Requirement of U12 snRNA for in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns

Science

Volumn 271, Issue 5256, 1996, Pages 1716-1718

  Hall, Stephen L ^a,b   Padgett, Richard A ^b  

^a UNIVERSITY OF TEXAS SOUTHWESTERN MEDICAL CENTER   (United States)

^b LERNER RESEARCH INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

MESSENGER RNA PRECURSOR; SMALL NUCLEAR RNA;

ANIMAL CELL; ARTICLE; BASE PAIRING; EUKARYOTE; INTRON; MOLECULAR INTERACTION; MUTATION; NONHUMAN; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; RNA SPLICING;

ANIMALIA; EUKARYOTA;

EID: 0029989519     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5256.1716     Document Type: Article

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22. The human P120 gene expression construct was prepared by PCR amplification from a genomic clone of the human P120 gene (supplied by H. Busch) by using primers in exons 5 and 8 to generate a fragment containing nucleotides 6775 to 7632 (numbering from GenBank accession number M33132). This fragment contained exons 5 through 8 and introns E, F, and G, where intron F is the minor class intron. This fragment was cloned between the Kpn I and Hind III sites of the pCB6 mammalian expression vector [S Andersson et al , J. Biol Chem 264, 8222 (1989)], which supplies a CMV promoter and termination and polyadenylation sites. Mutations were made either by site-directed mutagenesis of an M13 clone of this fragment followed by cloning in pCB6, or by PCR primer mutagenesis and subsequent cloning of the product in pCB6. All mutations were confirmed by sequence analysis
23. S. L Hall and R A Padgett, data not shown
24. The human U12 snRNA expression construct was produced as described [U. Bond et al., Genes Dev 5, 1709 (1991)] Briefly, a PCR fragment of the human U12 snRNA gene (supplied by J Steitz) (5) was cloned into the pUC13-U1 vector (7) such that the mature U12 snRNA sequences replaced the U1 snRNA sequences U12 snRNA mutants were produced by substituting oligonucleotides containing the appropriate mutations for the PCR pnmer at the 5′ end of the U12 sequence and by cloning the fragments into the pUC13-U1 vector. Both the wild-type and mutant constructs have been sequenced through the 5′ and 3′ U1 snRNA gene sequences and the U12 snRNA sequences and are identical except for positions 24 and 25.
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32. 6 CHO cells in 5 ml of growth medium containing hexadimethrine bromide (20 μg/ml). The cells were incubated with the DNA for 6 to 8 hours followed by a 5-min shock with 5 ml of complete medium containing 30% dimethyl sulfoxide The plates were then washed, the medium replaced, and the plates incubated for 36 to 40 hours RNA was then extracted from the cells with the Trizol reagent (Life Technologies) following the supplier's protocol.
33. 32P]deoxycytidine triphosphate (dCTP). PCR conditions were 94°C for 5 min followed by 30 cycles of 94°C, 57°C, and 72°C for 1 min each.
34. Supported by NIH grant RO1 GM45371 We thank J. Wei for assistance in the construction of the P120 mutants and H. Busch, J. Steitz, A. Weiner, and M. Roth for supplying clones, vectors, and cell lines. We also thank J Steitz and W.-Y Tam for communicating their results before publication.