The global shortening of mRNAs through alternative polyadenylation (APA) that occurs during enhanced cellular proliferation represents an important, yet poorly understood mechanism of regulated gene expression1,2. The 3′UTR truncation of growth promoting mRNA transcripts that relieves intrinsic microRNA- and AU-rich element-mediated repression has been observed to correlate with cellular transformation3; however, the importance to tumorigenicity of RNA 3′ end processing factors that potentially govern APA is unknown. Here, we have identified CFIm25 as a broad repressor of proximal poly(A) site usage that, when depleted, increases cell proliferation. Applying a regression model on standard RNA-seq data for novel APA events, we identified at least 1,450 genes with shortened 3′UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNA in HeLa cells. Dramatic increases in expression of several known oncogenes including Cyclin D1 are observed as a consequence of CFIm25 depletion. Importantly, we identified a subset of CFIm25-regulated APA genes with shortened 3′UTRs in glioblastoma (GBM) tumors that have reduced CFIm25 expression. Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumor size while CFIm25 overexpression reduces these properties and inhibits tumor growth. These findings identify a pivotal role of the CFIm25 in governing APA and reveal a previously unknown connection between CFIm25 and glioblastoma tumorigenicity.
CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcripts. The FF repeat domains of CA150 bind directly to the phosphorylated carboxyl-terminal domain of the largest subunit of RNAPII. We determined that this interaction is required for efficient CA150-mediated repression of transcription from the ␣ 4 -integrin promoter. Additional functional determinants, namely, the WW1 and WW2 domains of CA150, were also required for efficient repression. A protein that interacted directly with CA150 WW1 and WW2 was identified as the splicing-transcription factor SF1. Previous studies have demonstrated a role for SF1 in transcription repression, and we found that binding of the CA150 WW1 and WW2 domains to SF1 correlated exactly with the functional contribution of these domains for repression. The binding specificity of the CA150 WW domains was found to be unique in comparison to known classes of WW domains. Furthermore, the CA150 binding site, within the carboxyl-terminal half of SF1, contains a novel type of proline-rich motif that may be recognized by the CA150 WW1 and WW2 domains. These results support a model for the recruitment of CA150 to repress transcription elongation. In this model, CA150 binds to the phosphorylated CTD of elongating RNAPII and SF1 targets the nascent transcript.A complex array of general transcription factors, DNA binding activators and repressors, and a multitude of coregulators mediate transcription in eukaryotes (32,48,49). Regulation of the frequency of transcription initiation is a well-documented means of controlling gene expression (32,48,64), but many genes are also controlled by modulation of the ability of RNA polymerase II (RNAPII) to elongate transcripts (11, 15-17, 37, 53, 82, 93). Although the mechanisms controlling RNAPII elongation efficiency are not completely understood, it is clear that the interplay of multiple protein factors regulates RNAPII elongation efficiency (21,70,75). Some of these elongation factors have been identified, and they belong to two classes: positive transcription elongation factors (P-TEFs), such as positive elongation factor b (P-TEFb) (38,47,67,68), and negative transcription elongation factors (N-TEFs), such as DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) (28,84,90). In addition to trans-acting elongation factors, nucleic acid sequences in the template and transcript can modulate elongation (11,44,82). A topic central to elongation control is the role of the carboxyl-terminal domain (CTD) of the largest subunit of RNAPII (25). The CTD contains 52 repeats of a 7-amino-acid sequence with the consensus YSPTSPS and is the substrate for several kinases, including P-TEFb (25,68,99). Phosphorylation of the CTD occurs during a transitional step of the transcription cycle, the switch from the initiation phase to the elongation phase. Hypophosphorylated RNAPII (designated RNAPIIA) is preferentially recruited to a promoter and initiates transcription. Subsequently, the RNAPII becomes hyper...
and has received compensation from these companies in the form of stock; A.R.K. is a research collaborator of Ionis Pharmaceuticals and has received royalty income from Ionis through his employer, Cold Spring Harbor Laboratory. O.A.-W. has served as a consultant for H3 Biomedicine, Foundation Medicine Inc., Merck, and Janssen; O.A.-W. has received personal speaking fees from Daiichi Sankyo. O.A.-W. has received prior research funding from H3 Biomedicine unrelated to the current manuscript. D.I., R.K.B. and O.A.-W. are inventors on a provisional patent application (patent number FHCC.P0044US.P) applied for by Fred Hutchinson Cancer Research Center on the role of reactivating BRD9 expression in cancer by modulating aberrant BRD9 splicing in SF3B1 mutant cells.
Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3-end formation and found that processing was strongly dependent upon nucleotides located within the 3 stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3 box. Substitution of the actin promoter for the snRNA promoter abolished proper 3-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.In eukaryotes, the major transcripts produced by RNA polymerase II (RNAPII) include the polyadenylated [poly (A) ϩ ] mRNAs, the replication-dependent histone mRNAs, and the Sm class of small nuclear RNAs (snRNAs). The 3Ј ends of these three general classes of RNAs are all formed by cotranscriptional cleavage, but each one has a distinct mechanism for 3Ј-end formation (for reviews, see references 29 and 32). In poly(A) ϩ and histone pre-mRNAs there are conserved upstream and downstream sequences that flank the cleavage site; factors bind to these sites and then recruit additional factors that initiate cleavage (53). In the case of poly(A) ϩ pre-mRNA, the upstream element is the canonical AAUAAA polyadenylation signal (PAS) and the downstream sequence is the G/Urich downstream element (DSE). Recognition of the PAS is carried out by the cleavage and polyadenylation specificity complex (CPSF) component CPSF160 via its RNA recognition motifs (RRM) (36), whereas the DSE is bound by the RRM of the cleavage stimulation factor (CstF) component CstF64 (28). Subsequent to this recognition event is recruitment of additional factors that activate the endonucleolytic cleavage between the PAS and the DSE.Histone pre-mRNA contains a distinct set of flanking elements. Upstream of the cleavage site is a conserved stem-loop structure (SL) and downstream a purine-rich element called the ...
In epithelial cells, alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts leads to the expression of the FGFR2(IIIb) isoform, whereas in mesenchymal cells, the same process results in the synthesis of FGFR2(IIIc). Expression of the FGFR2(IIIc) isoform during prostate tumor progression suggests a disruption of the epithelial character of these tumors. To visualize the use of FGFR2 exon IIIc in prostate AT3 tumors in syngeneic rats, we constructed minigene constructs that report on alternative splicing. Imaging these alternative splicing decisions revealed unexpected mesenchymal-epithelial transitions in these primary tumors. These transitions were observed more frequently where tumor cells were in contact with stroma. Indeed, these transitions were frequently observed among lung micrometastases in the organ parenchyma and immediately adjacent to blood vessels. Our data suggest an unforeseen relationship between epithelial mesenchymal plasticity and malignant fitness.alternative splicing ͉ mesenchymal-epithelial transitions ͉ tumor plasticity R egulation of alternative splicing is essential for normal gene expression (1), and alterations of this regulation are linked to disease (2), as illustrated by the association between cancer and splicing defects (3-7). An elegant example of this is provided by the splicing of transcripts encoding fibroblast growth factor receptor 2 (FGFR2). The status of FGFR2 alternative splicing depends on the interplay between several cis-acting elements in the FGFR2 premRNA and transacting factors, some of which are cell-type-specific (8). In mesenchymal cells, exon IIIb is silenced by the action of an exonic splicing silencer (9) and two flanking intronic splicing silencers (10-12). This silencing is mediated by Polypyrimidine tract-binding protein (PTB), hnRNP A1, and heretofore unknown factors. In epithelial cells, exon IIIb silencing is countered by several intronic elements. The best characterized are the intronic activating sequence 2, the intronic splicing activator and repressor (ISAR, also known as IAS3), and several GCAUG repeats (13-18). These elements have a dual function in epithelial cells, because they are also involved in silencing exon IIIc (14-18).FGFR2 splicing has been studied in tumors derived from the R-3327 Dunning rat prostate tumor, which arose spontaneously from the dorsal lobe of the prostate in a Copenhagen rat (19). Some R-3327-derived tumors (DT or DT3) express FGFR2(IIIb), which is consistent with their epithelial phenotype (20), whereas AT tumors (e.g., AT3), which have lost epithelial markers and display many mesenchymal indicators (21), express FGFR2(IIIc) (20). The significance of these alternative decisions for tumor behavior is underscored by the fact that forced expression of FGFR2(IIIb) suppresses tumor progression of AT3 tumors (22). Most importantly, however, the differential splicing of FGFR2 transcripts in these two cell types highlights broad differences in gene expression programs. Arguably, monitoring alternative sp...
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