We have identified the AML-associated protein DEK as a factor that interacts in vitro and in vivo with SR proteins involved in pre-mRNA splicing. DEK associates efficiently with splicing complexes through interactions mediated by SR proteins. Its association with a pre-mRNA is stimulated by the presence of an ESE that binds to SR family and SR-related proteins. Moreover, its association with a pre-mRNA is stimulated by the addition of purified SR proteins to an SR protein-depleted reaction. After splicing, DEK remains associated preferentially with exon-product complexes. Importantly, this interaction requires prior splicing since DEK does not associate with an exon-product RNA that has not been generated by splicing. The results indicate that interactions involving DEK may distinguish a transcript that has been through splicing from a transcript that has not. Moreover, they suggest that DEK could function to communicate between splicing and one or more steps downstream in gene expression.
Increasing evidence indicates that the nuclear history of a transcript can influence its subsequent metabolism. For example, it has been demonstrated in many studies that the presence of an intron is important for efficient gene expression. These intron-mediated effects have been attributed to influences on mRNA stability, transport and translation. For example, prior splicing is important for translation-dependent degradation of mRNA by the nonsense-mediated decay (NMD) pathway (reviewed by Maquat 1995
, Maquat 1996
; Hentze and Kulozik 1999
). In a recent study, it was shown that an exon-product RNA generated by splicing assembles into a complex that is significantly larger and more efficiently exported to the cytoplasm than the complex formed on the equivalent RNA synthesized from an intron-less transcript (Luo and Reed 1999
). The presence and location of an intron, whether at the 5′ or 3′ end of a transcript, can also influence its translation in the cytoplasm. For example, Xenopus
maternal mRNAs transcribed in vivo are normally translationally masked, but can be relieved from translational repression by the insertion of an intron at the 5′, but not at the 3′ end, of a transcript (Matsumoto et al. 1998
). All of the above observations indicate the existence of factors that package an mRNP dependent on its passage through the spliceosome and that modulate downstream steps in gene expression. However, the factors that specifically tag mRNPs dependent on prior splicing are not known.
hnRNPs such as hnRNP-A1 are candidate factors for influencing the composition and activity of an mRNP dependent on prior splicing. HnRNP-A1 associates with nascent pre-mRNA, influences splicing activity, and associates with spliced mRNA in the nucleus and cytoplasm; it has also been detected in association with translationally engaged mRNA (Mayeda and Krainer 1992
; Visa et al. 1996
). However, neither hnRNP-A1 or other hnRNP proteins have been detected in purified spliceosomes and it is also not known whether their association with mRNPs is dependent on prior splicing. In contrast, it has been demonstrated that specific SR proteins, including the SRm160/300 splicing coactivator subunits and ASF/SF2, associate with splicing complexes through both steps of the splicing reaction and remain preferentially bound to the exon-product RNA (Blencowe et al. 1998
, Blencowe et al. 2000
; Hanamura et al. 1998
). Moreover, a Chironomas tentans
SR family protein, hrp45, which is related to mammalian ASF/SF2, associates with intron-containing Balbiani ring transcripts and remains bound to spliced mRNP until the time the mRNP is translocated through the nuclear pore complex, upon which it appears to be released (Alzhanova-Ericsson et al. 1996
). Very recently, Le Hir and colleagues have used an elegant cross-linking strategy to detect proteins that cross-link at or near spliced exon junctions, dependent on prior splicing. In this study, splicing-dependent cross-links were identified for SRm160, the U5 snRNP protein hPRP8/p220, and several unidentified proteins (Le Hir et al. 2000
). The results in the present study are consistent with these findings. In particular, since the association of DEK with pre-mRNA is promoted by interactions with SR proteins, it is possible that its association with spliced exons is mediated by SRm160. The results further suggest that interactions involving factors that define mRNP composition occur as early as the commitment stage of splicing complex formation, when U1 snRNP and SR family proteins bind to the pre-mRNA and facilitate the association of factors including SRm160/300 and DEK with the substrate (Blencowe et al. 1998
; present study).
The results of the present study, taken together with a previous finding indicating that DEK associates with the pets site within the HIV-II promoter (Fu et al. 1997
; see Introduction), reveals an interesting relationship with a growing number of factors that are associated with both RNA processing and malignant transformation. For example, it has been reported that certain isoforms of the Wilm's tumor protein (WT-1) bind to DNA at specific promoter sequences and concentrate within transcription factor domains in vivo, whereas other isoforms specifically associate with splicing components, including snRNPs and the 65-kD subunit of the U2 snRNP auxiliary factor (U2AF-65), and concentrate within splicing factor domains in the nucleus (Larsson et al. 1995
; Davies et al. 1998
). In another case, the oncoprotein TLS/FUS, which becomes fused to the CHOP protein in liposarcomas, or to the ERG protein in a subset of myeloid leukemias, binds to both DNA and RNA and has recently been identified as hnRNP-P2, a component of hnRNP complexes (Calvio et al. 1995
). Intriguingly, also similar to DEK, it has been reported that TLS/FUS interacts with SR protein splicing factors (Yang et al. 1998
). Another factor with dual RNA and DNA binding properties is the Ets-related transcription factor Spi-1/PU.1. This factor, besides its role as a hematopoietic-specific transcription factor, has been reported to interact with both TLS/FUS and the polypyrimidine tract-binding protein NonO/p54nrb, and to influence splicing activity (Hallier et al. 1996
, Hallier et al. 1998
). NonO/p54nrb, which also has the ability to bind to both DNA and RNA, and a closely related protein, PSF (the polypyrimidine binding protein–associated splicing factor), were both identified recently in fusions with the transcription factor TFE in papillary renal cell carcinomas (Clark et al. 1997
). Thus, all of the RNA processing-associated factors implicated in different human malignancies to date, like DEK, have associations with transcription components, as well as with pre-mRNA processing. While the significance of these similar features is not known, it is tempting to speculate that malignancies involving alterations to DEK and these other proteins could affect functions in transcription, RNA processing, or perhaps interactions that coordinate these steps in gene expression (see Introduction). However, it is also possible that AMLs involving the DEK/CAN fusion involve an alteration to the normal function of CAN (Fornerod et al. 1995
). Further work will be required to determine the normal function of DEK and whether its function is altered by fusion to CAN in AML.
The results in the present study suggest that DEK has a function in association with pre-mRNA metabolism. In particular, its association with spliced exons dependent on prior splicing represents one of the first examples of a protein with this property. It is possible that this splicing-dependent association is important for facilitating one or more steps downstream in gene expression that are influenced by the prior removal of an intron in pre-mRNA. Thus, based on the previous and present findings, DEK is a candidate for mediating interactions between transcription and pre-mRNA processing, as well as between splicing and one or more subsequent splicing-dependent steps in the gene expression pathway.