Supplemental Information

Transcription

1 Supplemental Information ATP-dependent unwinding of U4/U6 snrnas by the Brr2 helicase requires the C-terminus of Prp8 Corina Maeder 1,3, Alan K. Kutach 1,2,3, and Christine Guthrie 1 1 Department of Biochemistry and Biophysics University of California, San Francisco th Street, Genentech Hall San Francisco, CA 94143, USA 2 Current address: Roche Palo Alto, 3431 Hillview Ave, Palo Alto, CA these authors contributed equally Correspondence: christineguthrie@gmail.com (415) (fax)

2 Supplemental Figure 1. Prp8 protease sensitivity mapping. A. C-terminally tagged Prp8 protease resistant fragments of Prp8. Yeast splicing extracts were made using a strain expressing a triple-ha-tagged Prp8 as the sole source of Prp8. Approximately 500 µg of protein were incubated at room temperature with (+) or without (-) endoprotease Glu-C. At the indicated times, a small sample was removed and protease activity halted by the addition of SDS-PAGE loading dye. The protein samples were boiled and resolved by SDS-PAGE, western blotted and detected by anti-ha antibodies and enhanced chemiluminescence. Thus each fragment visualized by the western blot retains an intact C-terminus. The sites of proteolytic hypersensitivity were deduced by creating a series of N-terminal deletions of the triple-ha-tagged Prp8 in which the last deleted residue of each clone was a glutamic acid residue estimated to be in the vicinity of a protease hypersensitive site. The cleavage reaction products were then resolved in lanes adjacent to the N-terminal deletion clones to compare their apparent sizes. The hypersensitive sites at aa 1684 (83 kda), 2020 (56 kda), and 2142 (40 kda) were unambiguously identified. The hypersensitive site(s) resulting in the 175 kda fragment could only be limited to the amino acids B. Diagram of Prp8. Prp8 is oriented with the C-terminus down to parallel the western blot shown in part A. The Jab1/MPN domain and RP region of Prp8 are indicated within the rectangle

3 corresponding to Prp8. Numbers on the left indicate the mapped protease hypersensitive sites. The Prp8-CTF boundaries are indicated with the light gray rectangle. The N-terminus of Prp8-CTF was determined by mass spectrometry analysis. A plasmid encoding C-terminal 6-His tagged Prp8 amino acids was used to drive expression in bacteria. The full protein was insoluble, but a truncated form with an intact C-terminus was highly expressed. The protein was purified by immobilized metal affinity chromatography, dialyzed against 50 mm NaCl, and then its mass was determined by MALDI-TOF mass spectrometry. Multiple mass peaks corresponding to truncations at amino acids were identified. Since amino acid 1806 is a methionine and one of the most C-terminal of cleaved residues, it was chosen as the N- terminus for several sub-clones of Prp8 including Prp8-CTF that extends to the natural C- terminus of Prp8.

4 Supplemental Figure 2. Prp8-CTF and Prp8-CTF-R2388G do not unwind U4/U6. Unwinding assays were performed by combining U4/U6 duplex with Prp8-CTF alone or Prp8-CTF-RP alone in a 5 minute pre-incubation. The reactions were started with the addition of ATP (2mM final). Aliquots were withdrawn at the time points and quenched. B indicates an aliquot of the reaction boiled. A fluorescently labeled oligo was subsequently annealed to these quenched reactions to detect U4 snrna on both U4/U6 duplex or free U4. Native gel analysis was performed on the aliquots. Shown here are unwinding assays for 200 nm U4/U6 duplex in the presence of 250 nm Prp8-CTF or 250 nm Prp8-CTF-R2388G. Similar assays for F2382L and R2388G show that these mutants do not unwind U4/U6 in the absence of Brr2.

5 Supplemental Table 1. Summary of Plasmids constructed for use in this study. Name Vector Encoded Protein Oligonucleotides Template pak299 pentr/sd/d-topo CTF-C oak1& 2 pju197 pak302 pentr/sd/d-topo CTF-C-F2382L oak 3, 9, 10, & 12 a N/A pak303 pentr/sd/d-topo CTF-C-H2387P oak 4, 9, 10, & 13 a N/A pak304 pentr/sd/d-topo CTF-C-H2387R oak 5, 9, 10, & 14 a N/A pak305 pentr/sd/d-topo CTF-C-R2382G oak 6, 9, 10, & 15 a N/A pak306 pentr/sd/d-topo CTF-C-R2382K oak 7, 9, 10, & 16 a N/A pak310 pju197 Prp8-F2382L oak 1, 17, & 18 pak302 pak311 pju197 Prp8-H2387P oak 1, 17, & 18 pak303 pak312 pju197 Prp8-H2387R oak 1, 17, & 18 pak304 pak313 pju197 Prp8-R2388G oak 1, 17, & 18 pak305 pak314 pju197 Prp8-R2388K oak 1, 17, & 18 pak306 pak319 pdonr201 CTF oak19, 20, & 21 pju197 pak322 pdonr201 CTF-RP-F2382L oak19, 20, & 21 pak310 pak323 pdonr201 CTF-RP-H2387P oak19, 20, & 21 pak311 pak324 pdonr201 CTF-RP-H2387R oak19, 20, & 21 pak312 pak325 pdonr201 CTF-RP-R2382G oak19, 20, & 21 pak313 pak326 pdonr201 CTF-RP-R2382K oak19, 20, & 21 pak314 pak pdest15 CTF N/A b N/A pak pdest15 CTF-RP-F2382L N/A b N/A pak pdest15 CTF-RP-H2387P N/A b N/A pak pdest15 CTF-RP-H2387R N/A b N/A pak pdest15 CTF-RP-R2382G N/A b N/A pak pdest15 CTF-RP-R2382K N/A b N/A a Oligos were annealed and ligated into EcoRI and ClaI digested pak299. b Clones were made by Gateway recombination cloning.

6 Supplemental Table 2. Oligonucleotides used in this study Name oak1 oak2 oak3 oak4 oak5 oak6 oak7 oak9 oak10 oak11 oak12 oak13 oak14 oak15 oak16 oak17 oak18 oak19 oak20 oak21 Sequence CACCATGAGTCAAACGTTTAGTTCAAAG GCTATTAGCTAAACTGTAAAAAGTGTACTGG AATTGTATAATGAGATGCATCGTCCAGTACACTTT AATTTTATAATGAGATGCCTCGTCCAGTACACTTT AATTTTATAATGAGATGCGTCGTCCAGTACACTTT AATTTTATAATGAGATGCATGGTCCAGTACACTTT AATTTTATAATGAGATGCATAAACCAGTACACTTT TTACAGTTTAGCGAATTGGCGGGGGACGAAGAGTTAGAGGCCGAACAAAT CGATTTGTTCGGCCTCTAACTCTTCGTCCCCCGCCAATTCGCTAAACTG TAAAAAGTGTACTGG CGATTTGTTCGGCCTCTAACTCTTCGTCCCCCGCCAATTCGCTAAACTG TATAGAGTGTACTGG ACGATGCATCTCATTATAC ACGAGGCATCTCATTATAA ACGACGCATCTCATTATAA ACCAGGCATCTCATTATAA TTTATGCATCTCATTATAA TGAAATAACAGATTCCAGTTTATTGGGGAATATATTCAGCTAAATACAT CGATTTGTTCG CGTTGAATATTATACATACAAACTTATACTTACATATATATCTATGAAA TAACAGATTCC GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATAGAACCA TGGACTACAAGGATGACGATGACAAG GGATGACGATGACAAGAAAGCTAACCCAGCATTATATG GGGGACCACTTTGTACAAGAAAGCTGGGTCTATCAGCTAAATACATCGA TTTGTTCGGC Mutations incorporated into the oligonucleotides are indicated with an underline

7 Supplemental Methods. PRP8 and bacterial expression plasmid cloning First expression plasmids were created by PCR amplification of sequences encoding Prp8 amino acids (Prp8-CTF-C) from pju197, a PRP8-harboring plasmid, with primers that encode an N-terminal CACC and methionine, and two stop codons at the C-terminus. The PCR product was topo-cloned into pentr/sd/d-topo (Invitrogen) generating pak299, and the nucleotide sequence of the insert was confirmed. RP mutant versions were made by replacing the sequences between the unique EcoRI and ClaI sites of pak299 with four annealed DNA oligonucleotides harboring the RP mutations and a silent mutation to destroy the EcoRI site for screening purposes. Next full-length RP mutants were generated by gap repair of the PRP8-harboring plasmid pju197. NotI restriction enzyme was used to linearize pju197 which was then co-transformed into yeast with PCR products spanning the RP region from the Prp8-CTF- C-RP clones. The upstream primer (oak1) is the same as for the original cloning of the expression plasmids, and the downstream priming is by two partially overlapping downstream primers (oak17 & oak18) encoding 82 nucleotides of the 3 UTR and the natural stop site to provide homology to PRP8 on both sides of the NotI site to facilitate homologous recombination. Plasmid DNA was recovered from the yeast transformants and sequenced to confirm the presence of the RP mutations and absence of additional mutations in the region spanning the PCR products used for recombination. Finally, Prp8-CTF expression clones were made by PCR using the full-length wild-type and PRP8 RP mutants as templates. The final PCR products encode amino acids including a second stop codon, and Gateway attb cloning sequences on both ends. The PCR products were cloned into pdonr201 by in vitro recombination and the sequences were confirmed. The Prp8-CTF sequences were subsequently transferred to the expression vector pdest15 that fuses bacterial glutathione-s transferase (GST) to the Prp8-CTF sequences with a 15 amino acid linker. All steps were performed following the Gateway cloning system methods..