Our work provides insight in the role of yLuc7p in splicing, which we find remarkably similar to that of Nam8p. We previously showed (13
) that Nam8p, a U1 snRNP component, binds the pre-mRNA during commitment complex formation in a region directly downstream of the 5′ splice site, where it stabilizes pre-mRNA–U1 snRNP interaction and helps in the formation of commitment complexes. We also showed that changes in the pre-mRNA sequence introduced in the non-conserved region contacted by Nam8p produced a striking Nam8p-dependent effect in splice site recognition and selection. Thus, Nam8p by stabilizing U1 snRNP–pre-mRNA interaction modulates splicing (13
). We now propose that yLuc7p acts in a similar way. yLuc7p contacts non-conserved sequences in the exon, and in the specific context of our synthetic constructs, in a region within 23 nt from the cap. We also showed that this contact occurs mainly through its first zinc finger. Interestingly, similarly to what happens with Nam8p, changes in the non-conserved pre-mRNA sequence bound by yLuc7p affect splice site selection, and this effect is dependent on the presence of its first zinc finger too. Fortes et al.
) showed that extracts from yLuc7p mutant strains display defects in all steps of splicing in vitro
, and these defects can be rescued by adding recombinant yLuc7p. They also showed that yLuc7p mutants exhibit reduced splicing activity in vivo
, and that yLuc7p is required for CBC–U1snRNP interactions. Based on our data, and in agreement with the data from Fortes et al.
), we propose a model where yLuc7p contacts the pre-mRNA in a region close to the cap, where it is more likely to establish an interaction with CBC. yLuc7p acts by binding the pre-mRNA upstream the 5′ splice site and stabilizing pre-mRNA–U1 snRNP interaction in commitment complexes. Therefore, yLuc7p could directly or indirectly interact with CBC and mediate the CBC effect in splice site selection. Zhang and Rosbash (14
) showed that another U1snRNP component, U1-C, contacts the pre-mRNA and stabilizes its interaction with the U1snRNA. Altogether, yLuc7p, U1-C, Nam8p, CBC and maybe other U1 snRNP proteins (ySnp1p/U1-70K, SmD1, SmD3 and Snu56p) (14
) would interact with the pre-mRNA and produce a network of protein–RNA interactions keeping the pre-mRNA stably bound to the U1 snRNP.
yLuc7p has two zinc finger motifs. We show that the first one cross-links the pre-mRNA and it is required for yLuc7p splicing activity. What is then the role for the second zinc finger? The U1 snRNP particle purified from a yLuc7p mutant strain appears completely disrupted, missing several proteins and it is inactive in splicing (17
). It is possible that yLuc7p acts as a bridge between the pre-mRNA and the U1 snRNA through its two zinc fingers, the first one binding to the pre-mRNA and the second one binding to U1 snRNA. We tried to generate a yeast strain lacking the second zinc finger but we did not succeed. Diploid cells integrated the mutation but after sporulation none of the spores harboring the deletion were viable. This indicates that the integrity of the second zinc finger is necessary for viability. Perhaps it is required to keep a minimal structure of the U1 snRNP. It is also possible that the second zinc finger contributes to stabilize globally the snRNP–pre-mRNA interaction by cooperatively helping the first zinc finger bind the pre-mRNA. Further experiments will be necessary to clarify this point.
Three U1 specific proteins co-purify with human U1 snRNP (U1-A, U1-C and U1-70k) and all three have counterparts in yeast U1 snRNP. In contrast, yeast U1 snRNP has seven additional proteins (Snu71p, Snu65p, Snu56p, Prp39p, Prp40p, Nam8p and yLuc7p). However, a human homolog has been described only for Nam8p, the apoptotic factor TIA-1 (19
). Here we demonstrate that hLuc7A is a new splicing factor, homolog to yeast yLuc7p. Human hLuc7A is a nuclear protein expressed in several human cell lines ( and ). Importantly, antibodies against hLuc7A specifically precipitate U1 snRNA (), indicating that it is a bona fide
U1snRNP component. Interestingly, hLuc7A affects splice site selection by activating splicing from the distal 5′ splice site (). Supporting our results, a recent report identified the new SR protein, SRrp53, as a protein interacting with hLuc7A (36
). In addition, hLuc7A was purified with the supraspliceosome, a macromolecular complex involved in pre-mRNA splicing (37
). Therefore, we conclude that hLuc7A is a new splicing factor.
hLuc7 has three isoforms (hLuc7A, hLuc7B1 and hLucB2) derived from different genes, suggesting that it could be tissue specific or developmentally regulated. ESTs analysis shows that the pre-mRNA for hLuc7A undergoes alternative splicing, although we do not know yet the biological meaning of this variability. It is possible that hLuc7B1 and hLuc7B2 are also regulated by alternative splicing. The abundance of isoforms present in mammalian cells is remarkable when compared to the simplicity of the yeast system. This underscores the degree of complexity in the regulation of splicing in mammalian systems.
hLuc7A has a C-terminal tail rich in Asp, Ser and Arg repeats. These repeats are present in the splicing factors known as SR proteins where they have been shown to act as protein–protein interaction modules, or to influence RNA–RNA interactions. The SR repeats in hLuc7A could act by recruiting other splicing factors to the pre-mRNA, or by stabilizing U1snRNP–pre-mRNA interactions. The fact that hLuc7A does not co-purify with human U1 snRNP (38
) indicates that it remains loosely associated to this particle. This situation allows for more flexibility in the regulation of splice site selection. Thus, hLuc7A could first bind the pre-mRNA independently from U1 snRNP and, perhaps through interaction with U1-70k (via their RS domains) or by direct binding to U1 snRNA, could subsequently recruit the U1 snRNP to form the E-complex. In this sense, hLuc7A would act in a similar way than TIA-1, the human homolog of yeast Nam8p (19
). Interestingly, human and yeast Luc7p share 50% identity in the zinc finger region suggesting that the mechanism of action might be similar. More experiments will be necessary to confirm this hypothesis.