A fraction of the transcription factor TAF15 participates in interactions with a subset of the spliceosomal U1 snRNP complex

Abstract

RNA/ssDNA-binding proteins comprise an emerging class of multifunctional proteins with an anticipated role in coupling transcription with RNA processing. We focused here on the highly related transcription factors of the TET sub-class: TLS/FUS, EWS and in particular the least studied member TAF15. An extensive array of immunoprecipitation studies on differentially extracted HeLa nuclei revealed the specific association of TAF15 with the spliceosomal U1 snRNP complex, as deduced by the co-precipitating U1 snRNA, U1-70 K and Sm proteins. Additionally, application of anti-U1 RNP autoantibodies identified TAF15 in the immunoprecipitates. Minor fractions of nuclear TAF15 and U1 snRNP were involved in this association. Pull-down assays using recombinant TAF15 and U1 snRNP-specific proteins (U1-70 K, U1A and U1C) provided in vitro evidence for a direct protein–protein interaction between TAF15 and U1C, which required the N-terminal domain of TAF15. The ability of TAF15 to directly contact RNA, most likely RNA pol II transcripts, was supported by in vivo UV cross-linking studies in the presence of α-amanitin. By all findings, the existence of a functionally discrete subset of U1 snRNP in association with TAF15 was suggested and provided further support for the involvement of U1 snRNP components in early steps of coordinated gene expression.

Introduction

It is becoming increasingly clear that an extensive network of interactions between structurally distinct macromolecular assemblies coordinates and couples all events of mRNA metabolism in higher eukaryotes [1], [2]. The interactions involve factors of the transcriptional machinery, as well as of the post-transcriptional apparatus that refer to 5′ capping, splicing and polyadenylation steps acting co-transcriptionally [3], [4]. In particular, the coupling between transcription and splicing has been extensively analyzed and shown to involve a network of interactions between components of the basal transcriptional machinery and a large macromolecular assembly, the spliceosome [5], [6].

With respect to the transcriptional machinery, special reference is made to the role of TFIID complex, composed of the TATA-binding protein (TBP) in association with general and specific transcription factors (TAFIIs) [7], [8], as well as to the RNA pol II and, in particular, the carboxy-terminal domain (CTD) of its largest subunit [9]. As for the splicing factors, these refer to an extensive array of RNA-binding proteins, including members of the SR and hnRNP protein family for both basal and alternative splicing events, as well as to the spliceosomal U-snRNP (uridine-rich small nuclear ribonucleoprotein) complexes (U1, U2, U4, U5 and U6), all being important constituents of the spliceosome [10], [11]. Each U-snRNP complex is composed of a single, relatively stable snRNA component in the range of 107–196 nucleotides and, with the exception of the U6 snRNP, a common core of seven proteins, called Sm proteins (D1, D2, D3, E, F, G and the splice variants B/B′), and few unique protein species. The Sm proteins are bound to a conserved single-stranded U-rich region (the Sm site) which is present in U1, U2, U4 and U5 snRNA. With respect to U1 snRNP complex, in addition to the 164 nucleotide long U1 snRNA and Sm proteins, it includes three specific polypeptides; U1A, U1C and U1-70 K. Of the latter, the U1A and U1-70 K bind directly to the U1 snRNA while U1C can only bind to the U1 snRNP particle when U1-70 K and Sm proteins are already associated with the U1 snRNA [12 and references therein]. Several protein components of the spliceosomal U-snRNP complexes are specific and frequent targets of autoantibodies detected in the sera of patients with systemic rheumatic diseases (most common, anti-Sm and anti-U1 RNP autoantibodies). During spliceosome formation, U1 snRNP is the first to associate with the pre-mRNA at the 5′ splicing site, a very critical early step in spliceosome assembly. Afterwards, U2 binding at the vicinity of the 3′ splice site and subsequent entrance of U4/U5/U6 tri-complex follows [10], [11], [12].

A specific class of multifunctional proteins with a potential to act in coupling transcription and splicing are those with ability to bind both RNA and ssDNA. These refer to some known transcription factors that include members of the TET sub-class, as well as splicing factors and hnRNP proteins [13]. The precise role of individual RNA/ssDNA binding proteins in establishing a specific network of interactions is currently not fully explored. The TET proteins, TLS/FUS, EWS and TAF15 (formerly named TAFII68) are relatively abundant nuclear species. They have a highly conserved domain structure that includes the presence of a zinc finger motif, an RNA-binding domain (RBD/RRM) and RGG regions that also participate in RNA binding, characteristic features of multifunctional proteins capable of interacting with both DNA and RNA [14], [15]. An important feature of the TET family members is their participation in chromosomal translocations that lead to expression of fused proteins. The N-terminal part of TLS/FUS, EWS and TAF15 alike can fuse to the DNA-binding domain of specific transcription factors (e.g. CHOP, FLI1, ERG) giving rise to oncoproteins with novel strong transcriptional activator/repressor properties, characteristic of particular cancer cell types [16], [17]. TLS/FUS has been identified as the hnRNP P2 protein, an associated component of hnRNP complexes, important players of the post-transcriptional machinery [18]. Currently, a diverse list of biological roles is ascribed to TLS/FUS that includes its functioning in DNA repair, genomic integrity, regulation of transcription and RNA splicing [19]. In contrast, relatively little is yet known concerning the role of the related EWS and, in particular, TAF15.

TAF15 represents a relatively new TET family member initially characterized as a putative RNA/ssDNA binding protein that was considered to be a specific transcription factor with a suggestive role in transcription initiation and/or elongation [20], [21]. It has been detected in stable association with a subset of TFIID (both TBP-free and TBP-containing) complexes, as well as with RNA pol II [20]. Distinct TFIID complexes containing TAF15, TLS/FUS or EWS proteins have been reported, indicating that these proteins have related but discrete functional properties [21]. As pointed out before [20], about 5–10% of the cellular TAF15 is associated with TBP-containing complexes, thereby raising the question of the extent of its participation in additional nuclear functions. This has led to the speculation that TAF15 might participate, in addition to the pre-initiation complex (PIC) formation with distinct TFIID complexes, in the elongating RNA pol II complex, as well as in a number of protein–RNA interactions with the growing nascent mRNA transcripts [20], [21].

We have conducted studies aiming at defining novel nuclear interactions of TAF15 in human (HeLa) cells, in particular with components of the post-transcriptional apparatus. Recently, we have contributed in a study showing the interaction of TAF15 with the spliceosomal U1 snRNA, in the absence of U1 snRNP specific protein components [22]. We now present new findings that demonstrate the ability of TAF15 to form a complex with a minor fraction of the spliceosomal U1 snRNP complex, as well. Our evidence was based on the application of an extensive range of immunoprecipitation assays (IPs) on nuclear fractions obtained under different extraction protocols, showing the co-precipitation of TAF15 and U1 snRNP specific components (namely, U1 snRNA, U1-70 K and Sm proteins). Moreover, TAF15 was shown to form protein–protein interactions with the U1C protein in in vitro pull-down assays employing purified protein components, and to make direct association with RNA in vivo. Overall, the present study provided additional information on the multifaceted participation of U1 snRNP components in the interplay of factors operating in transcriptional and post-transcriptional events in higher eukaryotes.