Proteins interacting with pre-mRNAs during early stages of spliceosome formation in a HeLa nuclear extract were investigated by photochemical RNA-protein crosslinking. The level of protein crosslinking to a beta-globin pre-mRNA was positively correlated with the presence of an intron. Proteins of 110,000, 59,000 and 39,000 mol. wt. were crosslinked to the beta-globin pre-mRNA, the latter of which was identified as the A1 hnRNP protein. Comparable experiments with an adenovirus pre-mRNA revealed crosslinked proteins of 110,000, 56,000 and 45,000 mol. wt., with the latter identified as belonging to the C group hnRNP proteins. Crosslinking of hnRNP proteins to both the beta-globin and adenovirus pre-mRNAs was eliminated by oligodeoxynucleotide-directed RNase H excision of an internal region (nt 28-42) of U2 RNA, but was not affected by oligo/RNase H cleavage of the 5'-terminal 15 nucleotides of U2 RNA. Cleavage of the 5'-terminal 15 nucleotides of U1 RNA preferentially eliminated crosslinking of the hnRNP A1 protein to both pre-mRNAs. The requirement of intact U1 snRNP for A1 protein crosslinking was further demonstrated by the fact that although micrococcal nuclease-treated extracts did not support crosslinking of A1 hnRNP protein to beta-globin pre-mRNA, crosslinking was restored by addition of a U1 snRNP-enriched fraction.