Here, we demonstrate that an intron silencer sequence, ras ISS1, acts as a negative regulator of IDX inclusion, mainly by preventing its 3′SS from being properly recognized, an effect that is clearly seen in the single-intron substrates, where ras ISS1 inhibits splicing of intron D1 completely (Fig. ) but not the splicing of intron D2. Interestingly, rasISS1 is conserved from mice to humans (Fig. ). By means of nuclear extract depletion and add-back experiments, hnRNP A1 has been identified as one factor mediating the rasISS1 negative effect. The amino-terminal form of hnRNP A1, UP1, containing the RNA-binding domain, formed two defined bands when interacting with rasISS1 (Fig. ), suggesting that two binding sites for A1 might be present in this RNA. Some RNA-binding sequences for hnRNP A1 have been previously described, namely, CUAGACUAGA in the ESS and AUAGAAGAAGAA in the Janus regulator of HIV tat exon 2 (10
); UACCUUUAGAGUAGG in the ISS of human hnRNP A1 pre-mRNA, which modulates 5′SS selection (17
); UUAGAUUAGA in the mouse hepatitis virus RNA transcription regulatory region (44
); and UAGGGCAGGC in an ESS in K-SAM exon of human FGF receptor 2 (23
). It is remarkable that together with the SELEX winner sequence identified previously (8
), all of these hnRNP A1-binding sequences contain unique or tandem repeats of the sequence UAG(G/A) at their core, unlike the rasISS1 core sequence reported in this work (GGCAGUGAGGGAGGCGAGGG). The whole rasISS1 sequence, however, rendered a gel shift interaction with recombinant hnRNP A1 (Fig. ), most likely through the binding to the three AGG motifs, which match three residues of the core A1 binding sequence. It is therefore clear that the spectrum of RNA sequences binding to A1 is broad, and only functional studies like these are capable of identifying new hnRNP A1 interactive elements.
To date, there have been three proposed mechanisms for inhibition by hnRNPs: (i) a direct competition for overlapping enhancer-binding sites that are recognized by stimulatory factors, which prevent SS recognition (48
); (ii) the binding of hnRNPs to several sites around or within a silenced exon, followed by dimerization of the bound proteins, which might cause relevant portions of the pre-mRNA to loop out, which would make them unavailable for splicing, as happens in hnRNP A1 pre-mRNA (3
) and the FGF receptor 2 pre-mRNA (14
); and (iii) initial binding of hnRNP A1 to a high-affinity site on an exon and then nucleating the cooperative assembly of inhibitory hnRNP complexes that coat the pre-mRNA. In this final case, the cooperative binding of hnRNP A1 molecules interferes directly with the initial steps of spliceosome assembly or antagonizes the action of nearby enhancers (49
). In our c-H-ras
system, although deletion of the whole rasISS1 sequence results in efficient splicing of the 1N substrate, we cannot exclude the possibility that in the complete pre-mRNA, some overlapping between enhancer and inhibitory sites might be present. In fact, hnRNP H has two putative binding sequences within rasISS1 (GGGA), indicating possible competition between hnRNPs H and A1 for this site. In addition, and although we have not detected binding of hnRNP A1 to other sites surrounding or within exon IDX, the formation of a stable stem-loop trapping IDX by rasISS1 cannot be completely ruled out.
We have shown that SC35 and SRp40 can antagonize the inhibitory action of hnRNP A1 on IDX inclusion, presumably through binding to ESEs within IDX. The consensus heptamer GUUC(G/C)AG described previously (16
) as putative binding sites for SC35 is observed in IDX (as GCUCCAG). Further, the functional consensus GRYY(C/A)CYR described by Liu et al. (45
) is also observed at three different IDX positions (GACCCCCC, GACCCAUG, and GGCCCCUC). The heptamer consensus UGGGAGC described by Tacke et al. (57
) as a putative binding site for SRp40 is also observed at three different IDX positions (UGGGACC, CGGGACC, and UGUGACC). Two distinct models have been proposed for the stimulation of splicing by ESE elements. While early studies supported a U2AF recruitment model according to which ESE-bound SR proteins facilitate U2AF recruitment to the polypyrimidine tract and 3′SS by RS domain-mediated interactions (31
), another study suggests that the primary function of ESE elements is to somehow antagonize the effect of a juxtaposed splicing silencer (4
). From our study and others (18
), both functions could be present. SR proteins could stimulate the assembly of splicing factors and thus increase the splicing efficiency (in a RNA binding-dependent or -independent manner, therefore affecting 1N-ISS1Δ23 pre-mRNA), and as seen for the 1N substrate, they could bind to an ESE and counteract a nearby splicing repressor complex consisting of at least hnRNP A1. Our results, then, reveal the first mammalian gene which can be submitted to specific action of the antagonistic regulation of A1 and SR proteins.
We have also isolated three more proteins, namely, FUS/TLS, hnRNP H, and the ATP-dependent RNA helicase p68, that are linked to rasISS1 and/or IDX RNAs and which may be bridging the intronic and exonic sequences. FUS/TLS (also known as hnRNP P2) has been connected to RNA maturation (64
), among other nuclear processes. Its consensus RNA-binding sequence found by SELEX contains a conserved GGUG motif (43
), which is not present in either IDX or rasISS1. This points to a degeneration in its RNA binding sequences (as is common for other hnRNPs) or, alternatively, to an interaction with IDX mediated by other proteins. Interestingly, FUS/TLS has been found to bind to SC35 (64
) and also hnRNP A1 (67
), which we have demonstrated to be implicated in the regulation of IDX inclusion. hnRNP H was recently shown to regulate the inclusion of a regulated exon in the env
gene of HIV-1 in combination with SC35 and other SR proteins, through its binding to a GGGA motif (11
). In addition to the GGGA sequences in rasISS1, three other GGGA motifs are present in the IDX, suggesting that hnRNP H might play an important role in the alternative splicing of the IDX. Finally, p68 is a well-characterized ATP-dependent ATPase and RNA helicase, but its function and substrates are not well understood. A recent work has identified p68 as the unwinding factor necessary to destabilize U1snRNA-5′SS interaction prior to the first catalytic step of splicing (46
), but until now, no involvement of p68 helicase in alternative splicing regulation has been reported. Therefore, as far as we know, we have for the first time described a function for p68 in splicing control. Accordingly, it has also been shown that the closely related p72 RNA helicase plays a role in alternative splicing regulation of CD44 pre-mRNA (34
). The mechanism by which p68 helicase can inhibit IDX recognition and splicing remains obscure. One important observation is that rasISS1 exerts its function in the presence of IDX; when another exonic sequence like c-H-ras
exon 3 or exon 4a replaces IDX, rasISS1 is not active. In addition, we have demonstrated that the sequence IDXISS1, but not IDX or ISS1 separately, is capable of restoring splicing efficiency in vitro when present in competing amounts, reinforcing the plausibility of a secondary structure encompassing both the exonic and intronic regions. In this context, it is tempting to hypothesize the existence of a strong binding site for a helicase which could stabilize and destabilize the structure and thereby exert its function in splicing regulation. One attractive hypothesis is that IDX and rasISS1 might form several secondary structures that regulate this alternative splicing, with p68 helicase then being a factor that unwinds a more active secondary structure to a less active secondary structure (in terms of IDX inclusion).
Further analysis of the action and interplay between all of these regulatory factors and secondary structure elements will be necessary to elucidate the fine regulation of IDX inclusion in mature transcripts and, consequently, the expression of the alternative protein p19H-Ras.