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TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing [RNA-Seq]

ABSTRACT: TFIID is an essential basal transcription factor, crucial for RNA polymerase II (pol II) promoter recognition and transcription initiation. The TFIID complex consists of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs) that contain intrinsically disordered regions (IDRs) with currently unknown functions. Here, we show that a conserved IDR drives TAF2 condensation in nuclear speckles, independently of other TFIID subunits. Quantitative mass spectrometry analyses reveal that the TAF2 IDR specifically interacts with the nuclear speckle and spliceosome-associated protein SRRM2. Consequently, TAF2 recruits SRRM2 to TFIID to form non-canonical TFIID-SRRM2 complexes. Reduced SRRM2 recruitment elicits alternative splicing events in RNAs coding for proteins involved in transcription and transmembrane transport. Further, genome-wide binding analyses suggest TAF2 shuttling between nuclear speckles and pol II promoters. This study identifies an IDR of the basal transcription machinery as a molecular guide for protein partitioning into nuclear compartments, controlling protein complex composition and pre-mRNA splicing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE254060 | GEO | 2024/02/06

REPOSITORIES: GEO

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TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing [CUT&RUN]

Project description:TFIID is an essential basal transcription factor, crucial for RNA polymerase II (pol II) promoter recognition and transcription initiation. The TFIID complex consists of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs) that contain intrinsically disordered regions (IDRs) with currently unknown functions. Here, we show that a conserved IDR drives TAF2 condensation in nuclear speckles, independently of other TFIID subunits. Quantitative mass spectrometry analyses reveal that the TAF2 IDR specifically interacts with the nuclear speckle and spliceosome-associated protein SRRM2. Consequently, TAF2 recruits SRRM2 to TFIID to form non-canonical TFIID-SRRM2 complexes. Reduced SRRM2 recruitment elicits alternative splicing events in RNAs coding for proteins involved in transcription and transmembrane transport. Further, genome-wide binding analyses suggest TAF2 shuttling between nuclear speckles and pol II promoters. This study identifies an IDR of the basal transcription machinery as a molecular guide for protein partitioning into nuclear compartments, controlling protein complex composition and pre-mRNA splicing.

2024-02-06 | GSE254080 | GEO

TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing – data-independent acquisition MS

Project description:TFIID is an essential basal transcription factor, crucial for RNA polymerase II (pol II) promoter recognition and transcription initiation. The TFIID complex consists of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs) that contain intrinsically disordered regions (IDRs) with currently unknown functions. Here, we show that a conserved IDR drives TAF2 condensation in nuclear speckles, independently of other TFIID subunits. Quantitative mass spectrometry analyses reveal that TAF2 exhibits close proximities to RNA-binding proteins, but that the TAF2 IDR specifically interacts with the splicing factor SRRM2. Modulation of TAF2 protein levels elicits alternative splicing (AS) events in RNAs coding for proteins involved in transcription and transmembrane transport, some of which overlap with SRRM2-regulated AS events. This study identifies an IDR of the basal transcription machinery as a molecular guide for protein partitioning into nuclear compartments, controlling protein complex composition and pre-mRNA splicing.

2025-03-04 | PXD056099 | Pride

TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing

Project description:TFIID is an essential basal transcription factor, crucial for RNA polymerase II (pol II) promoter recognition and transcription initiation. The TFIID complex consists of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs) that contain intrinsically disordered regions (IDRs) with currently unknown functions. Here, we show that a conserved IDR drives TAF2 condensation in nuclear speckles, independently of other TFIID subunits. Quantitative mass spectrometry analyses reveal that TAF2 exhibits close proximities to RNA-binding proteins, but that the TAF2 IDR specifically interacts with the splicing factor SRRM2. Modulation of TAF2 protein levels elicits alternative splicing (AS) events in RNAs coding for proteins involved in transcription and transmembrane transport, some of which overlap with SRRM2-regulated AS events. This study identifies an IDR of the basal transcription machinery as a molecular guide for protein partitioning into nuclear compartments, controlling protein complex composition and pre-mRNA splicing.

2025-03-04 | PXD056095 | Pride

TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing

Project description:TFIID is an essential eukaryotic transcription factor, which is required for RNA polymerase II promoter recognition and activation. As a multiprotein complex, TFIID contains several intrinsically disordered regions (IDRs), but the functions of these IDRs are unknown. Here, we show that a conserved IDR drives the TFIID subunit TAF2 to nuclear speckles, where it forms biomolecular condensates, separate from the TFIID complex. Quantitative mass spectrometry analyses reveal that the TAF2 IDR is required for the interaction with the nuclear speckle and spliceosome-associated protein SRRM2, which is thereby recruited to TFIID. The formation of SRRM2-free TFIID complexes elicits a set of unique alternative splicing events. These include events in RNAs coding for proteins involved in transcription and transmembrane transport. Together, these data identify an IDR of the basal transcription machinery as a molecular switch between nuclear compartments to control protein complex composition and pre-mRNA splicing.

2024-04-26 | PXD049117 | Pride

Cytoplasmic complex comprising TAF2, TAF8, TAF10 provides evidence for nuclear holo-TFIID assembly from preformed submodules

Project description:General transcription factor TFIID is a cornerstone of RNA polymerase II transcription initiation in eukaryotic nuclei. Human TFIID is a megadalton-sized multiprotein complex composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). The cellular mechanism of TFIID assembly is poorly understood. In the cytoplasm of human cells, we discovered a heterotrimeric TFIID sub-complex consisting of the TAF2, TAF8 and TAF10 proteins. Native mass-spectrometry uncovered the interactions between the TAFs, defining a central role of TAF8 in nucleating the complex. X-ray crystallography revealed a non-canonical arrangement of the TAF8-TAF10 histone fold domains (HFDs). TAF2 binds to multiple motifs within the TAF8 C-terminal region, and these interactions dictate TAF2 incorporation into a core-TFIID complex that exists in the nucleus. Our results provide evidence for a step-wise assembly pathway of nuclear holo-TFIID, regulated by nuclear import of preformed cytoplasmic submodules.

2016-06-15 | PXD001427 | Pride

Importance of TAF8 regions for TFIID lobe B assembly, or for TAF2 interactions, are required for embryonic stem cell survival

Project description:The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B and C. Structural studies showed that TAF8 is forming a histone fold pair in lobe B and connecting lobe B to lobe C. In the present study, we have investigated the requirement of the different regions of TAF8 for in vitro TFIID assembly, and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a TAF8 region, different from the histone fold domain of TAF8, important for assembling with the 5TAF core complex in lobe B, and four regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, we show that the 5TAF core-interacting TAF8 domain, and the proline rich domain of TAF8 interacting with TAF2, are both required for mouse embryonic stem cell survival. Thus, our study demonstrates that TAF8 regions involving in the connection of lobe B to lobe C are crucial for TFIID function and consequent ESC survival.

2022-02-15 | PXD028352 | Pride

TAF8 regions important for TFIID lobe B assembly are required for embryonic stem cell survival

2021-10-02 | PXD026575 | Pride

TFIID enables RNA polymerase II promoter-proximal pausing

Project description:RNA polymerase II (pol II) transcribes all protein-coding and many non-coding RNAs in the human genome. Pol II transcription initiation is governed by the Pre-Initiation Complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, pol II, and Mediator. After initiation, pol II enzymes typically pause after transcribing less than 100 bases, and paused polymerases represent a common regulatory intermediate. Accordingly, paused pol II has been implicated in enhancer function, development and homeostasis, and diseases ranging from cancer to viral pathogenesis. Precisely how pol II promoter-proximal pausing is enforced and regulated remains unclear; however, protein complexes such as NELF and DSIF increase pausing whereas the activity of CDK9 (P-TEFb complex) correlates with pause release. To address specific mechanistic questions about pol II pausing and its regulation, we reconstituted human pol II promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pol II pausing in vitro, whereas P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pol II pausing, suggesting that pausing is an inherent property of the PIC. In agreement, pol II pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP); moreover, pausing was dependent upon TFIID subunits TAF1 and TAF2. TAF1/2 bind genomic DNA downstream of the pol II initiation site, invoking a “complex interaction” model for pausing. Consistent with this model, PRO-Seq experiments revealed increased transcription upon acute depletion (t=60 min) of TAF1 and TAF2 in human cells, and pol II pausing was disrupted at thousands of genes. Similar results were obtained in TAF1-depleted Drosophila S2 cells. Collectively, these data establish the general transcription factor TFIID as a genome-wide regulator of pol II promoter-proximal pausing.

2020-05-28 | GSE132764 | GEO

Interconversion between active and inactive TATA-binding protein transcription complexes in the mouse genome.

Project description:The conserved core domain of the TATA binding protein (TBP) interacts with multiple partners forming the complexes required for transcription by RNA Polymerases I, II and III. We use genetically modified mouse embryonic fibroblasts to show that many TBP core domain mutants complement loss of endogenous TBP, but this often results in a slow growth phenotype. Two TBP mutations, R188E and K243E, disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not affect global genomic distribution of TBP, but positively or negatively affects TBP and/or RNA Polymerase II (Pol II) recruitment to a selected set of promoters. We identify a set of promoters where wild-type TBP assembles a partial inactive preinitiation complex lacking Pol II and TAF1. Our results suggest that an exchange of the B-TFIID complex in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters recruits Pol II to activate their expression. We also observe that both Wt and mutant TBP can occupy promoters without concurrent Pol II recruitment and active transcription. Our data reveal a novel regulatory mechanism involving the formation of a partial preinitiation complex that primes the promoter for productive preinitiation complex formation in mammalian cells.

2012-03-01 | GSE27908 | GEO

TBP2/TFIIA complex drives an oocyte-type promoter usage grammar leading to female germ cell specific mRNA and endogenous retroviral element transcription

Project description:The transcriptional machinery involved in RNA polymerase II (Pol II) transcription during oogenesis is not well characterized. In somatic cells, the Pol II general transcription factor, TFIID, containing the TATA binding protein (TBP) and 13 TBP-associated factors, is the first to bind gene promoters to nucleate pre-initiation complex formation. During oocyte growth TBP is replaced by TBP2 and Tbp2-/- female mice are sterile. Here, we show that in oocytes TBP2 stably associates with TFIIA but does not assemble into a TFIID-type complex but stabily associates with TFIIA. we demonstrate that in female germ cells the specific TBP2-TFIIA-containing transcription machinery drives transcription from oocyte-specific core promoters, which are different from those occupied by TBP/TFIID in somatic cells.

2020-11-16 | PXD016347 | Pride

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