Alternative splicing of c-fos pre-mRNA: contribution of the rates of synthesis and degradation to the copy number of each transcript isoform and detection of a truncated c-Fos immunoreactive species

Abstract

Background: Alternative splicing is a widespread mechanism of gene expression regulation. Previous analyses based on conventional RT-PCR reported the presence of an unspliced c-fos transcript in several mammalian systems. Compared to the well-defined knowledge on the alternative splicing of fosB, the physiological relevance of the unspliced c-fos transcript in regulating c-fos expression remains largely unknown. This work aimed to investigate the functional significance of the alternative splicing c-fos pre-mRNA.

Results: A set of primers was designed to demonstrate that, whereas introns 1 and 2 are regularly spliced from primary c-fos transcript, intron 3 remains unspliced in part of total transcript molecules. Here, the two species are referred to as c-fos-2 (+ intron 3) and spliced c-fos (- intron 3) transcripts. Then, we used a quantitatively rigorous approach based on real-time PCR to provide, for the first time, the actual steady-state copy numbers of the two c-fos transcripts. We tested how the mouse-organ context and mouse-gestational age, the synthesis and turnover rates of the investigated transcripts, and the serum stimulation of quiescent cells modulate their absolute-expression profiles. Intron 3 generates an in-frame premature termination codon that predicts the synthesis of a truncated c-Fos protein. This prediction was evaluated by immunoaffinity chromatography purification of c-Fos proteins.

Conclusion: We demonstrate that: (i) The c-fos-2 transcript is ubiquitously synthesized either in vivo or in vitro, in amounts that are higher or similar to those of mRNAs coding for other Fos family members, like FosB, DeltaFosB, Fra-1 or Fra-2. (ii) Intron 3 confers to c-fos-2 an outstanding destabilizing effect of about 6-fold. (iii) Major determinant of c-fos-2 steady-state levels in cultured cells is its remarkably high rate of synthesis. (iv) Rapid changes in the synthesis and/or degradation rates of both c-fos transcripts in serum-stimulated cells give rise to rapid and transient changes in their relative proportions. Taken as a whole, these findings suggest a co-ordinated fine-tune of the two c-fos transcript species, supporting the notion that the alternative processing of the precursor mRNA might be physiologically relevant. Moreover, we detected a c-Fos immunoreactive species corresponding in mobility to the predicted truncated variant.

Figure 1

RT-PCR amplicons from c-fos and fosB transcripts. A) Schematic drawing of c-fos and fosB gene structures. Exons (E) are boxed and shaded in grey. Introns (I) are indicated by lines between exons, except for the alternatively spliced sequences that are indicated by brick filled in boxes. The beginning and end of exons are indicated by the corresponding nucleotide positions (NCBI/GeneBank accession numbers: V00727 and AF093624 for c-fos and fosB, respectively). B) Names, sequences and 5'-position of upper (U) and lower (L) primers. C) Agarose (1.5%) gel electrophoresis analysis of RT-PCR products generated by different primer pairs (identified at the top of each line). Total RNA from NIH 3T3 cells was used as template. Forty cycles of PCR were performed as detailed [16]. Genomic DNA was further amplified by the E1U-E2L primer pair to exclude the possibility that our PCR conditions were not optimal for the amplification of the longest theoretical fragment, i.e. the 933 nt amplicon that should be observed if intron 1 remained unspliced in the c-fos transcript population [see Additional file 7]. The molecular weight marker was a 100-nt ladder from Roche.

Figure 2

Organ-, embryo-, and cell type-associated differences in basal amounts of c-fos transcripts. Data are the numbers of transcript molecules per pg of total RNA. Regarding cultured cells, error bars (SEM) indicate transcript level variability among 13 (NIH 3T3) or 8 (Hepa1-6) independent cultures. Total RNA (pool of ~200 BALB/c mice) from animal organs and whole embryos were from Clontech. No error bars are shown for quantitations of these commercial samples of total RNA. E17 embryos are in late gestation (total of about 19 days). The percentage of c-fos-2 in total transcript amount (c-fos-2 plus spliced c-fos) is indicated for each of the sample examined.

Figure 3

Time-courses of c-fos transcripts in mouse kidney, lung and liver in response to AmD. Male BALB/c mice (see legend of Table 1) were intraperitoneally injected with 2 mg kg^-1 body weight of AmD dissolved in phosphate-buffered saline. Animals injected with phosphate-buffered saline served as vehicle controls. Total RNA was extracted at the indicated times. Data are the means of c-fos (triangles) or c-fos-2 (circles) molecules/pg of total RNA ± SEM (n = 3 mice). Data at 0 min represent the mean values ± SEM of 5 control mice. No time-related effect was noted in these vehicle controls. Some error bars are not visible because of small standard errors. Statistical significance was evaluated using analysis of variance followed by post hoc multiple comparison according to the Student-Newman-Keuls method. Significant differences relative to control animals are indicated by filled-in symbols. For each transcript, the maximal fold increment is given in parentheses.

Figure 4

Rates of transcript decay and synthesis in cultured cells. A) Decay rates of the c-fos-2 (open symbols) and c-fos (solid symbols) transcripts in NIH 3T3 (circles) and Hepa1-6 (triangles) cells. Data are the percentages of transcript molecules remaining after the addition of AmD. The half-life (t_1/2) values calculated from the resulting decay lines are given for comparisons. B) Rates of transcript decay and synthesis in NIH 3T3 cells. Transcript synthesis rates (in molecules/min) were calculated from the basal amounts of transcript molecules (per pg of total RNA) and their estimated half-lives (in minutes). These calculations are based on the assumption that though mRNA degradation is a complex process, it follows first-order kinetics[36]. The steady-state levels and rates of decay and synthesis of transcripts coding for both types of FosB protein (fosB and ΔfosB) and for heme oxygenase 1 (ho1), thioredoxin 1 (trx1) and superoxide dismutase 3 (sod3) are included for comparison.

Figure 5

Changes in transcript decay upon translation inhibition. Decay rates of c-fos-2 (circles) and c-fos (triangles) transcripts were determined as in Fig. 4, excepting that half of the plates were pretreated for 15 min with Cx (10 μg/ml). Then, AmD was added to all the plates and transcript decay was followed in the presence (solid symbols) and absence (open symbols) of Cx.

Figure 6

Time-course of c-fos (left) and fosB (right) transcript levels in serum-stimulated NIH 3T3 cells. Data are the means of transcript molecules per pg of total RNA ± SEM from independent cultures. Some error bars are not visible because of small SEM.

Figure 7

RT-PCR analysis of cytoplasmic and nuclear RNAs. Cytoplasmic (C) and nuclear (N) RNAs were isolated by using the PARIS™ Kit (Ambion) according to the manufacturer's protocol. RT-PCR conditions were as in Fig. 1. The RT-PCR product of 120 nt amplified by the E3U-I3L primer pair (see Fig. 1) indicates the presence of the c-fos-2 transcript in both cytoplasmic and nuclear fractions. Amplification of the unspliced fosB transcript (I4U-I4L primers) was included for comparison. Transcript coding for glutathione peroxidase 1 was amplified as control of efficient cellular fractionation. Primer U_GPX1 (5'-GCAGAAGCGTCTGGGACCTCGTG) was located in gpx1 exon 1, and primer L_GPX1 (5'-GGGAATTCAGAATCTCTTCATTCT TGCCA) in gpx1 exon 2 (the intron between both exons is 218 nt long). U_GPX1- L_GPX1 primers generated a single RT-PCR product of 101 nt when using the cytoplasmic RNA as template, indicating no detectable contamination with nuclear gpx1 pre-mRNA. In contrast when using the nuclear RNA as template, a fragment 319 nt long was detected in addition to the expected product from spliced gpx1 mRNA. Since the Ambion's PARIS™ Kit is designed for the isolation of both RNA and protein from the same sample, efficient cellular fractionation was further confirmed by Western blot analysis (botton panel) with an Ab specific for the cytoplasmic glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). GAPDH was present only in the cytoplasmic fraction, further indicating no detectable contamination with the nuclear fraction.

Figure 8

Detection of c-Fos immunoreactive species. A) Nucleotide sequence of c-fos intron 3 and flanking exons 3 and 4. Lower case letters refer to intronic nucleotides and upper case letters to exonic nucleotides. Minimal functional sequences within the mCRD are underlined. The encircled triplets encode the aa residues that contact to DNA. Triplets encoding two out of the five-leucine residues of the LZ motif are indicated in italics. The stop codon in intron 3 is boxed and shaded in grey. B) Western blot analysis of c-Fos proteins purified by immunoaffinity chromatography. A crude extract from NIH 3T3 cells stimulated with serum for 30 min was loaded onto the immunoaffinity column. The column eluate (E-30), as well as the input crude extract (CE-30) and the crude extract from serum-starved (CE-0) cells were subjected to inmunoblot analysis as described under "Methods". The positions of molecular size standards are indicated on the left in kDa. GAPDH was used for loading control.