Ordered splicing of thymidine kinase pre-mRNA during the S phase of the cell cycle.

Abstract

Concomitant with the onset of S phase, a series of thymidine kinase (TK) splicing intermediates as well as mature TK mRNA accumulates in the nucleus of BALB/c 3T3 cells. Most of the TK splicing intermediates are retained by oligo(dT)-cellulose chromatography, and, therefore, 3' end formation and polyadenylation probably precede the splicing of TK pre-mRNAs. We have further characterized the TK pre-mRNAs that are present in the nuclei of S-phase cells by using specific probes derived from each of the six TK intervening sequences. Based on the sizes of the pre-mRNAs and their patterns of hybridization with these intron probes, we propose a pathway for intron removal from nascent TK transcripts. Intron excision occurred by a preferred, but not necessarily obligatory, order which appears to have been conserved in mouse and Chinese hamster cells.The cytosolic enzyme thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) is of particular interest for the study of cell cycle-dependent regulatory mechanisms, because the mRNA encoding this enzyme accumulates to high levels just prior to the onset of S phase (12,15). The induction of TK mRNA at this point during the cell cycle involves transcriptional (4,22,24,35), posttranscriptional (4, 10, 14, 21, 22, 26, 35) and translational (8, 9, 33) controls.To obtain insight into the mechanism(s) involved in the posttranscriptional regulation of TK mRNA accumulation, we examined nuclear RNA from quiescent BALB/c 3T3 cells that had been restimulated to proliferate by the addition of fresh serum. Very little TK mRNA was detected in the nuclei of cells harvested during Go and Gl. At the onset of S phase, however, we observed a dramatic change in the processing of TK mRNA precursors (pre-mRNA) that was characterized by the appearance of a series of high-molecular-weight TK mRNA precursors in addition to mature TK mRNA. These high-molecular-weight TK mRNAs could be chased in the presence of the RNA synthesis inhibitor dactinomycin, suggesting a precursor-product relationship (11).We proposed that the high-molecular-weight TK transcripts were splicing intermediates which retained various intervening sequences (IVSs). Most eucaryotic genes contain IVSs or introns that are excised from nuclear premRNA by the RNA splicing machinery (1,25,27,32). To gain insight into the nature of the putative high-molecularweight TK pre-mRNAs present in G,/S-and S-phase nuclei, as well as to determine whether there is a preferential order of IVS removal from the TK gene, we further characterized the TK pre-mRNA splicing intermediates present in S-phase cells by Northern (RNA) blot analysis.Mammalian TK genes, which contain seven coding exons and six introns, have been well conserved during evolution (6,20,23,31). We isolated unique sequence DNA probes from each of the six TK introns and used them to character-* Corresponding author.ize the splicing intermediates in the nuclei. The human TK gene has the highest density of Alu-like repetitive elements within its intervening sequences of an...

Lack of an Effect of the Efficiency of RNA 3′-End Formation on the Efficiency of Removal of Either the Final or the Penultimate Intron in Intact Cells

“…The abnormally high level of intron-free RNA that is a consequence of inefficient RNA 3Ј-end formation indicates that splicing can take place on a nascent transcript, which is consistent with earlier studies of both cellular and viral RNAs (1-3, 16, 17, 22, 27), including human TPI RNA (33). Evidence that splicing can also take place on a polyadenylated RNA exists as well (12,15,23,34,35,41). However, it is unclear whether the process of 3Ј-end formation can take place in an intact cell after all of the introns have been removed.…”

Co-transcriptional splicing of constitutive and alternative exons

“…This indicates that it will be difficult to determine the order of intron removal from studies of full-length or partially spliced pre-mRNAs in total nuclear RNA samples, as has been suggested (Hatzoglou et al 1985;Lang and Spritz 1987;Shiels et al 1987;Gudas et al 1990;Kessler et al 1993;Neel et al 1993). While it is likely that some of these retained, nucleoplasmic introns are spliced post-transcriptionally, some nucleoplasmic transcripts may never mature and instead be degraded.…”

“…Analyses of endogenous and minigene-encoded intron-containing RNAs from isolates of mammalian total nuclear RNA indicate that intron removal will sometimes occur in a 59-to-39 fashion, consistent with a pattern of concurrent co-transcriptional intron removal (Hatzoglou et al 1985;Lang and Spritz 1987;Neel et al 1993). However, deviations from this pattern are also described (Gudas et al 1990;Kessler et al 1993). Moreover, it is not clear whether this intron excision occurs before or after transcript release from the template.…”

Splicing Signals Are Required for S-Phase Regulation of the Mouse Thymidylate Synthase Gene

“…Introns 1 and 4 of the proliferating cell nuclear antigen gene appear to be important for proper down-regulation of the gene (1,44). Alterations in splicing mechanisms also appear to be important for controlling thymidine kinase gene expression (23,24).…”