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Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing.
Author(s) -
Bringmann P.,
Appel B.,
Rinke J.,
Reuter R.,
Theissen H.,
Lührmann R.
Publication year - 1984
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1984.tb01977.x
Subject(s) - snrnp , ribonucleoprotein , rna , biology , ribonucleoprotein particle , small nuclear rna , small nuclear ribonucleoprotein , guanosine , biophysics , biochemistry , rna dependent rna polymerase , gene
Small nuclear ribonucleoprotein particles (snRNPs) from eucaryotic cells can be fractionated on affinity columns prepared with antibodies of high affinity for 2,2,7‐trimethyl‐guanosine (m3G), which is present in the 5′‐terminal caps of the snRNAs. While the snRNPs U1, U2 and U5 are eluted with the nucleoside m3G in the presence of 0.1 M salt, the snRNP species U4 and U6 are only desorbed when the salt concentration is increased. The same fractionation pattern was likewise observed for snRNPs from HeLa or Ehrlich ascites tumor cells. Since U6 RNA lacks the m3G residue and its RNA does not react with anti‐m3G, its co‐chromatography with U4 RNP on anti‐m3G affinity columns suggests either that discrete snRNPs U4 and U6 are intimately associated in nuclear extracts or that both RNAs are organized in one ribonucleoprotein particle. Further evidence for a U4/U6 RNP particle is obtained by sedimentation studies with purified snRNPs in sucrose gradients. Gel fractionation of RNAs shows identical distributions of snRNAs U4 and U6 in the gradient, and the U4/U6 RNP particle sediments faster than the snRNPs U1 or U2. Physical association between snRNPs U4 and U6 during sedimentation is shown by their co‐precipitation with anti‐m3G IgG from the gradient fractions. Finally, experimental evidence is provided that snRNAs U4 and U6 are associated by intermolecular base pairing in the U4/U6 RNP particle, as demonstrated by our finding that anti‐m3G IgG co‐precipitates U6 RNA with U4 RNA following phenolization of U4/U6 RNPs at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)