Magnesium cations are required for the association of U small nuclear ribonucleoproteins and SR proteins with pre-mRNA in 200 S large nuclear ribonucleoprotein particles.
In previous studies we have shown that specific nuclear pre-mRNAs and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are found packaged in 200 S large nuclear ribonucleoprotein (lnRNP) particles that represent the splicing machinery in vivo. The lnRNP particles contain all U small nuclear ribonucleoproteins (snRNPs) required for splicing, as well as several proteins including non-snRNP splicing factors. Here we show that upon addition of EDTA to sucrose gradient-fractionated 200 S particles, part of their components (e.g. part of the U snRNPs) are no longer associated with pre-mRNAs, which are now packaged in 70 S particles. This 200 S to 70 S transition makes the pre-mRNA more susceptible to digestion by RNase. The effect of EDTA is reversible, as back addition of Mg2+ results in the reconstitution into 200 S lnRNP particles of: (1) all five snRNPs required for splicing; (2) the SR proteins; and (3) CAD mRNA, as a representative of nuclear RNA polymerase II transcripts. Remarkably, electron microscopy of the reconstituted particles shows a compact structure, 50 nm in diameter, that is indistinguishable from the original undissociated particles. We conclude that Mg2+ is required for the integrity of the 200 S lnRNP particles.
Miriami
E
E
Department of Genetics, Hebrew University of Jerusalem, Israel.
Angenitzki
M
M
Sperling
R
R
Sperling
J
J
D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000096:biosynthesis; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D004080:Dihydroorotase / Q000096:biosynthesis; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008647:Mesocricetus; D008854:Microscopy, Electron; D009097:Multienzyme Complexes / Q000096:biosynthesis; D012319:RNA Polymerase II / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism* / Q000648:ultrastructure*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis; D012260:Ribonucleases; D017411:Ribonucleoproteins, Small Nuclear / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure*; D014158:Transcription, Genetic
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D012322:RNA Precursors; D012333:RNA, Messenger; D017411:Ribonucleoproteins, Small Nuclear; D004492:Edetic Acid; D001221:Aspartate Carbamoyltransferase; D012319:RNA Polymerase II; D012260:Ribonucleases; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D008274:Magnesium