Negative autoregulation is an effective mechanism for homeostatic control of gene expression. SF2/ASF is an abundant and highly conserved RNA-binding protein with multiple functions and oncogenic potential, whose expression level needs to be precisely controlled for normal cell physiology. Post-transcriptional regulation of splicing factors can be complex, involving multiple layers of control. For example, PTB antagonizes the expression of its paralog, nPTB, by promoting an NMD-targeted alternative splicing isoform, and possibly also by inhibiting translation of correctly spliced mRNA through an unkown mechanism36,37
. During neuronal differentiation, PTB expression is repressed by the neuron-specific miR-124, resulting in increased nPTB protein37
. nPTB expression is also repressed during myoblast differentiation by the muscle-specific miR-13338
. Our study shows that multiple levels of post-transcriptional and translational control are likewise involved in fine-tuning SF2/ASF expression.
We identified and characterized six alternatively spliced mRNA isoforms of SF2/ASF in HeLa cells, of which isoforms IV and VI are not shown in the UCSC or ENSEMBL browsers. The major isoform, I, encodes full-length protein, and has a long 3′UTR25,26
. Isoform II, which retains the third intron, was previously reported25,39
. A third isoform was also described in these studies, involving an alternative 3′ splice site in the third intron. We used a specific primer to amplify that isoform, but did not detect it in the cell lines we tested. Isoforms II and III retain the third intron, which changes the reading frame and results in a stop codon shortly after exon 3; this would result in a truncated protein without the C-terminal RS domain. However, we found that these two isoforms are retained in the nucleus, and are therefore not translated. This explains why our SF2/ASF antibody, which recognizes an epitope near the N-terminus, fails to detect any smaller protein isoforms by Western blotting7
In general, intron-containing pre-mRNAs are retained in the nucleus, and only mature mRNAs are exported to the cytoplasm, preventing translation of incompletely processed messages40
. Interestingly, isoform IV retains one intron, compared to isoform V, and it remains nuclear; however, the major isoform I retains that plus one additional intron, but somehow is compatible with efficient nuclear export, which might involve potential RNA cis-acting elements that are recognized as export signals. Many retroviruses and some cellular mRNAs, such as Tap, employ this mechanism40,41
Isoforms III, IV, V, and VI are generated by splicing that removes one or two introns in the 3′UTR. Among these, isoforms V and VI are exported to the cytoplasm and accumulate after cycloheximide treatment, suggesting that they are NMD targets. Isoform V encodes the same full-length protein as isoform I, whereas isoform VI encodes a truncated protein lacking the RS domain. SF2/ASF overexpression upregulates the unproductive isoforms III and VI, and decreases the protein-encoding major isoform I, but only modestly. Quantitation of the changes at the mRNA and protein levels indicates that alternative splicing associated with NMD or nuclear retention only partly explains the autoregulation of SF2/ASF.
By co-transfecting a V5-tagged genomic SF2/ASF construct with a T7-tagged SF2/ASF cDNA, we recapitulated the autoregulation seen with endogenous SF2/ASF. Co-transfection experiments with different mutants showed that RRM2 is required, and the 3′UTR is the only critical cis-element for the regulation. The length of the 3′UTR affects basal expression, but is not responsible for autoregulation.
Post-transcriptional regulation is frequently mediated by RNA-protein interactions in the UTRs42
, and this is also where the two UCRs are located in SF2/ASF ()20-23
. We tried to map cis-element(s) required for downregulation, but were unable to narrow them down to well-defined sequences. First, when the 3′UTR was divided into four fragments, three still showed downregulation by SF2/ASF overexpression (Supplementary Fig. 5
). Second, when each of the functional fragments was further subdivided, each subfragment gave much less or no repression (not shown). It appears that multiple elements in the 3′UTR are involved in SF2/ASF autoregulation, and the signals are dispersed and partially redundant. The roles of the two UCRs remain unclear, especially considering that the entire 3′UTR of SF2/ASF is ~95% conserved between human and mouse.
A recent quantitative-proteomics study showed that each miRNA has hundreds of target genes, but individual genes are only modestly repressed by a single miRNA43
. Therefore, several miRNAs might target multiple regions in this 3′UTR, with their combined action resulting in downregulation. However, the experiments with Dicer-disrupted or -knockout cell lines suggest that miRNA-mediated repression is not the main mechanism of SF2/ASF autoregulation, although it may contribute to some extent. Indeed, miR7 was recently found to reduce SF2/ASF levels through a single binding site in the 3′UTR (Jun Zhu, personal communication).
Using a sucrose-gradient assay, we found that SF2/ASF overexpression reduces the translational efficiency of an SF2/ASF-3′UTR-containing mRNA reporter. However, we could not recapitulate the translation inhibition by adding purified SF2/ASF protein to a cell-free translation system. Possible reasons for this include: i) a component(s) required for translation inhibition might be lost during extract preparation; ii) SF2/ASF does not repress translation directly, but could instead affect alternative splicing of a translational regulator; iii) the substrate for translational regulation might be a 3′UTR in the form of mRNP generated by a defined pathway, involving transcription, processing, and export.
Translation is a cytoplasmic event, but surprisingly, a nuclear-retained version of SF2/ASF was still able to autoregulate (). Perhaps nuclear SF2/ASF affects the mRNP composition of its own transcript, which in turn affects how efficiently it is translated in the cytoplasm. Nuclear events often determine the downstream cytoplasmic fate of mRNAs44
. It is also possible that SF2/ASF regulates translational control indirectly through its nuclear functions, such as splicing of a putative translational regulator's pre-mRNA. Finally, nuclear retention of the SF2/ASF-NRS variant might be slightly leaky. However, SF2/ASF can enhance translation of reporter mRNAs in a binding-site-dependent manner, which can be recapitulated in the cell-free system6
; this effect, which is reproducible in our hands (not shown), requires the shuttling activity of SF2/ASF, and the nuclear-retained mutant is no longer active6
Our experiments with viral IRES elements suggest that SF2/ASF translational autoregulation is cap-independent, and that eIF2 and/or eIF3 are important, although the exact mechanism remains unknown. On the other hand, SF2/ASF enhances cap-dependent translation by repressing the activity of 4E-BP, an inhibitor of eIF4E, and no enhancement was observed for IRES-dependent translation45
. Therefore, we believe that these two opposite effects of SF2/ASF in translation involve distinct mechanisms, and are not contradictory.
A recent study showed that SF2/ASF binds to its own transcript within the second UCR in the cytoplam, and enhances polysome association46
. Although we observed neither translational repression nor activation by in vitro
translation of a reporter with the SF2/ASF 3′UTR, it is possible that the long 3′UTR mediates complex positive as well as negative regulation, and that different mechanisms are dominant depending on the context.
SF2/ASF autoregulation is a complex process involving multiple mechanisms operating at different levels. We found that both alternative splicing and translation have contributing roles, and SF2/ASF translation itself may be negatively regulated at different steps by different factors. Multi-level regulation presumably serves to control SF2/ASF homeostasis more precisely. The relative contribution of each control mechanism might vary in different tissues or physiological states. Conversely, particular control mechanisms may be disrupted in different tumors associated with SF2/ASF upregulation11