Previously, we determined that the disruption of normal lamin organization blocked the elongation phase of DNA synthesis. To begin to determine if other nuclear functions are also dependent upon lamins, we examined the distribution of RNA splicing factors in BHK 21 cells within 2 h after ΔNLA, a dominant-negative lamin mutant, was microinjected into the cytoplasm. Consistent with previous findings (Spann et al., 1997
), immunofluorescence revealed that after the microinjection of ΔNLA, the organization of A- and B-type lamins was altered. This alteration was seen as a loss of the normal distribution of lamins in the lamina and the nucleoplasmic lamin veil. Instead, both types of lamins were found together with the mutant lamin in abnormal nucleoplasmic aggregates (, compare A and B with C and D). Under normal conditions, immunofluorescence using an antibody directed against B′′, a U2-specific binding protein, indicates that splicing factors are distributed in a characteristic pattern of speckles and interconnecting material within the nucleoplasm. After the disruption of lamin organization with ΔNLA, this pattern is significantly altered with the majority of speckles and the interconnecting material replaced with fewer and distinctly separated speckles (). A similar reorganization of splicing factors was observed after microinjection of ΔNLA and staining with Y12, an antibody that reacts with snRNPs that contain the SM antigen (unpublished data). In addition, a comparison of the distribution of B′′ and the lamin aggregates revealed that these proteins do not colocalize in the ΔNLA-treated cells. The dramatic alteration of the distribution of splicing factors in response to disruption of lamin organization is reminiscent of changes observed after treating cells with α-amanitin, an inhibitor of RNA polymerase II (Spector, 1993
). Together, these observations raise the possibility that within the nucleus, transcription depends upon the normal organization of lamins.
Figure 1. Disruption of lamin organization alters the distribution of splicing factors. The organization of A- and B-type lamins in untreated BHK 21 cells (A and B) and cells microinjected with ΔNLA (C and D) were examined by immunofluorescence with antibodies (more ...)
To determine whether the disruption of lamin organization affects transcription, ΔNLA was microinjected into the cytoplasm of BHK 21 cells, and 1–4 h later, transcription was assayed by in situ incorporation of BrU into RNA. After a 10-min labeling period, sites of BrU incorporation were detected by immunofluorescence (Huang et al., 1998
). Cells injected with ΔNLA displayed greatly reduced BrU staining when compared with the bright, somewhat punctate staining found throughout the nucleus of neighboring uninjected cells (). The most dramatic reductions in BrU incorporation were seen in cells where lamin organization was most affected, suggesting a dose–response relationship (, compare large and small arrows). Mouse 3T3 and human HeLa cells injected with ΔNLA displayed virtually identical reductions in BrU incorporation, demonstrating that the inhibition of transcription was not limited to a particular cell type (unpublished data). Additionally, BrU labeling in cells injected with an equimolar concentration of wild-type LA in the injection buffer was indistinguishable from uninjected cells (unpublished data). Although the overall levels of BrU incorporation were greatly reduced in the nuclei of cells microinjected with ΔNLA, no matter what the degree of disruption of lamin organization, a few clusters of BrU staining remained evident (). Phase contrast observations revealed that these clusters were associated with nucleoli (). The intensity of the BrU staining in the nucleolar regions after disruption of lamin organization is comparable to that observed in the nucleolar region of the uninjected cell (, arrowheads). This retention of BrU staining in nucleolar regions suggests that lamin disruption does not block RNA polymerase I activity. In addition, a normal nucleolar distribution of fibrillarin was detected in nuclei treated with ΔNLA, providing additional evidence that this disruption does not block RNA polymerase I activity (unpublished data; Ochs et al., 1985
Figure 2. Disruption of lamin organization results in reduced incorporation of BrUTP. After microinjection of ΔNLA (arrows in A and B), cells were assayed for transcriptional activity by in situ incorporation of BrUTP. Cells were fixed and stained by immunofluorescence (more ...)
Using the microinjection technique, only a few cells can be studied in each experiment. Therefore, we developed a cell-free system to simultaneously treat large numbers of transcriptionally active nuclei with ΔNLA. Xenopus
embryos are transcriptionally inactive until the midblastula transition (division 12, ~8 h after fertilization) when RNA polymerases II and III become active (Newport and Kirschner, 1982
). We found that early Xenopus
gastrulae (~10 h after fertilization) could be lysed by centrifugation, yielding intact nuclei in an embryonic extract.
The embryonic nuclei possessed a prominent lamina and typical nucleoplasmic lamin structures comprised of the endogenous lamin, LB3 (). However, 1 h after the addition of ΔNLA, instead of typical lamin structures, abnormal nucleoplasmic aggregates containing both ΔNLA and LB3 were observed in these nuclei (, E–G). The addition of BrUTP revealed that transcriptional activity was dramatically reduced in the ΔNLA-treated nuclei (, H–J), as compared with untreated nuclei (, C–D). The size of the transcription products synthesized by the embryonic nuclei was examined by adding [α32
P]UTP to the extract. After 15 min, RNA was prepared and resolved by denaturing agarose gel electrophoresis. In untreated nuclei, 32
P was incorporated into tRNA-sized products and higher molecular weight species of RNA ( A). Newly synthesized rRNA-sized products were not detected in these extracts, as RNA polymerase I is not active during early gastrulation (Newport and Kirschner, 1982
; Verheggen et al., 2000
). The synthesis of the upper molecular weight products was selectively inhibited by the addition of α-amanitin (10 μg/ml), indicating that these are products of RNA polymerase II ( B). ΔNLA treatment of embryonic nuclei also resulted in a dramatic inhibition of the synthesis of upper molecular weight RNA, whereas the synthesis of tRNA-sized products was not detectably altered (). These results indicate that disruption of lamin organization specifically inhibited RNA polymerase II activity.
Figure 3. Disruption of lamin organization in embryonic nuclei inhibits the incorporation of BrU. Buffer (A–D) or ΔNLA (E–J) was added to embryonic extracts. After 60 min, BrUTP was added, and 15 min later, the cells were fixed. Chromatin (more ...)
Figure 4. Disruption of lamin organization inhibits the synthesis of mRNA-sized products in embryonic nuclei. The size of transcription products synthesized in the embryonic extract was determined by the addition of [α32P]UTP. After 15 min, total RNA was (more ...)
To control for possible nonspecific effects on transcription resulting from the introduction of exogenous proteins into the nucleus, an equivalent concentration of full-length human LA was added to embryonic extracts. No alterations in the distribution of endogenous lamins or in the incorporation of BrU were observed (unpublished data). To eliminate the possibility that the inhibition of transcription was due to nonspecific effects of nuclear aggregates of IF proteins, Xenopus
vimentin, a cytoplasmic IF protein engineered to contain a nuclear localization signal, was added to embryonic extracts. This protein was imported into nuclei where it formed nucleoplasmic aggregates (Moir et al., 2000a
; Reichenzeller et al., 2000
) that did not alter the distribution of LB3 and did not inhibit the incorporation of BrU (unpublished data). Furthermore, chromatin distribution appeared normal in ΔNLA-treated nuclei (). We have previously demonstrated that ΔNLA disruption of lamin organization does not detectably alter nuclear transport properties (Moir et al., 2000a
To identify potential mechanisms underlying the inhibition of polymerase II that accompanies the disruption of lamin organization, we examined the distribution of Sp1, a gene-specific transcription factor that binds the GC box found in some polymerase II promoters. In uninjected BHK 21 cells, Sp1 was found throughout the nucleus ( B). Similarly, after injection of ΔNLA and the disruption of lamin organization, Sp1 remained distributed throughout the nucleus in a pattern indistinguishable from that observed in uninjected neighboring cells ( B). Therefore, the disruption of lamin organization does not appear to alter the distribution of the gene-specific transcription factor Sp1.
Figure 5. Disruption of lamin organization does not alter the distribution of Sp1. BHK 21 cells were microinjected with ΔNLA. Cells were stained with rabbit anti–human LA (A) and a monoclonal anti–human Sp1 (B). The cell on the left in each (more ...)
We also examined the effect of the disruption of lamin organization on the distribution of the TATA binding protein (TBP), a transcription factor for RNA polymerases I, II, and III (Hernandez, 1993
). TBP interacts with a number of TBP-associated factors to form the TFIID complex (Albright and Tjian, 2000
). This complex is a basal transcription factor thought to be required for forming preinitiation complexes on RNA polymerase II promoters containing a TATA box. We found that in BHK 21 cells, disruption of lamin organization altered the distribution of TBP. Instead of its normal punctate distribution throughout the nucleus (), most of the TBP colocalized with the nucleoplasmic lamin aggregates (). However, TBP staining often remained apparent in the nucleolar region of the ΔNLA-treated nuclei, most likely reflecting its involvement in RNA polymerase I–dependent transcription ( D, arrows). In Xenopus
embryonic nuclei, the disruption of lamin organization also resulted in an alteration of TBP distribution, such that TBP was found in nucleoplasmic lamin aggregates (, compare G and H with E and F). In contrast, LA did not alter the distribution of TBP in either BHK 21 cells or embryonic nuclei, and TBP did not colocalize with the NLS-vimentin aggregates in Xenopus
nuclei (unpublished data).
Figure 6. Disruption of lamin organization alters the distribution of TBP. BHK 21 cells (A–D) and embryonic nuclei (E–H) stained with a rabbit anti-TBP (B, D, F, and H), rat anti–human LA (A and C), and a monoclonal anti–Xenopus (more ...)
Therefore, the change in the distribution of TBP accompanying lamin disruption does not appear to simply be the result of adding an exogenous protein that forms nuclear aggregates. The specificity of the interaction between lamins and TBP is also supported by the findings that the transcription factor Sp1 and splicing factors do not colocalize with lamins in the ΔNLA-induced aggregates ( and ). Splicing factors are very abundant in the nucleus and are not tightly associated with chromatin (Spector, 1993
). Therefore, these findings argue against nonspecific trapping as a mechanism to explain the association of TBP with the lamin aggregates. Based on these results, we propose that the distribution of TBP in normal nuclei depends upon the maintenance of normal lamin organization. However, the lack of inhibition of RNA polymerases I and III suggests that lamins may not bind TBP directly. Instead, lamins may interact with TBP through other factors specific for RNA polymerase II, such as the TBP-associated factors of the TFIID complex.
In conclusion, our results demonstrate that disruption of normal lamin organization inhibits RNA polymerase II activity. We believe that these findings provide the first experimental evidence that specific nuclear structural proteins, the lamins, are involved in the synthesis of mRNA. Together with reports that lamins are distributed throughout the nucleoplasm, these findings raise the possibility that lamins may act as a scaffold upon which the basal transcription factors required for RNA polymerase II transcription are organized.