Project description:Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of a subset of small nucleolar RNAs (snoRNAs) and tRNAs transcribed by RNA Polymerase (Pol) III. Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral nuclear pore components, and importin-ß IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and snoRNA genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for Pol III recruitment. Instead, NPP-13 is required for cleavage of tRNA and snoRNA precursors into mature RNAs, whereas Pol II transcript processing occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and processing of Pol III transcripts in C. elegans.
Project description:Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of a subset of small nucleolar RNAs (snoRNAs) and tRNAs transcribed by RNA Polymerase (Pol) III. Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral nuclear pore components, and importin-ß IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and snoRNA genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for Pol III recruitment. Instead, NPP-13 is required for cleavage of tRNA and snoRNA precursors into mature RNAs, whereas Pol II transcript processing occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and processing of Pol III transcripts in C. elegans.
Project description:Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of a subset of small nucleolar RNAs (snoRNAs) and tRNAs transcribed by RNA Polymerase (Pol) III. Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral nuclear pore components, and importin-M-CM-^_ IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and snoRNA genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for Pol III recruitment. Instead, NPP-13 is required for cleavage of tRNA and snoRNA precursors into mature RNAs, whereas Pol II transcript processing occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and processing of Pol III transcripts in C. elegans. Genome-wide ChIP-seq and ChIP-chip were performed in mixed-stage C. elegans embryos for nuclear pore proteins NPP-13, NPP-3, IMB-1 and chromatin proteins Pol III (RPC-1), TBP-1, TFC-1 (SFC-1), TFC-4 (TAG-315), and Pol II (AMA-1). For RPC-1 and TBP-1 ChIP-seq, embryos depleted for NPP-13 were also used. Total RNAs from wild-type, NPP-13 RNAi, and IMB-1 RNAi embryos were analyzed by RNA-seq.
Project description:Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of a subset of small nucleolar RNAs (snoRNAs) and tRNAs transcribed by RNA Polymerase (Pol) III. Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral nuclear pore components, and importin-M-CM-^_ IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and snoRNA genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for Pol III recruitment. Instead, NPP-13 is required for cleavage of tRNA and snoRNA precursors into mature RNAs, whereas Pol II transcript processing occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and processing of Pol III transcripts in C. elegans. Genome-wide ChIP-seq and ChIP-chip were performed in mixed-stage C. elegans embryos for nuclear pore proteins NPP-13, NPP-3, IMB-1 and chromatin proteins Pol III (RPC-1), TBP-1, TFC-1 (SFC-1), TFC-4 (TAG-315), and Pol II (AMA-1). For RPC-1 and TBP-1 ChIP-seq, embryos depleted for NPP-13 were also used. Total RNAs from wild-type, NPP-13 RNAi, and IMB-1 RNAi embryos were analyzed by RNA-seq.
Project description:Pol III functions in the nucleus for transcription and cytoplasm as a viral-DNA sensor, but how cells maintain the cytoplasmic-to-nuclear partitioning of Pol III activity is unknown. Here, we found that Pol III forms transcription-dependent protein clusters in the nucleus and preferentially associates with assembly factors in the cytoplasm. Acute protein degradation of Pol III subunits followed by transient protein labeling showed that nuclear and cytoplasmic Pol III response differently after the loss of their subunits.
Project description:Transcription is regulated by a multitude of activators and repressors, which bind to the RNA polymerase II (Pol II) machinery and modulate its progression. Death-inducer obliterator (DIDO) and PHD finger protein 3 (PHF3) are paralogue proteins that regulate transcription elongation by docking onto phosphorylated serine-2 in the C-terminal domain (CTD) of Pol II through their SPOC domains. Here we show that DIDO3 and PHF3 form a complex that bridges the Pol II elongation machinery with chromatin and RNA processing factors, and tethers Pol II in a phase-separated microenvironment. Their SPOC domains and C-terminal intrinsically disordered regions are critical for transcription regulation. The dataset contains 3' Sequencing of WT, PHF3 KO, and PHF3 dSPOC mutant HEK293 cell line. Cytoplasmic and nuclear fractions were profiled separately.
Project description:Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with its Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing at the Mediator level. In sum, we provide insight into how NPC-associated adaptor complexes can access the core transcription machinery. RNAseq was performed from WT, sac3∆, cdk8∆ and Sac3 R288D mutant cells. For each strain triplicates were analyzed. WT strain was sac3∆ transformed with pRS315 SAC3 WT
Project description:C. elegans nuclear pore protein NPP-13 associates with small RNA genes transcribed by RNA Polymerase III. To test if the nuclear pore-chromatin interactions play a role in large-scale chromatin organization, we determined nuclear membrane-genome interactions and RNA Polymerase II localization in C. elegans embryos depleted for NPP-13.
Project description:C. elegans nuclear pore protein NPP-13 associates with small RNA genes transcribed by RNA Polymerase III. To test if the nuclear pore-chromatin interactions play a role in large-scale chromatin organization, we determined nuclear membrane-genome interactions and RNA Polymerase II localization in C. elegans embryos depleted for NPP-13.