Todd R. Albrecht

The Transcription Elongation Factor CA150 Interacts with RNA Polymerase II and the Pre-mRNA Splicing Factor SF1

ABSTRACT 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 α4-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.

CFIm25 links alternative polyadenylation to glioblastoma tumour suppression

The global shortening of messenger RNAs through alternative polyadenylation (APA) that occurs during enhanced cellular proliferation represents an important, yet poorly understood mechanism of regulated gene expression. The 3′ untranslated region (UTR) truncation of growth-promoting mRNA transcripts that relieves intrinsic microRNA- and AU-rich-element-mediated repression has been observed to correlate with cellular transformation; however, the importance to tumorigenicity of RNA 3′-end-processing factors that potentially govern APA is unknown. Here we identify CFIm25 as a broad repressor of proximal poly(A) site usage that, when depleted, increases cell proliferation. Applying a regression model on standard RNA-sequencing data for novel APA events, we identified at least 1,450 genes with shortened 3′ UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNAs in human cells. Marked increases in the 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 tumours that have reduced CFIm25 expression. Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumour size, whereas CFIm25 overexpression reduces these properties and inhibits tumour growth. These findings identify a pivotal role of CFIm25 in governing APA and reveal a previously unknown connection between CFIm25 and glioblastoma tumorigenicity.

Alternative inclusion of fibroblast growth factor receptor 2 exon IIIc in Dunning prostate tumors reveals unexpected epithelial mesenchymal plasticity

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.

A Subset of Drosophila Integrator Proteins Is Essential for Efficient U7 snRNA and Spliceosomal snRNA 3-End Formation†

ABSTRACT 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.

snRNA 3′ End Formation Requires Heterodimeric Association of Integrator Subunits

ABSTRACT The Integrator Complex is a group of proteins responsible for the endonucleolytic cleavage of primary small nuclear RNA (snRNA) transcripts within the nucleus. Integrator subunits 9 and 11 (IntS9/11) are thought to contain the catalytic activity based on their high sequence similarity to CPSF100 and CPSF73, which have been shown to be components of both the poly(A)+ and histone pre-mRNA cleavage complex. Here we demonstrate that the specific heterodimeric interaction between IntS9 and IntS11 is mediated by a discrete domain present at the extreme C terminus of IntS9 and within the C terminus of IntS11, adjacent to the predicted active site of this endonuclease. This domain is highly conserved within IntS11 but conspicuously absent in CPSF73. Using a cell-based complementation assay that measures Integrator activity, we determined that the IntS9 interaction domain within IntS11 is required for its ability to restore snRNA 3′ end processing after RNA interference (RNAi)-mediated depletion of IntS11. Moreover, overexpression of these interaction domains alone elicits snRNA misprocessing through a dominant-negative titration of endogenous Integrator subunits. These data collectively explain the mechanism by which the IntS11/9 and, by analogy, the CPSF73/100 heterodimeric cleavage factors distinguish themselves from each other and demonstrate that the heterodimeric interaction is functionally required for snRNA 3′ end formation.

Cytomegalovirus: Sodium entry and development of cytomegaly in human fibroblasts

A possible relationship between net Na+ entry and the development of CMV-induced cytomegaly (cell enlargement) was investigated in human fibroblasts derived from skin-muscle and thyroid tissue. We found that inhibiting cellular Na+ uptake, either by pharmacological means (amiloride, an inhibitor of Na+/H+ exchange) or by replacement of extracellular Na+ (by N-methyl-D-glucamine or choline), inhibited the development of cytomegaly. Furthermore, we noted a temporal parallelism between the development of cytomegaly and enhancement of ouabain-sensitive (O-S) 86Rb+ uptake. O-S 86Rb+ uptake is a monitor for the activity of the sodium pump resident in the plasmalemma of the fibroblasts. The enhanced O-S 86Rb+ uptake reflects either an increased intracellular Na+ concentration or an increased number of sodium pump complexes per fibroblast. Amiloride inhibited the enhancement of O-S 86Rb+ uptake, as well as cytomegaly development. Addition of amiloride at selected times after infection suggested that the same phase of virus replication was sensitive to the inhibitory effect of this drug on the enhancement of O-S 86Rb+ uptake and on the development of cytomegaly. There was also a similar pattern of inhibition of O-S 86Rb+ uptake and cytomegaly with increasing concentrations of amiloride. Thus, there may be a relationship between CMV-induced Na+ entry through activation of the Na+/H+ exchanger and development of cytomegaly.

Imaging alternative splicing in living cells.

We have developed an in vivo reporter of alternative splicing decisions that allows for the determination of FGF-R2 splicing patterns without the destruction of cells. This method has broad applications, including the study of other alternatively spliced genes in tissue culture and in whole animals, and may be useful in creating imaging markers for the study of tumor progression and metastasis. In this chapter, the authors present one example of this method using fluorescence reporters. As with any new assay, a series of experiments were performed to validate the method. This chapter documents some of these experiments.

Amiloride Inhibition of Human Cytomegalovirus Replication

Abstract Amiloride, an inhibitor of Na+/H+ exchange, interfered with cytomegalovirus (CMV) DNA synthesis, blocked the formation of nuclear inclusions, and reduced CMV infectious yields. The reduction of CMV infectious yields was concentration dependent with an ED90 of 46 μM. Amiloride at a concentration of 150 μM reduced CMV yields by about 100-fold. Reduction of infectious yields appeared to be related to interference with the formation of nuclear inclusions and to inhibition of CMV DNA synthesis. Nuclear inclusions were much reduced in size and demonstrated poorly defined cellulae in the amiloride-treated cells. CMV DNA synthesis was inhibited by approximately 70% when cells were treated with 150 μM amiloride. The reduction in CMV yields could not be related to the reported inhibitory effect of amiloride on protein synthesis. In amiloride (150 μM)-treated, CMV-infected cells, late, yet not immediate-early or early, protein synthesis was markedly decreased relative to untreated, CMV-infected cells. Accordingly, CMV DNA synthesis and the replication of CMV may be related to Na+ entry through an amiloride-sensitive pathway.

Nuclear-localized Asunder regulates cytoplasmic dynein localization via its role in the Integrator complex

A pool of dynein anchored to the nuclear surface mediates many processes at G2/M, although its spatial and temporal regulation is poorly understood. Asunder, a critical regulator of dynein recruitment to the nuclear envelope, works in the nucleus as part of Integrator, an snRNA-processing complex, to mediate this event.

Molecular basis for the interaction between Integrator subunits IntS9 and IntS11 and its functional importance

Significance The Integrator complex (INT) has important functions in the 3′-end processing of noncoding RNAs and RNA polymerase II transcription. The INT contains at least 14 subunits, but its molecular mechanism of action is still poorly understood. The endonuclease activity of INT is mediated by its subunit 11 (IntS11), which forms a stable complex with Integrator complex subunit 9 (IntS9) through their C-terminal domains (CTDs). Here, we report the crystal structure of the IntS9–IntS11 CTD complex at 2.1-Å resolution and detailed, structure-based biochemical and functional studies. Highly conserved residues are located in the extensive interface between the two CTDs. Yeast two-hybrid assays and coimmunoprecipitation experiments confirm the structural observations. Functional studies demonstrate that the IntS9–IntS11 interaction is crucial for INT in snRNA 3′-end processing. The metazoan Integrator complex (INT) has important functions in the 3′-end processing of noncoding RNAs, including the uridine-rich small nuclear RNA (UsnRNA) and enhancer RNA (eRNA), and in the transcription of coding genes by RNA polymerase II. The INT contains at least 14 subunits, but its molecular mechanism of action is poorly understood, because currently there is little structural information about its subunits. The endonuclease activity of INT is mediated by its subunit 11 (IntS11), which belongs to the metallo-β-lactamase superfamily and is a paralog of CPSF-73, the endonuclease for pre-mRNA 3′-end processing. IntS11 forms a stable complex with Integrator complex subunit 9 (IntS9) through their C-terminal domains (CTDs). Here, we report the crystal structure of the IntS9–IntS11 CTD complex at 2.1-Å resolution and detailed, structure-based biochemical and functional studies. The complex is composed of a continuous nine-stranded β-sheet with four strands from IntS9 and five from IntS11. Highly conserved residues are located in the extensive interface between the two CTDs. Yeast two-hybrid assays and coimmunoprecipitation experiments confirm the structural observations on the complex. Functional studies demonstrate that the IntS9–IntS11 interaction is crucial for the role of INT in snRNA 3′-end processing.