The anti-tumor drug E7107 reveals an essential role for SF3b in remodeling U2 snRNP to expose the branch point-binding region.

Duplex formation between the branch point-binding region (BBR) of U2 snRNA and the branch point sequence (BPS) in the intron is essential for splicing. Both the BBR and BPS interact with the U2 small nuclear ribonucleoprotein (snRNP)-associated SF3b complex, which is the target of the anti-tumor drug E7107. We show that E7107 blocks spliceosome assembly by preventing tight binding of U2 snRNP to pre-mRNA. E7107 has no apparent effect on U2 snRNP integrity. Instead, E7107 abolishes an ATP-dependent conformational change in U2 snRNP that exposes the BBR. We conclude that SF3b is required for this remodeling, which exposes the BBR for tight U2 snRNP binding to pre-mRNA.

SF3B1 and other novel cancer genes in chronic lymphocytic leukemia.

BACKGROUND: The somatic genetic basis of chronic lymphocytic leukemia, a common and clinically heterogeneous leukemia occurring in adults, remains poorly understood. METHODS: We obtained DNA samples from leukemia cells in 91 patients with chronic lymphocytic leukemia and performed massively parallel sequencing of 88 whole exomes and whole genomes, together with sequencing of matched germline DNA, to characterize the spectrum of somatic mutations in this disease. RESULTS: Nine genes that are mutated at significant frequencies were identified, including four with established roles in chronic lymphocytic leukemia (TP53 in 15% of patients, ATM in 9%, MYD88 in 10%, and NOTCH1 in 4%) and five with unestablished roles (SF3B1, ZMYM3, MAPK1, FBXW7, and DDX3X). SF3B1, which functions at the catalytic core of the spliceosome, was the second most frequently mutated gene (with mutations occurring in 15% of patients). SF3B1 mutations occurred primarily in tumors with deletions in chromosome 11q, which are associated with a poor prognosis in patients with chronic lymphocytic leukemia. We further discovered that tumor samples with mutations in SF3B1 had alterations in pre-messenger RNA (mRNA) splicing. CONCLUSIONS: Our study defines the landscape of somatic mutations in chronic lymphocytic leukemia and highlights pre-mRNA splicing as a critical cellular process contributing to chronic lymphocytic leukemia.

A model in vitro system for co-transcriptional splicing.

A hallmark of metazoan RNA polymerase II transcripts is the presence of numerous small exons surrounded by large introns. Abundant evidence indicates that splicing to excise introns occurs co-transcriptionally, prior to release of the nascent transcript from RNAP II. Here, we established an efficient model system for co-transcriptional splicing in vitro. In this system, CMV-DNA constructs immobilized on beads generate RNAP II transcripts containing two exons and an intron. Consistent with previous work, our data indicate that elongating nascent transcripts are tethered to RNAP II on the immobilized DNA template. We show that nascent transcripts that reach full length, but are still attached to RNAP II, are efficiently spliced. When the nascent transcript is cleaved within the intron using RNase H, both the 5' and 3' cleavage fragments are detected in the bound fraction, where they undergo splicing. Together, our work establishes a system for co-transcriptional splicing in vitro, in which the spliceosome containing the 5' and 3' exons are tethered to RNAP II for splicing.

Mammalian PERIOD2 regulates H2A.Z incorporation in chromatin to orchestrate circadian negative feedback.

Mammalian circadian oscillators are built on a feedback loop in which the activity of the transcription factor CLOCK-BMAL1 is repressed by the PER-CRY complex. Here, we show that murine Per-/- fibroblasts display aberrant nucleosome occupancy around transcription start sites (TSSs) and at promoter-proximal and distal CTCF sites due to impaired histone H2A.Z deposition. Knocking out H2A.Z mimicked the Per null chromatin state and disrupted cellular rhythms. We found that endogenous mPER2 complexes retained CTCF as well as the specific H2A.Z-deposition chaperone YL1-a component of the ATP-dependent remodeler SRCAP and p400-TIP60 complex. While depleting YL1 or mutating chaperone-binding sites on H2A.Z lengthened the circadian period, H2A.Z deletion abrogated BMAL1 chromatin recruitment and promoted its proteasomal degradation. We propose that a PER2-mediated H2A.Z deposition pathway (1) compacts CLOCK-BMAL1 binding sites to establish negative feedback, (2) organizes circadian chromatin landscapes using CTCF and (3) bookmarks genomic loci for BMAL1 binding to impinge on the positive arm of the subsequent cycle.

Bromodomain factors of BET family are new essential actors of pericentric heterochromatin transcriptional activation in response to heat shock.

The heat shock response is characterized by the transcriptional activation of both hsp genes and noncoding and repeated satellite III DNA sequences located at pericentric heterochromatin. Both events are under the control of Heat Shock Factor I (HSF1). Here we show that under heat shock, HSF1 recruits major cellular acetyltransferases, GCN5, TIP60 and p300 to pericentric heterochromatin leading to a targeted hyperacetylation of pericentric chromatin. Redistribution of histone acetylation toward pericentric region in turn directs the recruitment of Bromodomain and Extra-Terminal (BET) proteins BRD2, BRD3, BRD4, which are required for satellite III transcription by RNAP II. Altogether we uncover here a critical role for HSF1 in stressed cells relying on the restricted use of histone acetylation signaling over pericentric heterochromatin (HC).

[The mouse hairless gene: its function in hair root and at the heart of a subtle pleiotropy]. / Le gène hairless de la souris: fonctions à la racine du poil et au coeur d'une subtile pléiotropie.

The hairless gene in mammals encodes a nuclear factor that is highly expressed in skin and appears to control hair follicle integrity and cycling. In the absence of a normal and functional Hairless (Hr) protein, the hair bulb undergoes premature apoptosis during the first catagen stage of the hair cycle. The most striking effects of the mutation are loss of hair follicles and formation of epidermal utricles and dermal cysts. The hairless gene expression appears to be widespread and temporally regulated. The gene is strongly expressed in different compartments of the brain. Hairless mRNAs were detected in cartilage, gonads, thymus and colon. In addition to alopecia, hairless mice strains show subtle defects in the development and differentiation of various tissues and organs. The Hr protein is localised in cell nuclei and functions as a transcriptional regulator. Although its role has not been resolved in molecular terms, it was demonstrated that Hr is able to interact with multiple nuclear hormone receptors. Hr seems to be a part of a large multiprotein complex capable to repress transcription by its association to chromatin remodelling factors such as histone deacetylases. Recent experimental data suggest that Hr might be involved in Hox gene regulation, cell adhesion modulation and progenitor cells identity. At least in the skin, but probably in other organs, the Hr repressor seems to be responsible for the timing of epithelial cells differentiation.

In vitro systems for coupling RNAP II transcription to splicing and polyadenylation.

Studies over the past several years have revealed that steps in gene expression are extensively coupled to one another both physically and functionally. Recently, in vitro systems were developed for understanding the mechanisms involved in coupling transcription by RNA polymerase II to RNA processing. Here we describe an efficient two-way system for coupling transcription to splicing and a robust three-way system for coupling transcription, splicing, and polyadenylation. In these systems a CMV-DNA construct is incubated in HeLa cell nuclear extracts in the presence of (32)P-UTP to generate the nascent transcript. Transcription is then stopped by addition of α-amanitin followed by continued incubation to allow RNA processing.

Dynamics of the full length and mutated heat shock factor 1 in human cells.

Heat shock factor 1 is the key transcription factor of the heat shock response. Its function is to protect the cell against the deleterious effects of stress. Upon stress, HSF1 binds to and transcribes hsp genes and repeated satellite III (sat III) sequences present at the 9q12 locus. HSF1 binding to pericentric sat III sequences forms structures known as nuclear stress bodies (nSBs). nSBs represent a natural amplification of RNA pol II dependent transcription sites. Dynamics of HSF1 and of deletion mutants were studied in living cells using multi-confocal Fluorescence Correlation Spectroscopy (mFCS) and Fluorescence Recovery After Photobleaching (FRAP). In this paper, we show that HSF1 dynamics modifications upon heat shock result from both formation of high molecular weight complexes and increased HSF1 interactions with chromatin. These interactions involve both DNA binding with Heat Shock Element (HSE) and sat III sequences and a more transient sequence-independent binding likely corresponding to a search for more specific targets. We find that the trimerization domain is required for low affinity interactions with chromatin while the DNA binding domain is required for site-specific interactions of HSF1 with DNA.

Small-scale nuclear extracts for functional assays of gene-expression machineries.

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

The proteins PDIP3 and ZC11A associate with the human TREX complex in an ATP-dependent manner and function in mRNA export.

The conserved TREX complex, which contains UAP56, Aly, CIP29, and the multi-subunit THO complex, functions in mRNA export. Recently, several putative new components of the human TREX complex were identified by mass spectrometry. Here, we investigated the function of two of these, PDIP3 and ZC11A. Our data indicate that both of these proteins are components of a common TREX complex and function in mRNA export. Recently, we found that both CIP29 and Aly associate with the DEAD box helicase UAP56 and with the TREX complex in an ATP-dependent manner. We now show that this is also the case for PDIP3 and ZC11A. Thus, together with previous work, our data indicate that the TREX complex participates in multiple ATP-dependent interactions.

TREX exposes the RNA-binding domain of Nxf1 to enable mRNA export.

The metazoan TREX complex is recruited to mRNA during nuclear RNA processing and functions in exporting mRNA to the cytoplasm. Nxf1 is an mRNA export receptor, which binds processed mRNA and transports it through the nuclear pore complex. At present, the relationship between TREX and Nxf1 is not understood. Here we show that Nxf1 uses an intramolecular interaction to inhibit its own RNA-binding activity. When the TREX subunits Aly and Thoc5 make contact with Nxf1, Nxf1 is driven into an open conformation, exposing its RNA-binding domain, allowing RNA binding. Moreover, the combined knockdown of Aly and Thoc5 markedly reduces the amount of Nxf1 bound to mRNA in vivo and also causes a severe mRNA export block. Together, our data indicate that TREX provides a license for mRNA export by driving Nxf1 into a conformation capable of binding mRNA.

The "bald Mill Hill" mutation in the mouse is associated with an abnormal, mislocalized HR bmh protein.

We have previously identified a mutation in the mouse hairless locus-hairless rhino bald Mill Hill (Hr(rhbmh)). The genetic alteration in these mice consists in a large 296 bp deletion at the 3' part of the hairless gene (ID:MGI:3039558; J:89321). Here, we show that this deletion removes the stop codon and creates a new reading frame at the C terminus of the hairless protein, generating a larger mutant protein harboring an additional sequence of 117 amino acids. The mutant hairless gene mRNA is expressed during the embryonic and post-natal development of the hair follicle. The mutant protein is identified in bmh mouse skin at different stages of development by a specific antibody. We demonstrate that the HR bmh protein is able to interact with the vitamin D receptor (VDR), but is not able to repress VDR-mediated transactivation. Immunofluorescence analysis reveals that HR bmh protein displays an abnormal cellular localization in transfected cell lines, as well as in the epidermis and hair follicle of bmh mutant mice. We discuss the relevance of the hairless protein mis localization in cell signalling pathways and with respect to the specific skin phenotype of mouse hairless mutants.