Exactly how specific splice sites are recognized during the processing of complex precursor messenger RNAs is not clear. Small nuclear ribonucleoprotein particles (snRNPs) are involved, but are not sufficient by themselves to define splice sites. Now a human protein essential for splicing in vitro, called alternative splicing factor/splicing factor 2, is shown to cooperate with the U1 snRNP particle in binding pre-mRNA. This cooperation is probably achieved by specific interactions between the arginine/serine-rich domain of the splicing factor and a similar region in a U1 snRNP-specific protein.

A protein of molecular size 62,000 daltons (p62) was detected in HeLa cell nuclear extracts by UV cross-linking to mRNA precursors. p62 binds specifically to the polypyrimidine tract of the 3' splice site region of introns. p62 purified to homogeneity binds the polypyrimidine tract of pre-mRNAs. This binding does not require the AG dinucleotide at the 3' splice site. Alterations in the polypyrimidine tract that reduce the binding of p62 yield a corresponding reduction in the efficiency of formation of a U2 snRNP/pre-mRNA complex and splicing. The p62 protein is retained in the spliceosome, where it remains bound to the pre-mRNA. This polypyrimidine tract binding protein (pPTB) is proposed to be a critical component in recognition of the 3' splice site during splicing.

The polypyrimidine tract of mammalian introns is recognized by a 62-kD protein (pPTB). Mutations in the polypyrimidine tract that reduce the binding of pPTB also reduce the efficiency of formation of the pre-spliceosome complex containing U2 snRNP. The PTB protein was purified to homogeneity by affinity chromatography on a matrix containing poly(U), and peptide sequence was used to isolate several cDNAs. Because a variety of cell types express mRNA complementary to these cDNAs, PTB may be a ubiquitous splicing factor. Three classes of cDNAs were identified, on the basis of the presence of additional sequences at an internal position. This variation in sequence probably reflects alternative splicing of the PTB pre-mRNA and produces mRNAs encoding the prototype PTB protein, a form of PTB protein containing 19 additional residues, and a truncated form of PTB protein with a novel carboxyl terminus. A murine homolog of pPTB has been characterized previously as a DNA-binding protein. Sequence comparisons indicate that pPTB is distantly related to the hnRNP L protein and that these two proteins should be considered as members of a novel family of RNA-binding proteins. The splicing of nuclear pre-mRNAs is a highly regulated process in which introns are recognized and removed to yield mature mRNAs (Green 1986;Padgett et al. 1986;Breitbart et al. 1987;Maniatis and Reed 1987;Sharp 1988). Mammalian introns are characterized by three cisacting elements: the 5'-and 3'-splice site consensus sequences, and the poorly conserved sequences at the branch site. A polypyrimidine tract typically precedes the AG dinucleotide at the 3'-splice site or immediately follows the branch site. Assembly of the spliceosome begins with the recognition of the 5'-and 3'-splice sites and the branchpoint of the pre-mRNA by U small nuclear ribonucleoprotein particles (snRNPs; Black et al. 1985;Brody and Abelson 1985;Frendeway and Keller 1985;Grabowski et al. 1985;Konarska and Sharp 1986). Recognition of the branchpoint and 3'-splice-site region by U2 snRNP is enhanced by the presence of an adjacent polypyrimidine tract (Garcia-Blanco et al. 1989). The 62-kD polypyrimidine tract-binding protein (pPTB) binds the pyrimidine tract with specificity and probably facilitates the binding of U2 snRNP.pPTB was detected in HeLa cell nuclear extracts by UV cross-linking to pre-mRNAs (Garcia-Blanco et al. 1989). pPTB specifically bound to the introns of pre-mRNAs that are efficiently spliced in vitro. The binding of this 2Corresponding author. 3Present address:

We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the beta-subunit of human chorionic gonadotropin gene 6 and pre-trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant beta-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.