Prespliceosomal assembly on microinjected precursor mRNA takes place in nuclear speckles

Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. They have b een shown to serve neighboring active genes as a reservoir of these factors . In this study, we show that, in HeLa cells, the (pre)spliceosomal assembl y on precursor mRNA (pre-mRNA) is associated with the speckles. For this pu rpose, we used microinjection of splicing competent and mutant adenovirus p re-mRNAs with differential splicing factor binding, which form different (p re)spliceosomal complexes and followed their sites of accumulation. Splicin g competent pre-mRNAs are rapidly targeted into the speckles, but the targe ting is temperature-dependent. The polypyrimidine tract sequence is require d for targeting, but, in itself, is not sufficient. The downstream flanking sequences are particularly important for the targeting of the mutant pre-m RNAs into the speckles. In supportive experiments, the behavior of the spec kles was followed after the microinjection of antisense deoxyoligoribonucle otides complementary to the specific domains of snRNAs. Under these latter conditions prespliceosomal complexes are formed on endogenous pre-mRNAs. We conclude that the (pre)spliceosomal complexes on microinjected pre-mRNA ar e formed inside the speckles. Their targeting into and accumulation in the speckles is a result of the cumulative loading of splicing factors to the p re-mRNA and the complexes formed give rise to the speckled pattern observed .