Sequence elements in both the intergenic space and the 3' untranslated region of the Crithidia fasciculata KAP3 gene are required for cell cycle regulation of KAP3 mRNA

Abstract

mRNA levels of several Crithidia fasciculata genes involved in DNA metabolism have previously been found to cycle as cells progress through the cell cycle. Octamer consensus sequences in the 5' untranslated regions (5' UTRs) of these transcripts were shown to be required for cycling of these mRNAs. The KAP3 gene encodes a kinetoplast histone H1-like DNA binding protein, and its mRNA levels cycle in parallel with those of the kinetoplast DNA topoisomerase (TOP2), dihydrofolate reductase-thymidylate synthase (DHFR-TS), and the large subunit of the nuclear single-stranded DNA binding protein (RPA1). KAP3 mRNA contains two octamer consensus sequences in its 3' UTR but none in its 5' UTR. Mutation of these octamer sequences was not sufficient to prevent cycling of a sequence-tagged KAP3 mRNA expressed from a plasmid. Mutation of an octamer sequence contained on the precursor transcript but not on the mRNA, in addition to mutation of the two octamer sequences in the 3' UTR, was necessary to abolish cycling of the mRNA. The requirement for a sequence not present on the mature mRNA indicates that regulation of the mRNA levels by the octamer sequences occurs at or prior to splicing of the transcript. Incompletely processed RNAs containing octamer sequences were also found to accumulate during the cell cycle when the mRNA levels were lowest. These RNA species hybridize to both the KAP3 coding sequence and that of the downstream drug resistance gene, indicating a lack of processing within the intergenic region separating these genes. We propose a cell cycle-dependent interference in transcript processing mediated by octamer consensus sequences as a mechanism contributing to the cycling of such transcripts.

FIG. 1.

Physical map of the KAP3-GFP expression plasmid pMA1. Full-length EGFP was fused in frame near the 3′ end of the KAP3 coding sequence as a tag to distinguish the chimeric transcript from that of the KAP3 gene. The vector contains the HYG selectable marker flanked by the L. major 5′ and 3′ flanking regions of the DHFR-TS gene for expression of the HYG drug resistance marker. The 5′ and 3′ intergenic sequences flanking the C. fasciculata KAP3 gene are indicated. The locations of the KAP3 splice acceptor site and octameric sequences are also indicated. Mutated sequences are given in parentheses.

FIG. 2.

Expression and mitochondrial targeting of the KAP3-GFP fusion protein in C. fasciculata cells transformed with plasmid pMA1. (A) DAPI staining; (B) phase-contrast image with DAPI staining; (C) phase-contrast image with GFP fluorescence; (D) GFP fluorescence.

FIG. 3.

Expression of endogenous KAP3 and chimeric KAP3-GFP transcripts varies during the cell cycle. (A) Schematic structure (not to scale) of the KAP3-GFP gene and flanking sequences in pMA1. SA, the KAP3 splice acceptor site. Unlabeled open rectangles indicate the locations of the CATAGAAA octamer sequences. (B) Northern blot analysis of total RNA isolated from synchronized C. fasciculata cells carrying the pMA1 plasmid. Total RNA was isolated at the time of release from hydroxyurea arrest (0 min) and at 30-min intervals thereafter. Total RNA (8 μg per lane) was subjected to Northern blot analysis by hybridization to the KAP3 gene coding sequences to detect both chimeric KAP3-GFP transcripts and endogenous KAP3 transcripts. The same Northern blot was stripped and rehybridized to a 260-bp probe derived from a partial cDNA encoding the C. fasciculata homolog to the T. cruzi flagellar calcium binding protein (CaBP). (C) PhosphorImager quantitation of transcript levels in the Northern blot. Endogenous KAP3 (solid bars) and chimeric KAP3-GFP (dark shaded bars) RNA transcript levels have been normalized at each time point relative to the level of the CaBP transcript, which is expressed at a constant level during the cell cycle and serves as a loading control.

FIG. 4.

Sequence elements required for cycling of KAP3-GFP mRNA levels. Shown is a PhosphorImager quantitation of Northern blots of total RNA isolated from synchronized C. fasciculata cells carrying either pMA2 (A), pMA5.4 (B), or pMA5.1 (C). RNA samples were isolated at 30-min intervals after release from hydroxyurea arrest and analyzed as for Fig. 3. A schematic diagram of the relevant region of each plasmid, with open rectangles representing wild-type consensus octamer sequence elements (CATAGAAA) and filled rectangles representing mutated octamer sequences, is shown above the respective bar graph. Solid bars, KAP3 mRNA; dark shaded bars, KAP3-GFP mRNA.

FIG. 5.

RNase protection mapping of the KAP3 splice acceptor site. C. fasciculata total-cell RNA (15 μg) was hybridized to a ³²P-labeled RNA probe and digested with RNase A and RNase T1. The protected hybridized probe fragment was resolved on a 4% polyacrylamide gel containing 8 M urea. Lane 1, RNase A- and RNase T1-digested RNA; lanes 2 and 3, ³²P-labeled 1-kb Plus and 1-kb DNA markers (Invitrogen); lane 4, RNA probe. The probe and protected fragment are diagramed underneath the autoradiogram.

FIG. 6.

Accumulation of pre-mRNA species of the KAP3-GFP chimeric transcript during progression through the cell cycle in C. fasciculata cells carrying pMA5.1. (A) The number of cells (squares) and the percentage of cells with two nuclei (triangles) were measured every 30 min following release from the hydroxyurea block to examine synchrony and cell cycle position. (B) Northern blot analysis of total RNA isolated at 30-min intervals. The blot was probed with KAP3 coding sequences and then stripped and hybridized with the CaBP probe. (C) PhosphorImager quantitation of relative levels of chimeric KAP3-GFP and endogenous KAP3 transcripts. Transcript levels have been normalized at each time point relative to the level of the CaBP transcript, which is expressed at a constant level throughout the cell cycle.

FIG. 7.

Northern blot analysis of total RNA isolated at 30-min intervals from hydroxyurea-synchronized C. fasciculata cells carrying pMA5.1. (Right) The Northern blot was hybridized with HYG coding sequences. (Left) The same Northern blot was stripped and reprobed with KAP3 coding sequences as for Fig. 6. The endogenous KAP3, chimeric KAP3-GFP, and HYG mRNA bands are indicated on the sides of the autoradiogram. Arrowheads indicate incompletely processed transcripts.