Ex4-MS2. Exon 4. pcp Pre-mRNA Pre-mRNA

Transcript

1 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 U 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.

2 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.

3 Supplementary Table I: Protein composition of complexes assembled on various c-src transcripts*. Protein Name Acc. No. U1 snrnp specific proteins U1 70k NP_ U1 A NP_ U1 C NP_ U2 snrnp specific proteins SF3b 155, SF3b1 NP_ SF3b 145, SF3b2 NP_ SF3b 130, SF3b3 NP_ SF3b49 NP_ SF3b14a, P14 NP_ SF3b14b NP_ SF3b 10 NP_ SF3a120, SF3A1 NP_ SF3a60, SF3A3 NP_ SF3a66, SF3A2 NP_ U2A NP_ U2B NP_ U5 snrnp specific proteins 220k, hprp8 NP_ k, hbrr2 NP_ k, hsnu114 NP_ k, hprp6 NP_ k, hprp28 NP_ k, Prp8BP 40k NP_ U4/U6 snrnp specific proteins 90k, hprp3 NP_ k, hprp4 NP_ k, hprp31 NP_ CypH NP_ LSm2 NP_ LSm4 NP_ LSm8 NP_ Tri-snRNP specific protein SART1, hsnu66 NP_ Sm proteins SmB/B NP_ SmD1 NP_ SmD2 NP_

4 Protein Name Acc. No. SmD3 NP_ SmE NP_ SmF NP_ SmG NP_ Essential splicing factors CBP80, NCBP80 NP_ CBP 20, NCBP20 NP_ SF1, BBP, ZNF162 NP_ U2AF65, U2AF2 NP_ U2AF35, U2AF1 NP_ DEAD box proteins DDX1, DED1 NP_ DDX3 NP_ DDX 5, p68 NP_ DDX6, RCK/p54 NP_ DDX 15, Prp43 NP_ DDX 17, p72 NP_ DDX 36 NP_ DDX39, UAP56 NP_ DDX 46, Prp5 NP_ KIAA0052, SKIV2L2 NP_ SR proteins SRrp86, SFRS12 NP_ SRp75, SFRS4 NP_ SRp55, SFRS6 NP_ SRp54, SFRS11 NP_ ASF/SF2, SFRS1 NP_ SC35, SFRS2 NP_ SRp20, SFRS3 NP_ G8, SFRS7 NP_ htra-2α NP_ htra-2β, SFRS10 NP_ PRP19/CDC5 Complex hprp19 NP_ CDC5L NP_ Npw38BP NP_ SPF27 NP_ CCAP1, hsp70k8 NP_ CCAP2, hspc148 AAF PRL1 NP_

5 Protein Name Acc. No. Non-snRNP splicing proteins PUF60, SiahBP1 NP_ CAPER, HCC1, RBM35 NP_ CROP NP_ LUC7L NP_ LUC7L2 NP_ hprp40, FBP11 AAH SRm160 AAC SRm300 NP_ CARP-1, FLJ10839 NP_ Matrin 3 NP_ ARS2b NP_ SR140 NP_ BUB3 NP_ SF4 NP_ CHERP NP_ ZNF 207 NP_ RBM25, S164, fsap94 NP_ PCNP NP_ SPF45, RBM17 NP_ SPF31 NP_ TAT SF1 NP_ EWS NP_ RBM39 NP_ hprp38 NP_ MGC2803, fsap18 NP_ Acinus, fsap152 NP_ RNPS1 NP_ ZnF-CCHC 8 NP_ DNA-PKCS NP_ NHN1 NP_ CGI-99 NP_ DRBP1 NP_ RBM35B NP_ Xrn2 NP_ PRIC285 NP_ DEK NP_ Smu-1 homolog NP_ RED NP_ RBM22, FLJ0290 NP_ BCLTF NP_

6 Protein Name Acc. No. RBM42, MGC10433 NP_ Aquarius, IBP160 NP_ Prp4k NP_ RBM26, Se70-2 NP_ WD33 NP_ CAF1-p48, RbAp48 NP_ PEP19 NP_ BAT2 NP_ hnrnp proteins hnrnp U, SAF-A NP_ SAF-B NP_ hnrnp R NP_ hnrnp Q, NSAP 1 AAH hnrnp M NP_ hnrnp L-like NP_ hnrnp L NP_ hnrnp K NP_ FUS NP_ YB-1, NSEBP-1 NP_ hnrnp I, PTB NP_ nptb, PTB-2 NP_ hnrnp H1 NP_ hnrnp H2 NP_ hnrnp H3 NP_ hnrnp G, RBM-X NP_ hnrnp E2 NP_ hnrnp E1 NP_ hnrnp D-like NP_ hnrnp D, AUF 1 NP_ hnrnp C-like NP_ hnrnp C NP_ hnrnp A/B NP_ hnrnp A3-like XP_ hnrnp A3 NP_ hnrnp A2/B1 NP_ hnrnp A1-like XP_ hnrnp A1 NP_ hnrnp A0 NP_ IGF-II mrna BP3 NP_ IGF-II mrna BP1 NP_ FBP NP_

7 Protein Name Acc. No. CUGBP1 NP_ CUGBP2 NP_ MBNL3 NP_ MBNL2 NP_ MBNL1 NP_ Raver1 NP_ Raver2 NP_ hnrnp RALY, EBNA1 NP_ Musashi 2 NP_ HuR, ELAV-like 1 NP_ PSF NP_ p54nrb NP_ Transcription proteins CA150 NP_ NFAT-90 NP_ NFAT-45 NP_ NELF A, WHSC2 NP_ NELF B NP_ NELF E NP_ TAF 15 NP_ RuvBL1, TIP49a NP_ RuvBL2, TIP48 NP_ Ku70 NP_ Ku80 NP_ H4 member C NP_ H2 member X NP_ HMGB2 NP_ HMG-I NP_ SMC 1 NP_ SMC3 NP_ RNA processing/transport proteins PABPN1 NP_ CFI 68k, CPSF6 NP_ CF I 59k, FLJ39024 NP_ CF Im 25k, CPSF 5 NP_ CSF1 NP_ FIP1-like 1 NP_ DDX48, NUK-34 NP_ THOC4 NP_ ADAR-b NP_

8 Protein Name Acc. No. Translation/ribosomal proteins eef1 α1 NP_ eef1 α2 NP_ eef1 γ NP_ eif 4A NP_ eif 3 subunit 6 NP_ Other miscellaneous proteins ATP citrate lyase NP_ Aldolase A NP_ Aldolase C NP_ GAPDH NP_ PFK NP_ CSP A NP_ Sequestosome 1 NP_ α-tubulin NP_ β-tubulin NP_ Alpha 2 actin NP_ MAP 1B NP_ HSP90 NP_ HSP70 NP_ HSP40, DnaJ homolog NP_ HSP27 NP_ FLJ38348 NP_ FLJ38426 NP_ KIAA0082 NP_ KIAA1524 NP_ KIAA1554 XP_ Rho/rac GEF NP_ HP MGC5509 NP_ HP LOC23211 NP_ HP BC NP_ HP LOC79133 NP_ HP LOC23196 NP_ HP LOC55082 NP_ HP LOC51493 NP_ HP LOC79074 NP_ HP LOC22889 NP_ KIAA0406 NP_ DDB1, p127 NP_ p30 DBC NP_

9 Protein Name Acc. No. Palladin NP_ Annexin 2 NP_ PDI-associated 4 NP_ Calpactin I NP_ PHAX NP_ Gp96 NP_ Thymosin β 4 NP_ GRp58 NP_ α-synuclein NP_ Creatine Kinase NP_ MAP-tau NP_ FBP4 NP_ *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.