Crosslinking of hnRNP proteins to pre-mRNA requires U1 and U2 snRNPs.
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.
Mayrand
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SH
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Pederson
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T
D001483:Base Sequence; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012332:RNA, Heterogeneous Nuclear / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear
D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012322:RNA Precursors; D012332:RNA, Heterogeneous Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D005914:Globins