Sequential entry of components of the gene expression machinery into daughter nuclei

Abstract

In eukaryotic cells, RNA polymerase II (RNA pol II) transcription and pre-mRNA processing are coordinated events. We have addressed how individual components of the transcription and pre-mRNA processing machinery are organized during mitosis and subsequently recruited into the newly formed daughter nuclei. Interestingly, localization studies of numerous RNA pol II transcription and pre-mRNA processing factors revealed a nonrandom and sequential entry of these factors into daughter nuclei after nuclear envelope/lamina formation. The initiation competent form of RNA pol II and general transcription factors appeared in the daughter nuclei simultaneously, but prior to pre-mRNA processing factors, whereas the elongation competent form of RNA pol II was detected even later. The differential entry of these factors rules out the possibility that they are transported as a unitary complex. Telophase nuclei were competent for transcription and pre-mRNA splicing concomitant with the initial entry of the respective factors. In addition, our results revealed a low turnover rate of transcription and pre-mRNA splicing factors during mitosis. We provide evidence to support a model in which the entry of the RNA pol II gene expression machinery into newly forming daughter nuclei is a staged and ordered process.

Figure 1

Nuclear envelope/lamina is assembled before the entry of components of the gene expression machinery into daughter nuclei. Both the hypo- (b) and ser-5–phosphorylated form of RNA pol II (e) decorate the cytoplasm after the nuclear lamina formation (c and f). Similarly, colocalization studies with the ser-5–phosphorylated form of RNA pol II (h) and nuclear pore protein p62 (i) showed that the nuclear envelope was established before the nuclear entry of H14. Transcription factor TFIIE (k) and splicing factor B" (n) also remained in the cytoplasm until after nuclear envelope (l) and lamina (o) were assembled. Chromosomes were stained with DAPI (a, d, g, j, and m). Bar 5 μm.

Figure 2

Appearance of the transcription initiation-competent form of RNA pol II and associated transcription factors in postmitotic nuclei precedes pre-mRNA processing factors. The initiation-competent ser-5 phosphorylated form of RNA pol II (b and e), hypophosphorylated form of RNA pol II (c) and TFIIE (f) appeared in daughter nuclei almost simultaneously. Splicing factors B" (i) and SF2/ASF (l) were largely localized to the cytoplasmic MIGs (i and l, arrows) when H14 labeling was already apparent in daughter nuclei (h and k). During this same time period, the hnRNP C1/C2 proteins (o) were predominantly present in the cytoplasm, whereas H14 had already entered nuclei (n). However, CstF-64 (r) and H14 (q) appeared in daughter nuclei simultaneously. DNA was stained with DAPI (a, d, g, j, m, and p). Bar 5 μm.

Figure 3

Transcription elongation competent RNA pol II appears in daughter nuclei subsequent to the pre-mRNA processing machinery. Double-label immunofluorescence by using antibodies against the ser-2 phosphorylated, elongation-competent form of RNA pol II (H5) (b, e, h, k, and n) and hnRNP C1/C2 (c), hnRNP A1 (f), B" (i), and SF2/ASF (l and o), respectively, demonstrated that entry of the pre-mRNA processing machinery preceded the appearance of H5 labeling in daughter nuclei. Splicing factors (i, l, and o) colocalized with H5 in MIGs (k and l, see arrowhead). SF2/ASF was nearly absent from MIGs in G1 cells (o, arrow), whereas H5 labeling was largely retained in MIGs through G1 (n, arrow). DNA was stained with DAPI (a, d, g, j, and m). Bar, 5 μm.

Figure 4

Schematic representation of the order of events in the recruitment of the gene expression machinery into daughter nuclei. A consolidated overview of sequential entry of transcription and pre-mRNA processing factors into newly formed daughter nuclei.

Figure 5

Global transcription increases with accumulation of the ser-2 phosphorylated form of RNA pol II in daughter nuclei. Double-label immunofluorescence by using antibodies against ser-5 phosphorylated (H14) (b and e) or ser-2 phosphorylated RNA pol II (H5) (h and k) and bromo-UTP (c, f, i, and l) shows that there is no transcription in daughter nuclei prior to entry of RNA pol II (b and c). When ser-5 phoshorylated RNA pol II begins to enter daughter nuclei there is only a low level of nucleoplasmic bromo-UTP incorporation (e and f), similar to the presence of low levels of ser-2 phosphorylated RNA pol II (h and i). With further accumulation of the ser-2 phosphorylated RNA pol II, bromo-UTP incorporation dramatically increased (k and l). DNA was stained with DAPI (a, d, g, and j). Bar, 5 μm.

Figure 6

Splicing factors are functionally competent upon entry into daughter nuclei. RNA fluorescence in situ hybridization was performed on HeLa cells stably expressing a β-tropomyosin minigene. The locus is detected during interphase as a single dot (c, arrow) that recruits splicing factors such as SF2/ASF (b, arrow). During telophase, SF2/ASF (e) and B" (h) are recruited to the locus, which is detected by a splice junction probe (SJ) (f and i), demonstrating that splicing occurs at this stage. Control hybridization of 12-mer oligonucleotides (l) shows no hybridization signal at loci decorated with SF2/ASF (k, arrow indicates probable transcription site). DNA was stained with DAPI (a, d, g, and j). Bar, 5 μm.

Figure 7

MIGs are not protein degradation sites. Immunostaining with an antibody against ubiquitinated proteins (c) did not reveal any positive labeling of MIGs but predominantly labeled the midbody between daughter cells. MIGs were positively stained with the elongation competent form of RNA pol II, H5 (b). DNA was stained with DAPI (a). Bar, 5 μm.

Figure 8

Live cell observations of entry of SF2/ASF into daughter cell nuclei. HeLa cells were transiently transfected with YFP-SF2/ASF and live-cell observations were initiated 2 d posttransfection. Metaphase cells exhibit one to two MIGs near the metaphase plate (a, arrow). As the cell enters anaphase, MIGs become more abundant (b and c). SF2/ASF begins to enter daughter nuclei at telophase (d). Nuclear entry of YFP-SF2/ASF is nearly complete in ∼20 min (h). Time is indicated in min from the initiation of imaging the metaphase cell (see video, Mitosis.mov).

Figure 9

RNA processing factors exhibit a low turnover rate during mitosis. Cells blocked in prometaphase by nocodazole treatment were incubated with (N + C) or without (N) cycloheximide and pulsed with [³⁵S]methionine during release from the mitotic block. Aliquots of cells were collected at indicated times and efficacy of the protein synthesis block was analyzed by measuring incorporated [³⁵S]methionine by autoradiography (b) (see MATERIALS AND METHODS). The corresponding Coomassie-stained gel is shown in a. Lanes 1, 3, and 5 are cells treated with nocodazole for 14–18 h and collected at 1, 2, and 4 h postrelease, respectively. Lanes 2, 4, and 6 represent cells treated with nocodazole and cycloheximide at indicated time points. Lanes 7 and 8 represents nocodazole-arrested cells and an asynchronous population, respectively. DNA content of the respective cell populations indicated in d, 1–8, was determined by FACS analysis and is shown in c. Cells treated with or without cycloheximide were collected at indicated time points and extracted for immunoblot analysis of components of the gene expression machinery (d). The results show a low turnover of splicing factors (SF2/ASF and B"), CstF-64, hnRNP A1, hnRNPC1/C2, and various forms of RNA pol II during mitosis. Tubulin and cyclin A are shown as internal controls for protein loading and cell cycle progression, respectively.