Nuclear lamins are major architectural elements of the mammalian cell nucleus, and they have been implicated in the functional organization of the nuclear interior, possibly by providing structural support for nuclear compartments. Colocalization studies have suggested a structural role for lamins in the formation and maintenance of pre-mRNA splicing factor compartments. Here, we have directly tested this hypothesis by analysis of embryonic fibroblasts from knock-out mice lacking A- and C-type lamins. We show that the morphology and cellular properties of splicing factor compartments are independent of A- and C-type lamins. Genetic loss of lamins A/C has no effect on the cellular distribution of several pre-mRNA splicing factors and does not affect the compartment morphology as examined by light and electron microscopy. The association of splicing factors with the nuclear matrix fraction persists in the absence of lamins A/C. Live cell microscopy demonstrates that the intranuclear positional stability of splicing factor compartments is maintained and that the exchange dynamics of SF2/ASF between the compartments and the nucleoplasm is not affected by loss of lamin A/C. Our results demonstrate that formation and maintenance of intranuclear splicing factor compartments is independent of lamins A/C, and they argue against an essential structural role of lamins A/C in splicing factor compartment morphology.

• MeSH
• Cell Nucleus metabolism   MeSH
• Time Factors MeSH
• Cytoplasm metabolism   MeSH
• Microscopy, Electron MeSH
• Fibroblasts metabolism   MeSH
• Microscopy, Fluorescence MeSH
• Fluorescent Antibody Technique, Indirect MeSH
• Microscopy, Confocal MeSH
• Lamin Type A physiology   MeSH
• Lamins chemistry   MeSH
• RNA, Messenger metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• RNA-Binding Proteins chemistry   metabolism   MeSH
• RNA Splicing * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• lamin C MeSH Browser
• Lamin Type A MeSH
• Lamins MeSH
• RNA, Messenger MeSH
• RNA-Binding Proteins MeSH

Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. They have been shown to serve neighboring active genes as a reservoir of these factors. In this study, we show that, in HeLa cells, the (pre)spliceosomal assembly on precursor mRNA (pre-mRNA) is associated with the speckles. For this purpose, we used microinjection of splicing competent and mutant adenovirus pre-mRNAs with differential splicing factor binding, which form different (pre)spliceosomal complexes and followed their sites of accumulation. Splicing competent pre-mRNAs are rapidly targeted into the speckles, but the targeting is temperature-dependent. The polypyrimidine tract sequence is required for targeting, but, in itself, is not sufficient. The downstream flanking sequences are particularly important for the targeting of the mutant pre-mRNAs into the speckles. In supportive experiments, the behavior of the speckles was followed after the microinjection of antisense deoxyoligoribonucleotides 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 are formed inside the speckles. Their targeting into and accumulation in the speckles is a result of the cumulative loading of splicing factors to the pre-mRNA and the complexes formed give rise to the speckled pattern observed.

• MeSH
• Oligodeoxyribonucleotides, Antisense genetics   metabolism   MeSH
• HeLa Cells MeSH
• Humans MeSH
• RNA, Messenger chemistry   metabolism   MeSH
• Microinjections MeSH
• Mutation MeSH
• RNA Precursors chemistry   metabolism   MeSH
• RNA Splicing * MeSH
• Spliceosomes genetics   metabolism   MeSH
• Cell Nucleus Structures genetics   metabolism   ultrastructure   MeSH
• Temperature MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Oligodeoxyribonucleotides, Antisense MeSH
• RNA, Messenger MeSH
• RNA Precursors MeSH