A -MS2 A Exon 4 B N.E. N.E. pcp + + - + + - C N.E. N.E. pcp + + - + + - Pre-mRNA Pre-mRNA U2 U1 U4 U5 U6 U2 U1 U4 U5 U6 1 2 3 4 5 6 1 2 3 4 5 6 Supplementary Figure 1. The and complexes contain U1 and U2 snrnp. The and complexes assembled on the BS-MS2 RNA in and extracts were purified using the MS2 affinity tag method. (a) The BS-MS2 RNA was incubated at 30 C for 90 minutes in or extract in reactions lacking ATP. ATP was depleted from the extracts by pre-incubating at 30 C. The complexes were separated on 15-30% glycerol density gradient and purified on amylose resin. (b) The complexes were assembled on the BS-MS2 RNA in the presence of ATP in and extracts and purified as described above. RNA was extracted from the purified complexes and 3 -end labeled with 32P-pCp. Total RNA from nuclear extract (T) was used as markers for the U snrnas. The positions of the U snrnas and pre-mrna are indicated.
U1A NE NE U2B U2AF65 PTB nptb Supplementary Figure 2. The complexes do not bind PTB. Protein from the purified and complexes were separated by SDS-PAGE, blotted to nitrocellulose, and probed with antibodies against the indicated proteins. Total protein from and extracts (N.E.) was run in parallel lanes as controls.
Supplementary Table I: Protein composition of complexes assembled on various c-src transcripts*. Protein Name Acc. No. U1 snrnp specific proteins U1 70k NP_003080 7 7 4 23 15 10 12 8 U1 A NP_004587 5 11 2 6 7 3 4 U1 C NP_003084 2 2 5 7 2 2 2 3 U2 snrnp specific proteins SF3b 155, SF3b1 NP_036565 14 15 18 26 21 29 22 14 SF3b 145, SF3b2 NP_006833 4 11 8 23 10 22 20 11 SF3b 130, SF3b3 NP_036558 2 15 19 8 24 15 30 14 SF3b49 NP_005841 2 3 3 SF3b14a, P14 NP_057131 2 2 4 3 3 SF3b14b NP_116147 2 2 2 5 3 2 SF3b 10 NP_112577 2 2 3 2 SF3a120, SF3A1 NP_005868 7 17 10 19 20 11 24 16 SF3a60, SF3A3 NP_006793 4 5 9 7 9 2 10 SF3a66, SF3A2 NP_009096 2 2 3 5 9 3 7 U2A NP_003081 5 10 3 6 7 2 8 8 U2B NP_003083 2 2 3 2 3 U5 snrnp specific proteins 220k, hprp8 NP_006436 17 200k, hbrr2 NP_054733 11 116k, hsnu114 NP_004238 10 102k, hprp6 NP_036601 3 100k, hprp28 NP_004809 2 40k, Prp8BP 40k NP_004805 2 3 U4/U6 snrnp specific proteins 90k, hprp3 NP_004689 5 3 61k, hprp4 NP_004688 2 4 40k, hprp31 NP_056444 2 CypH NP_006338 2 LSm2 NP_067000 3 LSm4 NP_036453 2 LSm8 NP_057284 2 Tri-snRNP specific protein SART1, hsnu66 NP_005137 8 Sm proteins SmB/B NP_937859 3 10 6 14 7 9 10 9 SmD1 NP_008869 2 4 3 7 6 5 3 5 SmD2 NP_004588 4 2 10 9 9 9 8
Protein Name Acc. No. SmD3 NP_004166 3 7 4 10 5 6 8 4 SmE NP_003085 4 5 2 2 5 3 SmF NP_003086 2 3 2 5 2 SmG NP_003087 2 4 6 4 5 Essential splicing factors CBP80, NCBP80 NP_002477 8 12 12 25 2 5 2 5 CBP 20, NCBP20 NP_031388 3 4 8 2 2 4 SF1, BBP, ZNF162 NP_004621 4 4 2 7 9 7 8 2 U2AF65, U2AF2 NP_009210 8 3 8 3 7 2 7 2 U2AF35, U2AF1 NP_006749 4 6 4 5 5 4 DEAD box proteins DDX1, DED1 NP_004930 2 5 5 DDX3 NP_004651 2 2 DDX 5, p68 NP_004387 2 2 7 3 7 DDX6, RCK/p54 NP_004388 5 DDX 15, Prp43 NP_001349 6 11 6 10 13 8 DDX 17, p72 NP_006377 2 2 3 8 4 11 13 DDX 36 NP_065916 6 22 4 4 9 DDX39, UAP56 NP_004631 2 DDX 46, Prp5 NP_055644 2 19 11 19 31 12 20 8 KIAA0052, SKIV2L2 NP_056175 4 4 SR proteins SRrp86, SFRS12 NP_631907 2 5 5 3 3 3 SRp75, SFRS4 NP_005617 11 6 5 3 6 SRp55, SFRS6 NP_006266 2 3 3 SRp54, SFRS11 NP_004759 4 4 2 3 9 3 8 3 ASF/SF2, SFRS1 NP_008855 8 6 7 SC35, SFRS2 NP_003007 2 3 3 2 SRp20, SFRS3 NP_003008 2 4 8 5 2 6 9G8, SFRS7 NP_001026854 3 htra-2α NP_004584 3 htra-2β, SFRS10 NP_004584 5 PRP19/CDC5 Complex hprp19 NP_055317 7 CDC5L NP_001244 6 Npw38BP NP_057396 3 SPF27 NP_005863 3 CCAP1, hsp70k8 NP_006588 3 11 3 10 3 3 3 7 CCAP2, hspc148 AAF29112 7 PRL1 NP_002660 7
Protein Name Acc. No. Non-snRNP splicing proteins PUF60, SiahBP1 NP_055096 9 8 10 8 27 7 19 11 CAPER, HCC1, RBM35 NP_004893 2 4 4 6 9 6 15 7 CROP NP_057508 2 6 5 2 3 2 6 LUC7L NP_057508 2 2 2 2 LUC7L2 NP_057103 3 3 2 3 8 3 4 3 hprp40, FBP11 AAH67364 2 5 4 3 SRm160 AAC09321 3 SRm300 NP_057417 2 12 CARP-1, FLJ10839 NP_060707 7 12 6 5 12 9 7 Matrin 3 NP_061322 3 13 3 2 ARS2b NP_877952 10 15 8 39 11 13 17 15 SR140 NP_001073884 2 2 8 2 4 3 BUB3 NP_004716 3 4 4 6 12 9 8 5 SF4 NP_757386 2 2 2 3 CHERP NP_006378 3 2 5 3 ZNF 207 NP_003448 3 4 4 5 4 6 4 4 RBM25, S164, fsap94 NP_067062 5 9 10 4 5 6 PCNP NP_065090 3 3 6 SPF45, RBM17 NP_116294 2 4 7 2 SPF31 NP_055095 2 4 3 8 5 4 TAT SF1 NP_055315 3 5 4 3 2 EWS NP_005234 2 3 4 RBM39 NP_004893 4 hprp38 NP_060531 4 2 MGC2803, fsap18 NP_076943 3 2 5 4 3 2 Acinus, fsap152 NP_055792 4 RNPS1 NP_006702 2 ZnF-CCHC 8 NP_060082 2 2 DNA-PKCS NP_008835 6 8 3 2 NHN1 NP_653205 9 4 2 CGI-99 NP_057123 5 3 DRBP1 NP_694453 4 RBM35B NP_079215 3 Xrn2 NP_036387 2 PRIC285 NP_208384 2 DEK NP_003463 3 Smu-1 homolog NP_060695 5 RED NP_006074 3 RBM22, FLJ0290 NP_060517 2 BCLTF NP_055554 3
Protein Name Acc. No. RBM42, MGC10433 NP_077297 2 Aquarius, IBP160 NP_055506 3 Prp4k NP_003904 2 RBM26, Se70-2 NP_071401 2 WD33 NP_060853 2 CAF1-p48, RbAp48 NP_005601 2 2 PEP19 NP_006189 5 BAT2 NP_542417 2 hnrnp proteins hnrnp U, SAF-A NP_114032 20 40 5 60 13 35 3 13 SAF-B NP_002958 2 hnrnp R NP_005817 9 23 3 16 17 17 3 13 hnrnp Q, NSAP 1 AAH40844 7 21 3 15 12 12 6 hnrnp M NP_112480 4 7 3 5 hnrnp L-like NP_612403 2 hnrnp L NP_001524 4 6 8 3 5 8 hnrnp K NP_002131 2 6 2 3 18 11 7 10 FUS NP_004951 5 4 5 20 4 3 7 8 YB-1, NSEBP-1 NP_004550 4 7 2 9 5 5 2 2 hnrnp I, PTB NP_002810 27 36 4 5 nptb, PTB-2 NP_067013 2 3 hnrnp H1 NP_005511 2 4 3 hnrnp H2 NP_062543 2 hnrnp H3 NP_036339 7 hnrnp G, RBM-X NP_002130 2 3 2 7 8 hnrnp E2 NP_005007 4 14 2 3 hnrnp E1 NP_006187 2 5 hnrnp D-like NP_005454 4 6 4 hnrnp D, AUF 1 NP_002129 13 16 10 21 9 13 2 9 hnrnp C-like NP_001013653 2 5 3 hnrnp C NP_112604 4 11 3 13 8 2 8 10 hnrnp A/B NP_004490 10 9 6 3 hnrnp A3-like XP_371824 2 2 4 3 4 hnrnp A3 NP_919223 2 11 2 9 5 13 5 16 hnrnp A2/B1 NP_112533 5 17 2 17 15 18 15 17 hnrnp A1-like XP_208373 3 4 4 3 4 4 hnrnp A1 NP_112420 7 17 5 19 18 27 8 12 hnrnp A0 NP_006796 4 9 2 7 3 4 5 IGF-II mrna BP3 NP_006538 5 4 2 IGF-II mrna BP1 NP_006537 4 5 9 FBP NP_003893 2
Protein Name Acc. No. CUGBP1 NP_941989 8 11 CUGBP2 NP_006552 4 MBNL3 NP_597846 4 MBNL2 NP_659002 2 MBNL1 NP_066368 6 Raver1 NP_597709 4 11 Raver2 NP_060681 2 hnrnp RALY, EBNA1 NP_039313 7 5 3 4 Musashi 2 NP_620412 2 HuR, ELAV-like 1 NP_001410 4 3 3 PSF NP_005057 2 2 7 p54nrb NP_031389 3 Transcription proteins CA150 NP_006697 7 NFAT-90 NP_036350 2 17 13 5 15 7 NFAT-45 NP_004506 2 7 4 4 2 5 NELF A, WHSC2 NP_005654 2 4 2 NELF B NP_056271 3 NELF E NP_002895 4 4 3 TAF 15 NP_003487 2 RuvBL1, TIP49a NP_003698 2 RuvBL2, TIP48 NP_006657 2 Ku70 NP_001460 2 3 4 Ku80 NP_066964 3 H4 member C NP_003531 2 H2 member X NP_002096 3 HMGB2 NP_002120 4 HMG-I NP_665908 2 2 SMC 1 NP_006297 4 4 SMC3 NP_005436 2 RNA processing/transport proteins PABPN1 NP_004634 2 CFI 68k, CPSF6 NP_008938 2 3 2 CF I 59k, FLJ39024 NP_079087 2 7 3 2 CF Im 25k, CPSF 5 NP_008937 2 4 CSF1 NP_001315 2 FIP1-like 1 NP_112179 2 DDX48, NUK-34 NP_055555 2 THOC4 NP_005773 3 ADAR-b NP_056655 3
Protein Name Acc. No. Translation/ribosomal proteins eef1 α1 NP_001393 3 4 7 4 3 eef1 α2 NP_001958 3 5 2 12 3 4 3 eef1 γ NP_079215 2 2 2 eif 4A NP_001949 3 eif 3 subunit 6 NP_001559 2 Other miscellaneous proteins ATP citrate lyase NP_001087 20 55 9 45 35 19 18 13 Aldolase A NP_000025 7 3 4 Aldolase C NP_005156 2 GAPDH NP_002037 2 13 9 15 12 10 10 15 PFK NP_000280 2 3 CSP A NP_003642 12 16 7 16 4 11 2 Sequestosome 1 NP_003891 3 2 2 α-tubulin NP_005991 2 4 6 7 5 β-tubulin NP_115914 2 3 6 7 7 18 7 Alpha 2 actin NP_001604 8 2 MAP 1B NP_005900 6 2 5 2 HSP90 NP_005339 3 2 HSP70 NP_006588 2 11 3 4 2 HSP40, DnaJ homolog NP_006136 2 8 2 3 HSP27 NP_001531 2 3 FLJ38348 NP_777591 2 FLJ38426 NP_775882 2 3 KIAA0082 NP_055865 3 2 2 KIAA1524 NP_065941 2 KIAA1554 XP_290768 17 Rho/rac GEF NP_004714 3 HP MGC5509 NP_076943 3 HP LOC23211 NP_055983 10 4 3 HP BC001584 NP_653205 2 HP LOC79133 NP_077025 2 HP LOC23196 NP_055427 8 5 3 HP LOC55082 NP_060481 HP LOC51493 NP_055121 4 2 HP LOC79074 NP_076998 4 HP LOC22889 NP_055764 2 KIAA0406 NP_055472 2 2 DDB1, p127 NP_001914 5 p30 DBC NP_066997 2
Protein Name Acc. No. Palladin NP_057165 2 Annexin 2 NP_004030 3 16 4 PDI-associated 4 NP_004902 2 Calpactin I NP_002957 2 PHAX NP_115553 2 Gp96 NP_003290 4 Thymosin β 4 NP_066932 2 GRp58 NP_005304 2 α-synuclein NP_000336 2 Creatine Kinase NP_066270 3 MAP-tau NP_058518 2 FBP4 NP_056123 2 *The numbers of peptides for each identified protein are shown. At least two peptides are required to confirm the presence of a protein in a complex. a S stands for mixture of spliceosomal complexes. Proteins present in the repressed E and A complex but absent from the active spliceosomes are represented in red. Proteins present in the E complex and active spliceosome but absent from the repressed A are marked in blue.