A cytoplasmic 57-kDa protein that is required for translation of picornavirus RNA by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein ( ABSTRACTInitiation of translation of the RNA genomes of picornaviruses such as poliovirus and encephalomyocarditis vius is cap-independent and results from interaction of ribosomes with a segment of the 5' noncoding region of these mRNAs termed the internal ribosomal entry site. Genetic and biochemical studies have previously shown that a 57-kDa cytoplasmic RNA-binding protein (p57) plays an essential rol in this translation mechanism. We have now found that p57 shares physical, biochemical, and antigenic properties with the pyrimidine tract-binding protein (PTB), a nuclear protein that

We constructed and analyzed a synthetic poly(A) (SPA) site that was based on the highly efficient poly(A) signal of the rabbit I~-globin gene. By use of the SPA, we demonstrate that the minimum sequences required for efficient polyadenylation are the AATAAA sequence and a GT/T-rich sequence with the correct spacing of 22-23 nucleotides between them. When placed downstream of the poly(A) site of the human a2-globin gene, the SPA is used exclusively. We predict that the SPA, with its more extensive GT/T-rich sequence, is a more efficient poly(A) site than c~-globin. Also, we compared the use of the SPA when it is placed either in the exon 3 or intron 2 of the rabbit 13-globin gene. When in the exonic position, SPA is used 10-fold more than the regular poly(A) site of rabbit 13-globin. In contrast, when it is in the intronic location, no detectable use of SPA is observed; however, the deletion of the donor site of intron 2 reactivates the intronic positioned SPA. These results indicate that the splicing of intron 2 in the rabbit 13-globin gene occurs ahead of polyadenylation and have important implications for termination of transcription. Polyadenylation, although required for termination of transcription, is not sufficient; therefore, additional termination signals for RNA polymerase II must exist.

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:

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