Subcellular Localization Shapes the Fate of RNA Polymerase III
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus
SUBMITTER: 
Xiong Ji
PROVIDER: PXD037971 | iProX | Sun Nov 06 00:00:00 GMT 2022
REPOSITORIES: iProX
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Project description:RNA Pol III plays a vital role in transcription and as a viral-DNA sensor, but how it is assembled and distributed within cells remain poorly understood. Here, we show that Pol III is assembled with chaperones in the cytoplasm and forms transcription-dependent protein clusters upon transport into the nucleus. The largest subunit (RPC1) depletion through an auxin-inducible degron leads to rapid degradation and disassembly of Pol III complex in the nucleus and cytoplasm, respectively. This generates a pool of partially assembled Pol III intermediates, which can be rapidly mobilized into the nucleus upon the restoration of RPC1. Our study highlights the critical role of subcellular localization in determining Pol III's fate and provide insight into the dynamic regulation of nuclear Pol III levels and the origin of cytoplasmic Pol III complexes involved in mediating viral immunity.
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Project description:The packaging of the genetic material into chromatin imposes the remodeling of this barrier to allow efficient transcription. RNA polymerase II activity is associated with several histone modification complexes that enforce remodeling. How RNA polymerase III (Pol III) counteracts the inhibitory effect of chromatin is unknown. We report here that antisense RNA Polymerase II (Pol II) transcription is critical to prime and maintain nucleosome depletion at Pol III loci and allow efficient Pol III recruitment upon re-initiation of growth from stationary phase. Antisense Pol II is recruited by the Pcr1 transcription factor, which affects local histone occupancy through the associated SAGA complex and a Pol II phospho-S2 CTD / Mst2 pathway. These data expand the central role of Pol II in gene expression beyond mRNA synthesis.
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Project description:RNA polymerase III (Pol III) is specialized in the transcription of short, functional RNAs, including small nuclear RNAs (snRNAs). At snRNA genes, Pol III is recruited by the snRNA Activating Protein Complex (SNAPc)forming apreinitiation complex (PIC). Uniquely, SNAPc forms PICs with both Pol II and Pol IIIat their respectivepromoters. Increasingly, snRNA PIC structures arethe focus of structural characterization, however the mechanism of SNAPc cross-polymerase engagementand the role of the SNAPC2 and SNAPC5 subunits in transcription remain unclear. Here, we present CryoEM structuresoftheSNAPc-containing Pol III PIC assembled on the U6 snRNA promoterin the open and melting statesat 3.2-4.2Å resolution. Comparative structural analysis revealedunexpected differences with the corresponding yeast complex and revealedthe basis of SNAPc engagement with both Pol III and Pol IIPICs. Integrating crosslinkingmass spectrometry,we alsolocalizethe SNAPC2 and SNAPC5 subunitsin proximity to the bound promoter DNA, expanding upon existing descriptions of snRNA Pol III PIC structure.
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Project description:MAF1 represses Pol III-mediated transcription by interfering with TFIIIB and Pol III. Herein, we found that MAF1 knockdown induced CDKN1A transcription and chromatin looping concurrently with Pol III recruitment. Simultaneous knockdown of MAF1 with Pol III or BRF1 (subunit of TFIIIB) diminished the activation and looping effect, which indicates that recruiting Pol III was required for activation of Pol II-mediated transcription and chromatin looping. ChIP analysis after MAF1 knockdown indicated enhanced binding of Pol III and BRF1, as well as of CFP1, p300, and PCAF, which are factors that mediate active histone marks, along with the binding of TBP and POLR2E to the CDKN1A promoter. Simultaneous knockdown with Pol III abolished these regulatory events. Similar results were obtained for GDF15. Our results reveal a novel mechanism by which MAF1 and Pol III regulate the activity of a protein-coding gene transcribed by Pol II.
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Project description:MAF1 represses Pol III-mediated transcription by interfering with TFIIIB and Pol III. Herein, we found that MAF1 knockdown induced CDKN1A transcription and chromatin looping concurrently with Pol III recruitment. Simultaneous knockdown of MAF1 with Pol III or BRF1 (subunit of TFIIIB) diminished the activation and looping effect, which indicates that recruiting Pol III was required for activation of Pol II-mediated transcription and chromatin looping. ChIP analysis after MAF1 knockdown indicated enhanced binding of Pol III and BRF1, as well as of CFP1, p300, and PCAF, which are factors that mediate active histone marks, along with the binding of TBP and POLR2E to the CDKN1A promoter. Simultaneous knockdown with Pol III abolished these regulatory events. Similar results were obtained for GDF15. Our results reveal a novel mechanism by which MAF1 and Pol III regulate the activity of a protein-coding gene transcribed by Pol II. Knockdown assay was performed using siRNA obtained from MISSION®RNA (Sigma). Inhibition of expression of Pol III (SASI_Hs01_00046568) and MAF1 (SASI_Hs01_00135954) was achieved by transfection with LipofectamineTM RNAiMax (Invitrogen) according to the manufacturer’s protocol. MISSION® siRNA Universal Negative Control (Sigma) was used as knockdown control. Cells were transfected in serum-free medium. After 8 h, the siRNA containing medium was replaced with complete medium.
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