Julia C. Kenyon, Liam J. Prestwood and Andrew M.L. Lever

Transcription

1 A novel combined RNA- protein interaction analysis distinguishes HIV- 1 Gag protein binding sites from structural change in the viral RNA leader. Julia C. Kenyon, Liam J. Prestwood and Andrew M.L. Lever

2 Fig$S1$ Figure S1 The experimental set- up to calculate specific cross- linking TAR RNA or RNA- protein complexes were incubated with/without UV cross- linking, purified, reverse transcribed using 6FAM or VIC labeled primers, and analysed. Red/black traces are sequencing ladders used to align samples. a) Example trace for Fx control samples. RNA was cross- linked and reverse transcribed with either 6FAM or VIC labeled primer, shown in blue and green respectively. b) Experimental cross- linked RNA- protein sample. RNA only was reverse transcribed with a VIC labeled primer, RNA- protein with a 6FAM labeled primer. c) Fnox control sample. Non cross- linked RNA was reverse transcribed with either 6FAM or VIC labeled primer. D) Non cross- linked sample. RNA was incubated with/without protein; the RNA only was reverse transcribed with VIC labeled primer, the RNA- protein was reverse transcribed with 6FAM labeled primer.

3 Fig S2a Difference(in(cross+linking(reac0vity((Tat(data+HSV(data)( 7# 6# 5# 4# 3# 2# 1# 0#!1# Experiment(1( Experiment(2( Experiment(3( G#G#U#C#U#C#U#C#U#G#G#U#U#A#G#A#C#C#A#G#A#U#C#U#G#A#G#C#C#U#G#G#G#A#G#C#U#C#U#C#U#G#G#C#U#A#A#C#U#A#G#G#!2# Figure S2b Difference(in(cross+linking(reac0vity((Tat(data+HSV(data)( 8# 6# 4# 2# 0#!2#!4#!6# Experiment(1( Experiment(2( Experiment(3( Experiment(4( G#G#U#C#U#C#U#C#U#G#G#U#U#A#G#A#C#C# A#G#A#U#C#U#G#A#G#C#C#U#G#G#G#A#G#C#U#C#U#C#U#G#G#C#U#A#A#C#U#A#G#G#

4 Figure S2c 4# cross0linking$reac'vity$ 3# 2# 1# 0#!1# Tat$pep'de$ HSV$pep'de$ G#G#U#C#U#C#U#C#U#G#G#U#U#A#G#A#C# C# A#G#A#U#C#U#G#A#G#C# C#U#G#G#G#A#G#C#U#C#U#C#U#G#G#C#U#A#A#C#U#A#G#G#!2# Figure S2 Inter- experimental variation analysis a)cross- linking sensitivities for Tat peptide broken down into individual experiments. Data are presented as Tat peptide cross- linking minus HSV control peptide cross- linking at each nucleotide position. b) Cross- linking sensitivities for individual Tat peptide experiments as 2a, with the inclusion of a fourth experiment. c) Average cross- linking reactivities for Tat peptide and HSV peptide, taking into account four independent experiments. Stars represent those nucleotides showing reactivity in the top 20% of values and statistically significantly different from the HSV control.

5 3# 2.5# 20# 40# 2# 1.5# 5 # 3 # MS2#CBP# 1# BSA# 0.5# 0# %0.5# A" A" A" C" A" U" G" A" G" G" A" U" U" A" C" C" C" A" U" G" U" C" U" G" C" A" G" G" U" C" G" A" C" U" C" %1# %1.5# 20# 40# %2# Figure S3 Validation of the cross- linking experimental set- up using MS2 RNA and Coat binding protein. Cross- linking reactivity profile of the interaction of MS2 CBP, shown in black, or BSA, shown in yellow, with the MS2 RNA construct. Cross- linking reactivities were calculated as above, and those nucleotides where reactivity is statistically significantly higher than in the BSA control (p<0.05, paired t- test) are shown with stars. Inset: secondary structural model of the RNA, containing MS2 stem- loop (centre) and nonspecific stem- loops (5 and 3 ). Stars indicate the position of statistically significant cross- link sites as above. Data were not obtained for the small stem- loop at the 5 of the RNA. Nucleotide position (each 20 nt) is marked.

6 Figure S4 Expected migration of the dimer (c 800 nt) Expected migration of the monomer (c 400 nt) Figure S4 Agarose gel electrophoresis confirming dimeric nature of HIV RNA examined by XL- SHAPE HIV- 1 RNA was renatured in the same manner as for cross- linking experiments. EMSA buffer (binding buffer) with or without Gag buffer (protein elution buffer) was added to mimic conditions just before addition of Gag. The dimeric nature of the RNA was assessed by native agarose electrophoresis and ethidium bromide staining. The band of low molecular weight visible in lanes containing EMSA buffer is excess trna added to minimize nonspecific interactions between HIV RNA and protein. The distinct migration of the dimeric HIV RNA at around 800nt shows that the trna is not binding to the HIV RNA under these conditions.

7 S5a 12# 10# cross!linking#reac8vity# 8# 6# 4# 2# Gag# BSA# 0#!2#!4# C"A"C"U"G"C"U"U"A"A"G"C"C"U"C"A"A"U"A"A"A"G"C"U"U"G"C"C"U"U"G"A"G"U"G"C"U"U"C"A"A"G"U"A"G"U"G"U"G"U"G"C"C"C"G"U"C"U"G"U"U"G"U"G"U"G"A"C"U"C"U"G"G"U" 60# 80# 100# 120# S5b 12# 10# cross!linking#reac8vity# 8# 6# 4# 2# 0# A"A"C"U"A"G"A"G"A"U"C"C"C"U"C"A"G"A"C"C"C"U"U"U"U"A"G"U"C"A"G"U"G"U"G"G"A"A"A"A"U"C"U"C"U"A"G"C"A"G"U"G"G"C"G"C"C"C"G"A"A"C"A"G"G"G"A"C"C"U"G"A"A"A"G"C"G"A"A"A"G"G"G"A"A"A"C"C"A"G"A"G"G"A"G"C"U"C"U"C"U"C" Gag# BSA#!2# 140# 160# 180# 200# 220#!4#

8 S5c 12# 10# cross!linking#reac8vity# 8# 6# 4# 2# 0#!2#!4# Gag# BSA# G" A" C" G" C" A"G"G" A" C" U" C" G"G" C" U"U"G" C" U"G" A" A"G" C" G" C" G" C" A" C" G"G" C" A" A"G" A"G"G" C" G" A"G"G"G"G" C" 240# 260# 280# S5d 12# cross!linking#reac9vity# 10# 8# 6# 4# 2# 0#!2# G"G"C"G"A"C"U"G"G"U"G"A"G"U"A"C"G"C"C"A"A"A"A"A"U"U"U"U"G"A"C"U"A"G"C"G"G"A"G"G"C"U"A"G"A"A"G"G"A"G"A"G"A"G"A"U"G"G"G"U"G"C"G"A"G" Gag# BSA#!4# 300# 320# 340# Figure S5 Expanded cross- linking reactivity profiles of sections of the RNA leader, taken from Figure 3a. Scales have been adjusted so as to allow clearest visualization of lower peaks; refer to figure Figure 3a for the highest/lowest values. The other regions are shown approximately as: a) the Poly(A) stem- loop into the U5 region. b), the PBS region to SL1. c) SL1 and d), SL2- Gag AUG.

9 Table S1 NT number Sequence RNA- Gag XL RNA- BSA XL p value (t- test) 25 G A G C C U G G G A G C U C U C U G G C U A A C U A G G G A A C C C A C U G C U U A A G

10 69 C C U C A A U A A A G C U U G C C U U G A G U G C U U C A A G U A G U G U G U G C C C G U C

11 115 U G U U G U G U G A C U C U G G U A A C U A G A G A U C C C U C A G A C C C U U U U A G U C

12 161 A G U G U G G A A A A U C U C U A G C A G U G G C G C C C G A A C A G G G A C C U G A A A G

13 207 C G A A A G G G A A A C C A G A G G A G C U C U C U C G A C G C A G G A C U C G G C U U G C

14 253 U G A A G C G C G C A C G G C A A G A G G C G A G G G G C G G C G A C U G G U G A G U A C G

15 299 C C A A A A A U U U U G A C U A G C G G A G G C U A G A A G G A G A G A G A U G G G U G C G

16 345 A G Table S1 Numerical cross- linking data for RNA- Gag complexes and RNA- BSA complexes. The top 20% of values are coloured green. P value represents the statistical significance by paired t- test between RNA- Gag and RNA- BSA complexes. Those less than 0.05 are coloured purple.

17 Table S2 NT number Sequence RNA only RNA plus Gag 36 C U C U C U G G C U A A C U A G G G A A C C C A C U G C U U A A G C C U C A A U A A A G

18 80 C U U G C C U U G A G U G C U U C A A G U A G U G U G U G C C C G U C U G U U G U G U G A C

19 126 U C U G G U A A C U A G A G A U C C C U C A G A C C C U U U U A G U C A G U G U G G A A A A

20 172 U C U C U A G C A G U G G C G C C C G A A C A G G G A C C U G A A A G C G A A A G G G A A A

21 218 C C A G A G G A G C U C U C U C G A C G C A G G A C U C G G C U U G C U G A A G C G C G C A

22 264 C G G C A A G A G G C G A G G G G C G G C G A C U G G U G A G U A C G C C A A A A A U U U U

23 310 G A C U A G C G G A G G C U A G A A G G A G A G A G A U G G G U G C G A G A G C G U C A Table S2: Numerical SHAPE data Average acylation sensitivity measured for RNA- Gag and RNA- BSA complexes.