRS, 15 rue Hélène Brion 7525 Paris Cedex 13, France 4 Institut de Génétique Moléculaire de Montpellier, Universités de Montpellier I & II, Montpellier, France 7 Corresponding authors: yamina.bennasser@igh.cnrs.fr or bmonsef@igh.cnrs.fr Nature Structural & Molecular Biology: doi:1.138/nsmb.1987

3 1 2 3 D Scrambled 5 Mock O ic er b XP a 1.3 kb 5.7 kb Exportin-5 Tubulin 1.7 kb Actin c Primers 1 Primers Primers ed am bl sc r am bl sc r am bl sc r ed ed mrna (relative amount) 1. Supplementary Figure 1: inhibits DICER protein expression but has no effect on mrna levels. a) HeLa cells were transfected with three supplemental directed against three additional regions of mrna. 48 hours post transfection, cells were analyzed for DICER, and tubulin expression by western blot. b) DICER mrna levels are not affected by inhibition as assessed by northern blot. DICER mrna level was analyzed by northern blot following DICER or transfection. c) Quantitative RT-PCR of DICER mrna levels using 3 sets of primers recognizing different regions of DICER mrna following inhibition: primers 1 amplifies region , primers 2 region (also used in figure 1c) and primers 3 region Results are represented as DICER mrna levels compared to cells transfected with a control scramble. Nature Structural & Molecular Biology: doi:1.138/nsmb.1987

4 Supplementary Figure 2: Karyopherin immunoprecitation. HeLa cell extracts were prepared and subjected to immunoprecipitation using (control), anti-crm1, anti-, or anti-tap/p15 antibodies. After washing, a fraction of the unbound (FT) material and immunoprecipitates (IP) were analyzed by western blot using anti-crm1, anti-, or anti-tap/p15 antibodies. Nature Structural & Molecular Biology: doi:1.138/nsmb.1987

5 b a + Exportin-5 (ng) + RanQ69L-GTP mrna (arbitrary units) 3,5 3, 2 buffer alone Exportin-5 / RNA RNase resistant complex + RanQ69L-GTP 2,5 2, 1,5 1, 5 Free digested RNA Supplementary Figure 3: DICER mrna/ specific interaction requires RanGTP. a) Karyopherin immunoprecipitation analysis were realized as described in figure 3, in the absence (grey bars) or presence of RanQ69LGTP (black bars). b) EMSA was realized as described in Figure 2 in the absence (lanes 1 and 2) or presence of RanQ69L-GTP (lanes 3 and 4). Nature Structural & Molecular Biology: doi:1.138/nsmb.1987

6 a Pre-miR16 GAPDH 7, 6, 5, 4, 3, 2, 1, Pre-miR-3 (2 μg) 5, 4, 3, 2, 1, Pre-miR-3 (2 μg) CRM TAP CRM TAP U3 snrna Pre-miR-3 8, 6, 4, 2, Pre-miR-3 (2 μg) 3, 25, 2, 15, 1, 5, Pre-miR-3 (2 μg) CRM TAP CRM TAP Supplementary Figure 4 Nature Structural & Molecular Biology: doi:1.138/nsmb.1987

7 b Pre-miR16 GAPDH 7, 6, 5, 4, 3, 2, 1, pvv2 pva1 (2 μg) 12, pvv2 pva1 (2 μg) 1, 8, 6, 4, 2, CRM TAP U3 snrna VA RNA 2, 15, 1, 5, pvv2 pva1 (2 μg) 3,, 2,5, 2,, 1,5, 1,, 5, pvv2 pva1 (2 μg) Supplementary Figure 4: Pre-miR-3 or VA1 overexpression does not affect amounts of U3 snrna or GAPDH mrna recovered from CRM1 and TAP immunoprecipitates. HeLa cells were transfected with either 2 µg of empty vector (pvv2 or ) or plasmid expressing pre-mir-3 (A) or VA1 (B). 48 hours post transfection, cell extracts were prepared and subjected to immunoprecipitation using (control), anti-crm1, anti-, or anti-tap/p15 antibodies. Purified RNA were reverse transcribed and subjected to qrt-pcr using specific primers for U3 snrna, pre-mir-16, GAPDH mrna, pre-mir-3, VA and DICER mrna as in figure 2. Error bars are expressed as s.d. Nature Structural & Molecular Biology: doi:1.138/nsmb.1987