In this study we used an affinity purification assay to isolate human FCP1 interacting partners. We demonstrated that, in addition to RAP74, the FCP1-affinity materials contained the RNAPII, suggesting the presence of the FCP1/TFIIF/RNAPII complex in vivo
. Our data are in agreement with recent purification of a similar complex FCP1/TFIIF/PolII from S.pombe
). Our data suggest that FCP1 interacts with both RNAPIIo and RNAPIIa forms. The association of FCP1 with the hypophosphorylated RNAPII is consistent with the previous findings showing that FCP1 is a component of the mammalian RNAPII holoenzyme (8
). Likely, the association of FCP1 with RNAPII that is not engaged in transcription may prevent phosphorylation of the CTD by CTD-kinases. On the other hand, it has been recently shown that a transcription-independent CTD dephosphorylation occurs in X.laevis
, suggesting that free hyperphosphorylated RNAPII is a bona fide substrate for FCP1 activity (12
). Finally, it has recently shown that FCP1 is associated with polymerase during transcription elongation in vivo
). Thus, it appears that FCP1 has the inherent ability to interact with RNAPII irrespectively from the CTD-phosphorylation status.
By mass spectrometry of affinity purified FCP1-associated factors, we identified a novel FCP1-interacting protein, named MEP50. MEP50, together with PMRT5 and pICln, constitutes the 20S methylosome complex, which is responsible for diarginine methylation of the Sm protein and it is required for snRNPs assembly. FCP1 co-immunoprecipitation analysis supported the specific interaction between FCP1 and MEP50. We demonstrate that FCP1 and MEP50 complex is present into the nucleus, thus it represents a distinct entity from the 20S methylosome. Moreover, we did not detect any specific association between FCP1 and the dimethylarginine PMRT5 component of the 20S methylosome. Conversely, we found evidence that FCP1 interacts with Sm proteins, common components of the snRNPs. Because MEP50 binds the Sm proteins, we hypothesize that association between FCP1 and Sm protein might be mediated by MEP50. It has been suggested (16
) that the WD repeat protein MEP50 may provide a platform for multiple protein interactions. However, because FCP1 associates with RNAPII and RNAPII has been recently reported to associate the spliceosome complex (23
), it is conceivable if not likely, that interaction between FCP1 and common components of the spliceosome complex might be mediated by RNAPII. Moreover, we found no evidence of specific association between MEP50 and RNAPII by immunoprecipitation experiments (data not shown).
Other mammalian protein phosphatases have been described to have a role in pre-mRNA splicing (24
and references therein). Even if there is currently only limited information on the spliceosomal substrates of these protein phosphatases it is known that several splicing factors as the SF2/ASF and SAP155 undergo phosphorylation/dephosphorylation events during the process of splicing (25
). In contrast with other phosphatase, FCP1 is the first example of a phosphatase that is involved in transcription elongation.
Our findings add further support to the concept that there is functional intercommunication between the transcription and splicing machineries, and the RNAPII-CTD appears to play a pivotal role in coordinating transcription and pre-RNA processing (13
and references therein). Splicing factors have been detected in association with a transcriptionally active ‘holoenzyme’ containing polymerase. Furthermore, the evidence for reciprocal stimulation between splicing and transcription machineries has been reported (28
). The TAT-SF1 protein, able to interact with the P-TEFb CTD kinase, was shown to bind specifically splicing factors and this association positively influences transcription (28
). Interestingly, another snRNA, the 7SK RNA, was shown to associate with the P-TEFb complex and negatively modulates transcription (19
). Using the in vivo
cross-linking/chromatin immunoprecipitation assays it has been shown that FCP1 phosphatase cross-links to promoter and coding regions, suggesting that the FCP1 is associated with the elongating polymerase. Moreover, mutations in the FCP1 lead to increase levels of CTD-Ser2 phosphorylation (15
). Thus, it appears that FCP1 may regulate transcription elongation by modulating the levels of CTD-Ser2 phosphorylation. The ability of FCP1 to associate and to dephosphorylate the RNAPII CTD at specific stage of transcription elongation, and to interact in vivo
with MEP50, a protein associated with the common Sm factors, suggests the possibility that FCP1 might play a regulatory role in the coordination of transcription elongation and pre-mRNA splicing process. Alternatively, association between FCP1 and MEP50 might lead to de-phosphorylation of MEP50 or other components associated to MEP50. Clearly, additional studies are required to elucidate the functional significance of this association.