Recently, we identi®ed proteins that co-purify with the human spliceosome using mass spectrometry. One of the identi®ed proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5 + gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step. We also show that this complex is required for the second catalytic step of pre-mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly. The ®rst catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex. The puri®ed CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identi®ed. This work reports a ®rst puri®cation and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least ®ve additional protein factors. Keywords: CDC5L/mass spectrometry/pre-mRNA splicing/spliceosome IntroductionNuclear pre-mRNA splicing is the process by which the introns (intervening sequences) in the primary transcript are speci®cally removed and the coding sequences joined to form mature mRNA, which is subsequently transported into the cytoplasm for protein synthesis. Splicing takes place in the nucleus, via a two-step transesteri®cation mechanism, and this is catalysed by a large RNA±protein complex termed the spliceosome. The major subunits of spliceosomes are the U1, U2, U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs), each of which contains the corresponding snRNA and a set of snRNP proteins (reviewed by Kra Èmer, 1996;Will and Lu Èrhmann, 1997). In addition, other non-snRNP protein splicing factors are also required for spliceosome formation and splicing (Will and Lu Èrhmann, 1997;Staley and Guthrie, 1998). Spliceosome assembly occurs in a pathway that involves the sequential binding of snRNP and protein splicing factors to conserved intron sequences to form the active complex. This is a multistep process and separate ATP-dependent complexes designated A, B and C have been identi®ed in vitro (reviewed by Reed and Palandjian, 1997). The C complex contains the active spliceosome while the A and B complexes contain assembly intermediates. The detailed roles played by many spliceosomal proteins in the pre-mRNA splicing mechanism are not yet fully understood.The protein composition of mammalian spliceosomes has been studied using complexes puri®ed from HeLa nuclear extracts by a combination of gel ®ltration and af®nity chromatography (Reed, 1990;Bennett et al., 1992). More recently in our laboratories, a large-scale analysis of spliceosome-associated proteins has been carried out using mass spectrometry and database searches (Neubauer et al., 1998). One...

A crucial step in transcription is the recruitment of RNA polymerase to promoters. In the transcription of human rRNA genes by RNA Polymerase I (Pol I), transcription factor SL1 has a role as the essential core promoter binding factor. Little is known about the mechanism by which Pol I is recruited. We provide evidence for an essential role for hRRN3, the human homologue of a yeast Pol I transcription factor, in this process. We find that whereas the bulk of human Pol I complexes (I alpha) are transcriptionally inactive, hRRN3 defines a distinct subpopulation of Pol I complexes (I beta) that supports specific initiation of transcription. Human RRN3 interacts directly with TAF(I)110 and TAF(I)63 of promoter-selectivity factor SL1. Blocking this connection prevents recruitment of Pol I beta to the rDNA promoter. Furthermore, hRRN3 can be found in transcriptionally autonomous Pol I holoenzyme complexes. We conclude that hRRN3 functions to recruit initiation-competent Pol I to rRNA gene promoters. The essential role for hRRN3 in linking Pol I to SL1 suggests a mechanism for growth control of Pol I transcription.

Transcription of the ribosomal RNA genes (rDNA) that encode the three largest ribosomal RNAs (rRNA), is mediated by RNA Polymerase I (Pol I) and is a key regulatory step for ribosomal biogenesis. Although it has been reported over a century ago that the number and size of nucleoli, the site of ribosome biogenesis, are increased in cancer cells, the significance of this observation for cancer etiology was not understood. The realization that the increase in rRNA expression has an active role in cancer progression, not only through increased protein synthesis and thus proliferative capacity but also through control of cellular check points and chromatin structure, has opened up new therapeutic avenues for the treatment of cancer through direct targeting of Pol I transcription. In this review, we discuss the rational of targeting Pol I transcription for the treatment of cancer; review the current cancer therapeutics that target Pol I transcription and discuss the development of novel Pol I-specific inhibitors, their therapeutic potential, challenges and future prospects.

Efficient transcription elongation from a chromatin template requires RNA polymerases (Pols) to negotiate nucleosomes. Our biochemical analyses demonstrate that RNA Pol I can transcribe through nucleosome templates and that this requires structural rearrangement of the nucleosomal core particle. The subunits of the histone chaperone FACT (facilitates chromatin transcription), SSRP1 and Spt16, copurify and co-immunoprecipitate with mammalian Pol I complexes. In cells, SSRP1 is detectable at the rRNA gene repeats. Crucially, siRNA-mediated repression of FACT subunit expression in cells results in a significant reduction in 47S pre-rRNA levels, whereas synthesis of the first 40 nt of the rRNA is not affected, implying that FACT is important for Pol I transcription elongation through chromatin. FACT also associates with RNA Pol III complexes, is present at the chromatin of genes transcribed by Pol III and facilitates their transcription in cells. Our findings indicate that, beyond the established role in Pol II transcription, FACT has physiological functions in chromatin transcription by all three nuclear RNA Pols. Our data also imply that local chromatin dynamics influence transcription of the active rRNA genes by Pol I and of Pol III-transcribed genes.

Knowledge of the role of components of the RNA polymerase I transcription machinery is paramount to understanding regulation of rDNA expression. We describe key findings for the roles of essential transcription factor SL1 and activator upstream binding factor (UBF). We demonstrate that human SL1 can direct accurate Pol I transcription in the absence of UBF and can interact with the rDNA promoter independently and stably, consistent with studies of rodent SL1 but contrary to previous reports of human SL1. UBF itself does not bind stably to rDNA but rapidly associates and dissociates. We show that SL1 significantly reduces the rate of dissociation of UBF from the rDNA promoter. Our findings challenge the idea that UBF activates transcription through recruitment of SL1 at the rDNA promoter and suggest that the rate of pre-initiation complex (PIC) formation is primarily determined by the rate of association of SL1, rather than UBF, with the promoter. Therefore, we propose that SL1 directs PIC formation, functioning in core promoter binding, RNA polymerase I recruitment, and UBF stabilization and that SL1-promoter complex formation is a necessary prerequisite to the assembly of functional and stable PICs that include the UBF activator in mammalian cells.RNA polymerase (Pol) 1 enzymes themselves have no intrinsic ability to recognize and bind specifically to promoter DNA sequences, so pre-initiation complex formation in transcription calls for the recruitment of the Pol enzymes to the promoter via transcription factors. Basal transcription factors and (co-)activators of transcription cooperate in this function in eukaryotes where three classes of highly related enzymes, Pol I, Pol II, and Pol III, catalyze the transcription of specific sets of genes. With few exceptions, a complex of TBP and TBP-associated factor (TAF) proteins is required for the accurate initiation of transcription by all three polymerases (1). The particular complement of TAFs in each complex, although variable in Pol II transcription, is specific to each class of genes. There is evidence that binding of certain TAF subunits to TBP precludes the binding of TAFs from a different class (2). The precise roles of the TBP-TAF complexes in mediating a specific interaction between the polymerases and their respective promoters are distinct.In mammalian Pol II transcription, the TBP-TAF complex TFIID can bind at the promoter by virtue of the specific interaction of TBP with TATA boxes and the specific interactions of the TAF proteins with other promoter sequences, whereupon Pol II and other factors are recruited to form the pre-initiation complex (PIC) (3). The initial phases of mammalian Pol III transcription from the different types of Pol III promoters converge on the recruitment of the TBP-TAF complex TFIIIB and the Pol III enzyme in formation of the PIC (4, 5). Loading of promoter type-specific TFIIIB at the promoter DNA occurs with the assistance of the specific DNA binding capabilities of TFIIIA and TFIIIC or TFIIIC alone or the multisubunit compl...

Cyclin D1 expression represents one of the key mitogenregulated events during the G 1 phase of the cell cycle, whereas Cyclin D1 overexpression is frequently associated with human malignancy. Here, we describe a novel mechanism regulating Cyclin D1 levels. We find that SNIP1, previously identified as a regulator of Cyclin D1 expression, does not, as previously thought, primarily function as a transcriptional coactivator for this gene. Rather, SNIP1 plays a critical role in cotranscriptional or posttranscriptional Cyclin D1 mRNA stability. Moreover, we show that the majority of nucleoplasmic SNIP1 is present within a previously undescribed complex containing SkIP, THRAP3, BCLAF1, and Pinin, all proteins with reported roles in RNA processing and transcriptional regulation. We find that this complex, which we have termed the SNIP1/SkIPassociated RNA-processing complex, is coordinately recruited to both the 3 ¶ end of the Cyclin D1 gene and Cyclin D1 RNA. Significantly, SNIP1 is required for the further recruitment of the RNA processing factor U2AF65 to both the Cyclin D1 gene and RNA. This study shows a novel mechanism regulating Cyclin D1 expression and offers new insight into the role of SNIP1 and associated proteins as regulators of proliferation and cancer. [Cancer Res 2008;68(18):7621-8]

Ribosomal RNA gene transcription by RNA polymerase I (Pol I) is the driving force behind ribosome biogenesis, vital to cell growth and proliferation. The key activator of Pol I transcription, UBF, has been proposed to act by facilitating recruitment of Pol I and essential basal factor SL1 to rDNA promoters. However, we found no evidence that UBF could stimulate recruitment or stabilization of the pre-initiation complex (PIC) in reconstituted transcription assays. In this, UBF is fundamentally different from archetypal activators of transcription. Our data imply that UBF exerts its stimulatory effect on RNA synthesis, after PIC formation, promoter opening and first phosphodiester bond formation and before elongation. We provide evidence to suggest that UBF activates transcription in the transition between initiation and elongation, at promoter escape by Pol I. This novel role for UBF in promoter escape would allow control of rRNA synthesis at active rDNA repeats, independent of and complementary to the promoter-specific targeting of SL1 and Pol I during PIC assembly. We posit that stimulation of promoter escape could be a general mechanism of activator function.

The assembly, disassembly, and functional properties of transcription preinitiation complexes (PICs) of human RNA polymerase I (Pol I) play a crucial role in the regulation of rRNA gene expression. To study the factors and processes involved, an immobilized-promoter template assay has been developed that allows the isolation from nuclear extracts of functional PICs, which support accurate initiation of transcription. Immunoblotting of template-bound factors showed that these complexes contained the factors required to support initiation of transcription, SL1, upstream binding factor (UBF), and Pol I. We have demonstrated that, throughout a single round of transcription, SL1 and UBF remain promoter bound. Moreover, the promoterbound SL1 and UBF retain the ability to function in transcription initiation. SL1 has a central role in the stable association of the PIC with the promoter DNA. The polymerase component of the PIC is released from the promoter during transcription yet is efficiently recycled and able to reinitiate from "poised" promoters carrying SL1 and UBF, since the PICs captured on the immobilized templates sustained multiple rounds of transcription. Kinetic analyses of initiation of transcription by Pol I revealed that Pol I-dependent transcription is rate limited in a step subsequent to recruitment and assembly of Pol I PICs. The rate of RNA synthesis is primarily determined by the rates at which the polymerase initiates transcription and escapes the promoter, referred to as promoter clearance. This rate-limiting step in Pol I transcription is likely to be a major target in the regulation of rRNA gene expression.