Abstract
Incubation in HeLa nuclear extract of a 32P-labeled 61 nucleotide-long RNA corresponding to the lariat branch site/polypyrimidine tract/3' splice site of the first intron of human beta-globin pre-mRNA led to the crosslinking of a single protein of approximately 62,000 mol. wt. (p62). p62 corresponds to a polypyrimidine tract-binding protein recently described by Garcia-Blanco et al. (Genes & Dev. 3: 1874-1886, 1989). Crosslinking of p62 to the 61 nt RNA was highly sequence specific. No p62 crosslinking was observed with a 60 nt pGEM vector RNA, a 63 nt RNA antisense to the 61-mer or a 72 nt U2 RNA sequence. p62 crosslinking to the 61 nt RNA was competed by unlabeled 61 nt RNA, by beta-globin pre-mRNA containing intron 1, and by poly(U) and poly(C), but was competed to a lesser extent or not at all by pGEM RNA, a beta-globin RNA lacking intron 1, or poly(A). Experiments with mutated RNAs revealed that neither the lariat branch site adenosine nor the 3' splice site were required for p62 crosslinking to polypyrimidine tract-containing RNA. Elimination of the polypyrimidine tract reduced p62 crosslinking, as did mutation of a polypyrimidine tract UU dinucleotide to GA. However, replacement of a pyrimidine-rich tract immediately adjacent (3') to the lariat branch site with a 57% A + G pGEM vector RNA sequence also significantly reduced p62 crosslinking, indicating the involvement of both this pyrimidine-rich region and the classical polypyrimidine tract adjacent to the 3' splice site. The sites of protein interaction were further defined by RNase H protection experiments, the results of which confirmed the patterns of p62 crosslinking to mutant RNAs. p62 crosslinking was efficiently competed by a DNA oligonucleotide having the same sequence as the 61 nt RNA, showing that p62 requires neither ribose 2' OH groups nor uracil bases for its interaction with the polypyrimidine tract. p62 was not crosslinked to double-stranded 61 nt RNA. Q-Sepharose chromatography of HeLa nuclear extract yielded an unbound fraction (QU) in which p62 was the only polypyrimidine tract-crosslinkable protein and a bound fraction (QB) in which, surprisingly, several non-p62 proteins were crosslinkable to the polypyrimidine tract RNA. Yet, when the two Q-Sepharose fractions were combined, p62 strongly out-competed the otherwise-crosslinkable QB proteins for polypyrimidine tract RNA crosslinking. This indicates that p62 may have the highest affinity and/or crosslinking efficiency for the intron polypyrimidine tract of any HeLa nuclear protein.