Splicing of the c-src N1 exon is controlled by a splicing enhancer sequence in the downstream intron. The conserved core of this sequence (called the DCS) assembles a complex of regulatory proteins. We previously identified two proteins in this complex, hnRNP F and KSRP, and showed that they were needed for N1 splicing in vitro. We have now isolated a third major component of the DCS complex and identified it as hnRNP H. hnRNP H is needed for efficient splicing of the N1 exon in vitro and is likely to play a critical role in the assembly of the DCS complex. Thus, one cellular function for hnRNP H is in regulating alternative splicing patterns. This does not preclude other functions.
We isolated hnRNP H by Mono Q and anti-Sm (Y12) affinity chromatography. Since antibody Y12 had not previously been shown to bind hnRNP H, we thought it likely that hnRNP H coimmunoprecipitated with Sm proteins in the Mono Q peak fraction. However, Western blot analysis of the Mono Q fraction with antibody Y12 detected none of the standard Sm proteins. Instead, a band with the same mass as hnRNP H was observed (data not shown). After cloning the protein, we confirmed that hnRNP H cross-reacts with antibody Y12 by immunoprecipitation of the in vitro-translated hnRNP H. Interestingly, the in vitro-translated hnRNP F was not immunoprecipitable with antibody Y12 (data not shown). The Sm snRNPs share a sequence motif that could serve as the epitope to antibody Y12 (27
). However, we have not found this motif in the hnRNP H sequence, and thus the basis for the antibody Y12 reactivity is still not clear.
We previously showed that hnRNP F is in the DCS complex by using a monoclonal antibody to specifically precipitate hnRNP F cross-linked to the DCS RNA (48
). This antibody also had reactivity to hnRNP H by Western blot analysis (44
). However, this hnRNP H reactivity was much weaker than with hnRNP F, which may explain why we did not immunoprecipitate the H protein and identify it previously.
After comparing the sequences of hnRNPs F and H, we raised an antibody to the unique C-terminal peptide of hnRNP H. This antibody is specific to hnRNP H by both Western blotting and immunoprecipitation assays. Using this hnRNP H antibody, we depleted the hnRNP H and not hnRNP F from the nuclear extract. The immunodepleted nuclear extract was significantly reduced in N1 splicing. The addition of recombinant hnRNP H to the depleted extract restored the splicing activity, indicating a role for hnRNP H in N1 exon splicing. Because both the depletion of hnRNP H and the inhibition of splicing were only partial, there are limits to the interpretation of this experiment. We cannot say that hnRNP H is required for any splicing activity, only that it is needed for full activity. The residual splicing after the hnRNP H depletion does not likely result from the highly related hnRNP F protein substituting for hnRNP H, as addition of hnRNP F to the hnRNP H-depleted extract did not restore activity. Moreover, the residual 18% of the hnRNP H remaining in the depleted extract is still in excess of the pre-mRNA in the in vitro splicing reaction, and this depletion produces a fourfold reduction in splicing. It thus seems likely that hnRNP H is indeed essential to the reaction and that a complete depletion, if it were achieved, would show a complete inhibition of splicing.
Although they are very similar in protein sequence (78% identical), several studies have revealed functional distinctions between hnRNP F and hnRNP H. Phorbol ester treatment of cultured cells strongly down-regulates the expression of hnRNP F but not the expression of hnRNP H (30
). Yeast two-hybrid screening identified an interaction between hnRNP F and the nuclear cap-binding protein complex (CBC). Subsequent gel mobility shift assays showed that CBC-RNA complexes bind preferentially to hnRNP F over hnRNP H (22
). These authors also showed that partial immunodepletion of hnRNP F led to partial inhibition of splicing in vitro. Far-Western blotting analysis identified hnRNP F, but not hnRNP H, as interacting with transportin 1, a mediator of nucleocytoplasmic transport for certain hnRNPs (55
). Studies of the rat β-tropomyosin gene transcript have implicated hnRNP H in the regulation of alternative splicing in that system (26a
). These results imply important activity differences between the two proteins H and F.
Given that hnRNP H can bind directly to hnRNP F, hnRNPs H and F may at times function as a heterodimer. Gel mobility shift results indicate that both hnRNP F and hnRNP H are present in the DCS complex simultaneously. The complex is known to contain F and yet can be completely supershifted by the hnRNP H-specific antibody. Determining the stoichiometry and interactions of each protein in the DCS complex will be important in identifying any functional differences between these two highly homologous proteins.
The DCS is 33 nucleotides long and is composed of at least three different functional elements, GGGGGCUG, CUCUCU, and UGCAUG (14
). Additional elements outside the DCS are also required for enhancer function. The G tract in the DCS is likely to be the binding site for hnRNP H, since hnRNP H is known to bind tightly to poly(rG) (44
). A similar element GGGGGAUG is also present upstream of the DCS, and G-rich elements have been identified within several other intronic splicing regulatory elements (11
). In the chicken β-tropomyosin pre-mRNA, UV cross-linking experiments identified a 55-kDa protein binding to intronic (A/U)GGG enhancer elements that activate alternative splicing (61
). It is not clear yet whether this protein is hnRNP H.
Spliceosome assembly is a very dynamic process. The interactions between the different components of the splicing apparatus change at the different steps of assembly (64
). Some interactions with the pre-mRNA are transient, occurring only at a specific step in the pathway. It has been shown that many hnRNPs that initially bind to the pre-mRNA in splicing extract are displaced upon assembly of the spliceosome (63
). However, immunoprecipitation of splicing products and intermediates with anti-hnRNP H antibody indicates that hnRNP H is present in the spliceosome of the src
pre-mRNA. This enhancer protein evidently maintains its interaction with the src
pre-mRNA throughout splicing.
So far, we have identified hnRNP F, hnRNP H, and KSRP as components of the DCS complex, responsible for activating N1 exon splicing. All of these proteins are non-cell-type specific factors. This is not unexpected, as the enhancer has some activity in nonneural cells (50
). However, the enhancer is stronger in neuronal cells, and in vitro the assembly of the full-sized DCS complex is specific to neuronal extract. It is not clear what causes the neuron-specific assembly of the complex. The availability of recombinant hnRNPs F and H and KSRP will allow us to develop assays for the cooperative assembly of these proteins into a splicing enhancer complex. Ultimately, one would like to examine how these proteins interact with each other to assemble onto a specific RNA sequence and how the assembled enhancer complex interacts with the spliceosome.