| Abstrakt: |
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 machineryin 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. |
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