Identification of proteins that bind tightly to pre-mRNA during in vitro splicing.
Incubation of a human beta-globin pre-mRNA in a HeLa cell nuclear extract under conditions permissive for efficient splicing resulted in the assembly of the RNA into ribonucleoprotein (RNP) complexes. This RNP formation occurred largely within the characteristic lag period that precedes splicing. Two classes of RNP were detected by the criterion of their stability in Cs2SO4 gradients. One was unstable and contained mainly aberrant RNA cleavage products. The other class of RNP complexes comprised 50-85% of the beta-globin RNA, formed only under splicing-permissive conditions, was stable in Cs2SO4 gradients, and contained both unspliced pre-mRNA molecules and the lariat intron 1-exon 2 splicing intermediate. This latter class of RNP complexes banded at approximately equal to 1.30 g/cm3, a density very similar to that of native heterogeneous nuclear RNP particles that contain pre-mRNA. RNA-protein crosslinking revealed major proteins of Mr approximately equal to 38,000 and 41,000 in the stable class of RNP. The use of antibodies specific for heterogeneous nuclear RNP core proteins and for small nuclear RNA-associated proteins, in conjunction with [32P]RNA-protein crosslinking, revealed polypeptides having the molecular weights of both sets of antigens. These results show that both heterogeneous nuclear RNP particle core proteins and small nuclear RNA-associated proteins bind tightly to pre-mRNA during splicing in vitro.
Mayrand
S H
SH
Pedersen
N
N
Pederson
T
T
D001323:Autoantibodies / Q000276:immunology; D005260:Female; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D066298:In Vitro Techniques; D009694:Nucleic Acid Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000276:immunology / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*
D001323:Autoantibodies; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009694:Nucleic Acid Precursors; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D005914:Globins