Absmeieret al.S1

Supplemental Data

The large N-terminal region of the Brr2 RNA helicase

guidesproductive spliceosomeactivation

Eva Absmeier1, Jan Wollenhaupt1,#, Sina Mozaffari-Jovin2,#, Christian Becke1,6,#, Chung-Tien Lee3,4, Marco Preussner5, Florian Heyd5, Henning Urlaub3,4,Reinhard Lührmann2,*, Karine F. Santos1,*, Markus C. Wahl1,*

1Laboratory of Structural Biochemistry, FreieUniversität Berlin, Takustr. 6, D-14195 Berlin, Germany

2Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany

3Research Group Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany

4Bioanalytics Group, Institute for Clinical Chemistry, University Medical Center Göttingen, Georg-August-Universität, Robert-Koch-Straße 40, D-37099 Göttingen, Germany

5Laboratory of RNA Biochemistry, FreieUniversität Berlin, Takustr. 6, D-14195 Berlin, Germany

6Present address: Molox GmbH, Thielallee 63, D-14195 Berlin, Germany

#These authors contributed equally to this work

*Correspondence: (R.L.), (K.F.S.), (M.C.W.)

Supplemental Table 1

Supplemental Figure 1

Supplemental References

Supplemental Figures

SupplementalFigure1.Evolutionary conservation of the NTR.(A) Schematic representation of predicted secondary structure, surface exposure and disorder in human, C. thermophilum and yeast Brr2 NTRs. α-helices, red; β-strands, blue; buried residues, yellow; surface exposed residues, gray; intrinsically disordered regions, green.(B) Multiple sequence alignment of NTRs of C. thermophilum, human and yeast Brr2. The alignment was constructed using ClustalX(Chenna et al. 2003). Residue numbers above the alignment refer to C. thermophilumBrr2, below the alignment to yeast Brr2. Black background – invariant residues; gray background – residues identical in two of the three sequences or highly related residues in all three sequences. Blue, green, yellow and red background indicate start residues of the T1, T2, T3 and T4 truncations, respectively, of the different Brr2 orthologs (for each species, only those variants that could be produced recombinantly or that were used in in vivo experiments are highlighted).Lines below the alignment indicate the elements of yeast Brr2 NTR that could be traced in the electron density of the present FL Brr2-Jab1 crystals.

Supplemental Tables

SupplementalTable 1.Identified cross-links in isolated yeast Brr2

XL residue 1 / Domain / XL residue 2 / Domain / Crosslinking
reagent / Cα-Cα
distance (Å)
15 / N-ext.a / 28 / N-ext. / DMTMMb
25 / N-ext. / 50 / N-ext. / BS3b
25 / N-ext. / 1187 / HLHNC c / BS3
28 / N-ext. / 43 / N-ext. / DMTMM
28 / N-ext. / 50 / N-ext. / BS3
28 / N-ext. / 454 / NC-clamp / BS3
28 / N-ext. / 784 / RecA2NC / DMTMM
28 / N-ext. / 967 / WHNC / BS3
50 / N-ext. / 59 / N-ext. / BS3
50 / N-ext. / 74 / N-ext. / BS3
50 / N-ext. / 82 / N-ext. / BS3
50 / N-ext. / 85 / N-ext. / BS3
50 / N-ext. / 454 / NC-clamp / BS3
50 / N-ext. / 967 / WHNC / BS3
50 / N-ext. / 1158 / HLHNC / BS3
59 / N-ext. / 74 / N-ext. / BS3
74 / N-ext. / 85 / N-ext. / BS3
74 / N-ext. / 90 / N-ext. / BS3
74 / N-ext. / 91 / N-ext. / BS3
74 / N-ext. / 454 / NC-clamp / BS3
74 / N-ext. / 736 / RecA2NC / DMTMM
74 / N-ext. / 1023 / HBNC / DMTMM
75 / N-ext. / 85 / N-ext. / DMTMM
81 / N-ext. / 85 / N-ext. / DMTMM
81 / N-ext. / 90 / N-ext. / DMTMM
82 / N-ext. / 90 / N-ext. / BS3
82 / N-ext. / 91 / N-ext. / BS3
82 / N-ext. / 454 / NC-clamp / BS3
82 / N-ext. / 1021 / HBNC / DMTMM
82 / N-ext. / 1023 / HBNC / DMTMM
85 / N-ext. / 91 / N-ext. / BS3
85 / N-ext. / 454 / NC-clamp / BS3
85 / N-ext. / 717 / RecA2NC / BS3
85 / N-ext. / 1021 / HBNC / DMTMM
85 / N-ext. / 1023 / HBNC / DMTMM
85 / N-ext. / 1051 / HBNC / BS3
90 / N-ext. / 454 / NC-clamp / BS3
90 / N-ext. / 717 / RecA2NC / BS3
90 / N-ext. / 1055 / HBNC / BS3
91 / N-ext. / 450 / NC-clamp / DMTMM
91 / N-ext. / 454 / NC-clamp / BS3
91 / N-ext. / 1021 / HBNC / DMTMM
91 / N-ext. / 1023 / HBNC / DMTMM
91 / N-ext. / 1055 / HBNC / BS3
91 / N-ext. / 1067 / HBNC / DMTMM
91 / N-ext. / 1068 / HBNC / DMTMM
91 / N-ext. / 1070 / HBNC / DMTMM
91 / N-ext. / 1071 / HBNC / DMTMM
152 / Plug / 190 / Plug / BS3 / 11.5
156 / Plug / 168 / Plug / DMTMM / 16.9
158 / Plug / 168 / Plug / DMTMM / 12.2
159 / Plug / 168 / Plug / DMTMM / 12.0
168 / Plug / 717 / RecA2NC / BS3 / 10.0
168 / Plug / 720 / RecA2NC / BS3 / 11.3
181 / Plug / 748 / RecA2NC / BS3 / 25.4
271 / IC-clamp / 347 / PWI / DMTMM / 25.8
271 / IC-clamp / 351 / PWI / BS3 / 21.6
271 / IC-clamp / 1545 / RecA2CC c / DMTMM / 5.3
271 / IC-clamp / 1733 / RecA2CC / BS3 / 10.8
276 / IC-clamp / 331 / PWI / DMTMM / 28.4
281 / IC-clamp / 364 / PWI / DMTMM / 27.4
289 / PWI / 364 / PWI / DMTMM / 11.2
290 / PWI / 364 / PWI / DMTMM / 9.1
290 / PWI / 417 / NC-clamp / DMTMM
304 / PWI / 341 / PWI / DMTMM / 22.6
304 / PWI / 417 / NC-clamp / BS3
304 / PWI / 1357 / RecA1CC / DMTMM / 64.8
304 / PWI / 1364 / RecA1CC / DMTMM / 69.2
351 / PWI / 398 / NC-clamp / BS3
364 / PWI / 417 / NC-clamp / BS3
364 / PWI / 1529 / RecA1CC / BS3 / 53.1
390 / PWI / 398 / NC-clamp / BS3
390 / PWI / 417 / NC-clamp / BS3
390 / PWI / 1315 / Inter-cassette linker / BS3 / 34.9
398 / NC-clamp / 417 / NC-clamp / BS3
398 / NC-clamp / 1733 / RecA2CC / BS3
414 / NC-clamp / 417 / NC-clamp / BS3
414 / NC-clamp / 967 / WHNC / BS3
417 / NC-clamp / 967 / WHNC / BS3
546 / RecA1NC / 549 / RecA1NC / BS3 / 8.8
546 / RecA1NC / 1904 / HBCC / BS3 / 25.2
546 / RecA1NC / 1634 / RecA2CC / BS3 / 32.4
546 / RecA1NC / 2003 / HLHCC / BS3 / 34.6
549 / RecA1NC / 584 / RecA1NC / BS3 / 14.9
549 / RecA1NC / 1904 / HBCC / BS3 / 23.1
549 / RecA1NC / 1907 / HBCC / BS3 / 25.7
564 / RecA1NC / 591 / RecA1NC / DMTMM / 13.4
564 / RecA1NC / 610 / RecA1NC / DMTMM / 10.9
564 / RecA1NC / 611 / RecA1NC / BS3 / 9.4
564 / RecA1NC / 1138 / HBNC / BS3 / 21.0
564 / RecA1NC / 1150 / HLHNC / BS3 / 24.4
597 / RecA1NC / 1692 / RecA2CC / DMTMM / 22.2
597 / RecA1NC / 1896 / HBCC / BS3 / 17.1
597 / RecA1NC / 1904 / HBCC / BS3 / 21.3
611 / RecA1NC / 1138 / HLHNC / BS3 / 22.4
626 / RecA1NC / 659 / RecA1NC / DMTMM / 16.1
626 / RecA1NC / 1600 / RecA2CC / DMTMM / 18.6
659 / RecA1NC / 967 / WHNC / BS3 / 22.9
678 / RecA1NC / 688 / RecA1NC / DMTMM / 9.5
717 / RecA2NC / 720 / RecA2NC / BS3 / 4.9
720 / RecA2NC / 748 / RecA2NC / BS3 / 17.0
736 / RecA2NC / 772 / RecA2NC / DMTMM / 13.7
748 / RecA2NC / 758 / RecA2NC / BS3 / 15.6
748 / RecA2NC / 795 / RecA2NC / BS3 / 15.0
748 / RecA2NC / 1088 / HBNC / BS3 / 15.1
748 / RecA2NC / 1090 / HBNC / DMTMM / 10.7
758 / RecA2NC / 769 / RecA2NC / BS3 / 11.2
758 / RecA2NC / 772 / RecA2NC / BS3 / 11.2
758 / RecA2NC / 778 / RecA2NC / BS3 / 16.6
758 / RecA2NC / 782 / RecA2NC / BS3 / 16.8
758 / RecA2NC / 795 / RecA2NC / BS3 / 9.2
763 / RecA2NC / 778 / RecA2NC / DMTMM / 21.0
769 / RecA2NC / 784 / RecA2NC / DMTMM / 20.0
769 / RecA2NC / 778 / RecA2NC / BS3 / 13.2
772 / RecA2NC / 782 / RecA2NC / BS3 / 12.9
772 / RecA2NC / 784 / RecA2NC / DMTMM / 15.1
778 / RecA2NC / 784 / RecA2NC / DMTMM / 9.6
778 / RecA2NC / 795 / RecA2NC / BS3 / 13.6
782 / RecA2NC / 795 / RecA2NC / BS3 / 10.6
782 / RecA2NC / 798 / RecA2NC / DMTMM / 6.6
784 / RecA2NC / 795 / RecA2NC / DMTMM / 12.4
967 / WHNC / 971 / WHNC / DMTMM / 6.3
1039 / HBNC / 1042 / HBNC / DMTMM / 5.0
1051 / HBNC / 1055 / HBNC / BS3 / 5.8
1060 / HBNC / 1158 / HLHNC / BS3 / 22.3
1118 / HBNC / 1729 / RecA2CC / BS3 / 15.7
1138 / HBNC / 1150 / HLHNC / BS3 / 13.5
1138 / HBNC / 1158 / HLHNC / BS3 / 16.1
1158 / HLHNC / 1187 / HLHNC / BS3 / 16.1
1187 / HLHNC / 1197 / HLHNC / BS3 / 18.5
1392 / RecA1CC / 1504 / RecA1CC / BS3 / 13.2
1406 / RecA1CC / 1427 / RecA1CC / DMTMM / 10.2
1414 / RecA1CC / 1423 / RecA1CC / BS3 / 11.3
1414 / RecA1CC / 1653 / RecA2CC / BS3 / 21.7
1437 / RecA1CC / 2082 / IGCC / DMTMM / 38.1
1437 / RecA1CC / 2116 / IGCC / BS3 / 18.3
1437 / RecA1CC / 2121 / IGCC / BS3 / 16.6
1441 / RecA1CC / 2080 / IGCC / DMTMM / 20.3
1441 / RecA1CC / 2082 / IGCC / DMTMM / 16.1
1589 / RecA2CC / 1896 / HBCC / BS3 / 10.0
1589 / RecA2CC / 1904 / HBCC / BS3 / 18.9
1603 / RecA2CC / 1634 / RecA2CC / BS3 / 13.9
1896 / HBCC / 1904 / HBCC / BS3 / 14.8
2070 / IGCC / 2115 / IGCC / BS3 / 13.1
2070 / IGCC / 2122 / IGCC / DMTMM / 23.3
2070 / IGCC / 2127 / IGCC / DMTMM / 6.8
2070 / IGCC / 2129 / IGCC / DMTMM / 4.5
2078 / IGCC / 2121 / IGCC / DMTMM / 9.8
2080 / IGCC / 2121 / IGCC / DMTMM / 5.1
2082 / IGCC / 2121 / IGCC / DMTMM / 9.9
2090 / IGCC / 2097 / IGCC / DMTMM / 15.8
2095 / IGCC / 2110 / IGCC / DMTMM / 29.1
2108 / IGCC / 2110 / IGCC / DMTMM / 5.6
2109 / IGCC / 2116 / IGCC / BS3 / 20.3
2115 / IGCC / 2121 / IGCC / BS3 / 17.1

aN-ext. – N-terminal extension comprising the first 111 residues preceding the plug and removed in T1

bDMTMM – 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; BS3 – bis[sulfosuccinimidyl] suberate

cNC – N-terminal cassette; CC – C-terminal cassette

Supplemental References

Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD. 2003. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res31: 3497-3500.