depicts a recently proposed model for the regulation of alternative splicing of pre-mRNA H-Ras in which hnRNP A1 and p68 act as inhibitors and SC35 and SRp40 as stimulators of IDX inclusion 
. In this model, there are two distinguishing features driving IDX inclusion: (1) the silencer sequence (rasISS1) downstream of IDX that contains a hnRNP A1-binding site; and (2) the IDX itself also harbors putative sequences that can bind SC35 and SRp40. Previously, studies using RNA affinity columns containing either IDX, rasISS1 or the mutant ΔrasISS1 (see ) in presence of nuclear extract showed that p68, hnRNP H and FUS bound to IDX and rasISS1, but not to ΔrasISS1 
. Previous in vivo
studies found that RNAi-mediated depletion of p68 increased the level of endogenous p19 mature mRNA, thereby revealing a role for p68 RNA helicase as an inhibitor of IDX inclusion 
. Here we show that the addition of recombinant p68 to in vitro
splicing reactions () inhibits splicing of intron D1, (lanes 2, 3 and 4) and reverts the activation of SRp40 (compare lanes 1 to 2, 3 and 4) confirming previous results obtained in vivo 
RNA folding predictions suggested a stem-loop secondary structure comprising IDX and rasISS1 sequences, see 
and , that is conserved between hamster, mouse, rat and human 
. Interestingly, the addition of the stem-loop RNA sequence to in vitro
splicing reactions enhanced IDX inclusion 
, suggesting that the reaction can be activated by titrating out inhibitory factors that recognize this structure. To gain additional insights into the function of this putative stem-loop structure, we tested whether both individual IDX and rasISS1 sequences wind together in vitro
. clearly shows that double-stranded (dsRNA) structures were obtained for IDX and rasISS1 in RNA native gels (see lanes 2–5), but, very interestingly, not for the ΔrasISS1 mutant (lanes 6–9), thereby indicating that the“Δ” sequence is essential for the formation of this dsRNA structure. We next studied whether the IDX-rasISS1-linked sequence winds and forms a dsRNA in vitro
and found that the IDX-rasISS1 sequence wound in a dsRNA structure (, lane 1) that was rapidly reverted to ssRNA in the presence of p68 (lanes 2, 3 and 4). This finding demonstrates that IDX-rasISS1 winds in a dsRNA structure that is recognized and can be unwound by p68, pointing to a role for this secondary structure in the efficient inclusion of the alternative exon IDX.
P68 RNA helicase unwinds IDX-rasISS1 in vitro.
The intrinsic nature of the binding of hnRNP H and FUS/TLS to IDX and rassISS1 sequences was further studied using binding-shift assays. GST-fused hnRNP H (GST-H) showed a binding-shift in native gels in the presence of the rasISS1 sequence, reflecting the formation of high molecular weight complexes (, left lanes 2, 3 and 4), but not in the presence of ΔrasISS1 (, lanes 6, 7 and 8). Since hnRNP H showed no binding-shift with the IDX sequence (, lanes 10–12) we conclude that its previously reported association to IDX RNA-affinity 
columns is due to an indirect binding or it is a weak interaction that does not survive gel resolution in binding shift assays. Our studies further demonstrate that rasISS1 forms a protein-RNA complex in nuclear extract (, lane 5) that is stabilized and supershifted with anti-hnRNP H antibodies (lanes 2, 3 and 4), thereby indicating that hnRNP H is present in this rasISS1 complex and that it is highly accessible to the antibody. HnRNP H also showed a binding-shift with the stem-loop IDX-rasISS1 (, lanes 4–6) that was weakened by the unwinding activity of p68 (see lanes 7–9). Moreover, crosslinking assays between labeled hnRNP H and IDX-rasISS1, in the absence or presence of p68 under splicing conditions in vitro,
further confirmed that hnRNP H and IDX-rasISS1 directly bind (see band at around 50 kDa: , lane 2) and that this binding is inhibited in the presence of p68 (, lanes 3–7). Collectively, data from the binding-shift and cross-linking assays indicate that (1) hnRNP H directly binds to rasISS1 and that the “Δ” sequence is required for this binding (, lanes 1–8); (2) hnRNP H does not directly bind to the IDX sequence and therefore (), as it was seen to bind in previous IDX-RNA-affinity column studies 
, we propose that hnRNP H indirectly binds to IDX via at least one other IDX-bound protein in the complex. A second explanation is that this binding is weak and does not survive gel resolution in binding shift assays; (3) hnRNP H was seen to directly bind to the stem-loop IDX-rasISS1 that involves a dsRNA stem-loop structure (, lanes 4–9); and (4) p68 regulates hnRNP H-stem-loop binding since p68 reduces this interaction ().
Although FUS/TLS was found to bind to IDX and to rasISS1 RNA-affinity columns, this protein showed no binding-shift to any IDX or rasISS1 sequence (, right). These results indicate that FUS/TLS should therefore indirectly bind to IDX and rasISS1 through other IDX-bound proteins. Again, a second explanation is that the binding may be weak and does not survive gel resolution in gel shift assays. Furthermore, in vitro splicing assays showed that GST-FUS stimulates intron D1 splicing (, lanes 5 and 6), whereas GST-H did not (lanes 2, 3 and 4).
To better define the roles of hnRNP A1, hnRNP H, FUS/TLS and p68 in regard to p19 as well as other genes, we analyzed the effect of depleting these four splicing factors by means of RNAi-mediated depletion assays to determine how (1) endogenous p19 abundance is affected and (2) the expression of other genes is regulated. The RNAi-mediated depletion of the four splicing factors were obtained with specific shRNA oligonucletides (, lanes 2–5) using HeLa cell extracts. Previously, we have shown that RNAi-mediated depletion of p68 increases p19 mRNA levels 
. In this study, we found that RNAi-mediated depletions of FUS/TLS or hnRNP H decreased the p19 protein level as seen by Western blot quantification (see , respectively); a finding that corroborates with our observation that the efficient splicing of intron D1 is proportional to the FUS/TLS level (). No change in p19 mRNA abundance was seen during the RNAi-mediated depletion of hnRNP A1 albeit different interfering sequences were used (result not shown). As hnRNP A1 was previously reported to downregulate p19 
, we suggest that we did not observe this effect for two main reasons: (1) the level of hnRNP A1 is high and the remaining amount of hnRNP A1 is enough to maintain the level of p19 mRNA; and (2) other hnRNP proteins similar to A1, (e.g. A2/B1 and A3 have 57% and 85% homology, respectively), can also participate in and therefore compensate for lower levels of A1 in regards to regulation of alternative splicing of H-Ras pre-mRNA.
RNAi-mediated depletion of FUS and hnRNP H downregulates p19 H-Ras expression.
The RNA derived from the four RNAi-mediated depletion assays of the splicing factors in HeLa cells were individually incubated in microarrays containing 19,000 human ESTs. From the results obtained, we sorted the genes exhibiting modified expression into five groups showed in Table S1
. Group I contains genes whose expression varies with both the RNAi-mediated depletion of hnRNP A1 and p68, but not with the RNAi-mediated depletion of FUS/TLS and hnRNP H. Groups II to V have genes whose expression only varies with the RNAi-mediated depletion of one of the four splicing factors, but not with the RNAi-mediated depletion of the other three. Interestingly, only the ZNF462
gene was found to increase its expression in the presence of RNAi-mediated depletion of hnRNP A1 and p68. ZNF462 is a zinc finger protein that has been implicated in agenesis of the corpus callosum 
, a common brain anomaly. We suggest that ZNF462 and p19 H-Ras could be coordinately regulated by hnRNP A1 and p68. Microarrays analyzing the RNAi-mediated depletion of hnRNP A1 showed an increase and decrease of PDCD4
and BAG4 mRNA
levels, respectively; the proteins of which are thought to have anti-apoptotic effects 
. For the RNAi-mediated depletion of FUS/TLS, the microarray data showed a decrease in the tyrosine protein kinase EGFR
mRNA level; EGFR
gene mutations have been observed in lung cancers 
. Moreover, RNAi-mediated depletion of hnRNP H also produced a significant decrease in the ARAF, CABLES and PCBP4
mRNA levels. All three proteins are known to be involved in cell proliferation 
. Interestingly, the knockdown of p68 downregulated TOB2
that is known to be an endogenous activator of pro-proliferative pathways 
Since the SR proteins SC35 and SRp40 are known to enhance p19 H-Ras alternative splicing 
, we also analyzed whether the RNAi-mediated depletion of hnRNP A1, FUS/TLS, hnRNP H and p68 affect SR proteins mRNA levels. Selected results of these studies are provided in . Note that other SR proteins that showed no variation were not included in the table. The mRNA levels of SC35 and SRp40 showed no variation with any of the RNAi-mediated depletions here studied; however, interestingly, SRp20 and SFRS14 mRNAs were downregulated with RNAi-mediated depletion of p68. Although SC35 was not regulated by any of these RNAi-mediated depletions, a SR protein that specifically binds to SC35, namely SFRS2IP, was upregulated by RNAi-mediated depletion of hnRNP A1, and to a lesser extent by the RNAi-mediated depletions of FUS/TLS and hnRNP H. These findings suggest that these three proteins may regulate a feedback loop involving SC35 via SFRS2IP.
Log2 is the log2 value of the fold change measuring the effect of the RNAi on the ESTs expresión as compared with the negative control performed with the empty pSuper vector.
Using RNAi-mediated depletion assays, we next analyzed how the RNAi-mediated depletions of p68, hnRNP A1, hnRNP H and FUS/TLS affect SC35 localization inside the cell. We found that only RNAi-mediated depletion of p68 alters SC35 localization (, panel -p68) by increasing SC35 levels outside of the interchromatin granule cluster (IGC) in a diffuse nuclear pattern. This suggests that RNAi-mediated depletion of p68 can induce SC35 to be localized similar to that seen in actively transcribing cells where both splicing and transcription factors display a highly dynamic localization within the nucleus and may be recruited from speckles to the position of active transcription 
. Moreover, despite not affecting SC35 expression, the downregulation of p68 may increase the level of SC35 protein available for the splicing machinery, such as for p19 H-Ras alternative splicing.
Downregulation of p68 RNA helicase alters the dynamic localization of the SC35 splicing factor.
Whether p68 modifies the dynamic localization of other speckles was also studied in RNAi-mediated depletion assays. RNAi-mediated depletion of p68 was observed not to affect the localization of B” (U2snRNP), Coilin and SMN speckles; however, when p68 was knocked down, SMN bodies were more susceptive to anti-SMN antibodies (, compare SMN panels). Finally, RNAi-mediated depletion of p68 did not stimulate cell apoptosis ( PARP panels and 5C), nor did it activate the dsRNA-activated protein kinase PKR, since eIF2-α was not hyperphosphorylated (not shown).
Collectively, we have found that p68 regulates p19 H-Ras splicing by (1) affecting the dsRNA structure of the stem-loop IDX-rasISS1 (), (2) disrupting binding of hnRNP H to the stem-loop IDX-rasISS1 () and (3) may increase the level of SC35 protein available for the splicing machinery, such as for p19 H-Ras alternative splicing (). Moreover, (4) knockdown of either hnRNP A1, FUS/TLS or hnRNP H () led to upregulation of SFRS2IP, a SC35-binding protein. Currently, studies are underway to decipher whether SFRS2IP exerts an effect on IDX inclusion. Finally, (5) although FUS/TLS showed no direct binding to the stem-loop IDX-rasISS1 ( C, right), assays have indicated that FUS stimulated IDX inclusion ( and ). (6) Now, we know that hnRNP H binds to the stem-loop IDX ras-ISS1 (), but so far our in vitro splicing studies have not thrown light on its action as regards IDX inclusion. However, as the p68 (an inhibitor of IDX inclusion) weakened hnRNP H binding to the stem-loop (), it is likely that hnRNP H is necessary to stimulate IDX inclusion. This latter reasoning agrees with the in vivo downregulation of p19 by hnRNP H knock-down ( C).
There are to date only a few studies reporting on the synchronized action of all the splicing factors studied here. Interestingly, RNA helicase A was also found to associate with hnRNP proteins including hnRNP H 
. Additionally, RNAi-mediated depletion studies revealed that by suppressing the expression of RNA helicase A, the nuclear distribution of hnRNP C was altered 
. Van Herreweghe et al.
recently showed that a fraction of HeLa 7SK snRNA specifically interacts with RNA helicase A and heterogeneous nuclear ribonucleoprotein A1 
. Moreover, p68 was found to shuttle in and out of SC35 domains, forming fibers and granules in a cell-cycle dependent manner 
. Functional analysis demonstrated that SC35 and TASR, a FUS-associated protein with SR repeats, have antagonistic effects on adenovirus E1A pre-mRNA splicing and abrogate the influence of FUS on this splicing. Interestingly, protein-protein assays have revealed that FUS is also associated with helicases 
. The hnRNP A1 and SR proteins have also been reported to have antagonistic functions in splicing processes 
. In addition, a recent NMR study showed the competition between proteins SC35, SRp40 and heterogeneous nuclear ribonucleoprotein A1 at the HIV-1 Tat exon 2 splicing site 
All the factors implicated so far in the regulation of the alternative splicing of H-Ras seem to also be involved in regulating many other splicing processes and to act in concert. An observation that serves to emphasize that the alternative splicing model studied here is highly helpful in advancing our understanding of the interactions of all these splicing factors. This enhanced understanding will better enable us to define cis-element sequences and trans-acting factors as well as to determine their interactions in generating future models for other exon splicing processes. The number of cis-element sequences and trans-acting factors implicated in the alternative splicing of H-Ras pre-mRNA, as well as RNA secondary structures, indicates how complex and fine-tuned the regulation of this gene is. The pivotal role of p68 in the regulation of such an important signal transduction pathway further underlines the need to better understand winding/unwinding activities in cancer processes.