Direct cross-linking of snRNP proteins F and 70K to snRNAs by ultra-violet radiation in situ.
Protein-RNA interactions in small nuclear ribonucleoproteins (UsnRNPs) from HeLa cells were investigated by irradiation of purified nucleoplasmic snRNPs U1 to U6 with UV light at 254 nm. The cross-linked proteins were analyzed on one- and two-dimensional gel electrophoresis systems, and the existence of a stable cross-linkage was demonstrated by isolating protein-oligonucleotide complexes from snRNPs containing 32P-labelled snRNAs after exhaustive digestion with a mixture of RNases of different specificities. The primary target of the UV-light induced cross-linking reaction between protein and RNA was protein F. It was also found to be cross-linked to U1 snRNA in purified U1 snRNPs. Protein F is known to be one of the common snRNP proteins, which together with D, E and G protect a 15-25 nucleotide long stretch of snRNAs U1, U2, U4 and U5, the so-called domain A or Sm binding site against nuclease digestion (Liautard et al., 1982). It is therefore likely that the core-protein may bind directly and specifically to the common snRNA domain A, or else to a sub-region of this. The second protein which was demonstrated to be cross-linked to snRNA was the U1 specific protein 70K. Since it has been shown that binding of protein 70K to U1 RNP requires the presence of the 5' stem and loop of U1 RNA (Hamm et al., 1987) it is likely that the 70K protein directly interacts with a sub-region of the first stem loop structure.
Woppmann
A
A
Max-Planck-Institut für Molekulare Genetik, Otto Warburg Laboratories, Berlin, FRG.
Rinke
J
J
Lührmann
R
R
D003432:Cross-Linking Reagents; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008970:Molecular Weight; D009711:Nucleotides; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D012342:RNA, Small Nuclear / Q000302:isolation & purification / Q000528:radiation effects*; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism / Q000528:radiation effects*; D017411:Ribonucleoproteins, Small Nuclear; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D009711:Nucleotides; D010750:Phosphoproteins; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear