Spliceosomal Assembly on Precursor mRNA Takes Place in the Nuclear Speckles
Nuclear speckles (speckles), enriched in splicing factors, form a distinct nuclear compartment within the interchromatin space. It has been shown to serve vicinal active genes as a reservoir of splicing factors. We show that (pre-)spliceosomal assembly on pre-mRNA is associated with the speckles in HeLa cells. To this end, we microinjected several mutant pre-mRNAs, which allowed the binding of some factors only and thus gave rise to the assembly of certain (pre-)spliceosomal complexes and followed their sites of accumulation. Alternatively, we followed the behavior of the speckles after the microinjection of antisense deoxyoligoribonucleotides, complementary to the specific domains of snRNAs through the generation of (pre-)spliceosomal complexes on endogenous pre-mRNAs. We show that splicing competent pre-mRNAs are targeted into the speckles, but this requires an energy dependent step. The polypyrimidine tract (necessary, but insufficient condition) and particularly downstream flanking sequences are important for the targeting of mutant pre-mRNAs. In harmony with the biochemical findings, we conclude that the (pre-)spliceosomal complexes are formed in the speckles, and that the targeting to, as well as the accumulation in, the speckles results from the cumulative wrapping of splicing factors to the pre-mRNA, the accumulated (pre-)spliceosomal complexes giving rise to the observed speckled pattern. We speculate that (pre-)spliceosomal complexes generate a macromolecular network through multiple interaction sites which contributes to the formation and maintenance of the speckles.
This work was supported by the Wellcome Trust grant 049949/Z/97/Z/JMW/JPS/CG, by research grants of the Grant Agency of the Czech Republic 302/99/0587 and 304/00/1481, and the grant of the Ministry of Education, Youth and Sports VS 96129.
Ivo Melcak, Stepanka Cermanova and Ivan Raska
Department of Cell Biology, Institute of Experimental Medicine,