Nucleic Acids Research, 1990, Vol. 18, No. 11 3307-3318
© 1990
MOLECULAR BIOLOGY |
Crosslinking of hnRNP protiens to pre-mRNA requires U1 and U2 snRNPs
Cell Biology Group, Worcester Foundation for Experimental Biology Shrewsbury, MA 01545, USA
Received February 3, 1990. Accepted April 25, 1990.
Proteins interacting with pre-mRNAs during early stages of spliceosome formation in a HeLa nuclear extract were investigated by photochemical RNAprotein crosslinking. The level of protein crosslinking to a ß-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 ß-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 ß-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'-termlnal 15 nucleotides of U2 RNA. Cleavage of the 5'-termlnal 15 nucleotides of U1 RNA preferentially eliminated crosslinking of the hnRNP A1 protein to both premRNAs. 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 ß-globin pre-mRNA, crosslinking was restored by addition of a U1 snRNP-enriched fraction.
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