scholarly journals Mechanism of RNA polymerase III termination-associated reinitiation-recycling conferred by the essential function of the N terminal-and-linker domain of the C11 subunit

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saurabh Mishra ◽  
Shaina H. Hasan ◽  
Rima M. Sakhawala ◽  
Shereen Chaudhry ◽  
Richard J. Maraia
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Point Mutations ◽  
Cleavage Activity ◽  
Terminal Domain ◽  
Polymerase Iii ◽  
Essential Function ◽  
High Level ◽  
Pol Iii

AbstractRNA polymerase III achieves high level tRNA synthesis by termination-associated reinitiation-recycling that involves the essential C11 subunit and heterodimeric C37/53. The C11-CTD (C-terminal domain) promotes Pol III active center-intrinsic RNA 3′-cleavage although deciphering function for this activity has been complicated. We show that the isolated NTD (N-terminal domain) of C11 stimulates Pol III termination by C37/53 but not reinitiation-recycling which requires the NTD-linker (NTD-L). By an approach different from what led to current belief that RNA 3′-cleavage activity is essential, we show that NTD-L can provide the essential function of Saccharomyces cerevisiae C11 whereas classic point mutations that block cleavage, interfere with active site function and are toxic to growth. Biochemical and in vivo analysis including of the C11 invariant central linker led to a model for Pol III termination-associated reinitiation-recycling. The C11 NTD and CTD stimulate termination and RNA 3′-cleavage, respectively, whereas reinitiation-recycling activity unique to Pol III requires only the NTD-linker. RNA 3′-cleavage activity increases growth rate but is nonessential.

scholarly journals A Novel Upstream RNA Polymerase III Promoter Element Becomes Essential When the Chromatin Structure of the Yeast U6 RNA Gene Is Altered

2001 ◽  
Vol 21 (19) ◽  
pp. 6429-6439 ◽  
Author(s):  
Michael P. Martin ◽  
Valerie L. Gerlach ◽  
David A. Brow
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Point Mutations ◽  
Tata Box ◽  
Initiation Factor ◽  
Base Pairs ◽  
Polymerase Iii ◽  
U6 Rna

ABSTRACT The Saccharomyces cerevisiae U6 RNA gene,SNR6, possesses upstream sequences that allow productive binding in vitro of the RNA polymerase III (Pol III) transcription initiation factor IIIB (TFIIIB) in the absence of TFIIIC or other assembly factors. TFIIIC-independent transcription ofSNR6 in vitro is highly sensitive to point mutations in a consensus TATA box at position −30. In contrast, the TATA box is dispensable for SNR6 transcription in vivo, apparently because TFIIIC bound to the intragenic A block and downstream B block can recruit TFIIIB via protein-protein interactions. A mutant allele ofSNR6 with decreased spacing between the A and B blocks,snr6-Δ42, exhibits increased dependence on the upstream sequences in vivo. Unexpectedly, we find that in vivo expression of snr6-Δ42 is much more sensitive to mutations in a (dT-dA)7 tract between the TATA box and transcription start site than to mutations in the TATA box itself. Inversion of single base pairs in the center of the dT-dA tract nearly abolishes transcription of snr6-Δ42, yet inversion of all 7 base pairs has little effect on expression, indicating that the dA-dT tract is relatively orientation independent. Although it is within the TFIIIB footprint, point mutations in the dT-dA tract do not inhibit TFIIIB binding or TFIIIC-independent transcription ofSNR6 in vitro. In the absence of the chromatin architectural protein Nhp6, dT-dA tract mutations are lethal even when A-to-B block spacing is wild type. We conclude that the (dT-dA)7 tract and Nhp6 cooperate to direct productive transcription complex assembly on SNR6 in vivo.

scholarly journals Chromatin Structure and Expression of a Gene Transcribed by RNA Polymerase III Are Independent of H2A.Z Deposition

2008 ◽  
Vol 28 (8) ◽  
pp. 2598-2607 ◽  
Author(s):  
Aneeshkumar Gopalakrishnan Arimbasseri ◽  
Purnima Bhargava
Keyword(s):  
Rna Polymerase ◽  
Chromatin Structure ◽  
Rna Polymerase Iii ◽  
Nutritional Deprivation ◽  
U6 Snrna ◽  
Polymerase Iii ◽  
Downstream Box ◽  
Pol Iii

ABSTRACT The genes transcribed by RNA polymerase III (Pol III) generally have intragenic promoter elements. One of them, the yeast U6 snRNA (SNR6) gene is activated in vitro by a positioned nucleosome between its intragenic box A and extragenic, downstream box B separated by ∼200 bp. We demonstrate here that the in vivo chromatin structure of the gene region is characterized by the presence of an array of positioned nucleosomes, with only one of them in the 5′ end of the gene having a regulatory role. A positioned nucleosome present between boxes A and B in vivo does not move when the gene is repressed due to nutritional deprivation. In contrast, the upstream nucleosome which covers the TATA box under repressed conditions is shifted ∼50 bp further upstream by the ATP-dependent chromatin remodeler RSC upon activation. It is marked with the histone variant H2A.Z and H4K16 acetylation in active state. In the absence of H2A.Z, the chromatin structure of the gene does not change, suggesting that H2A.Z is not required for establishing the active chromatin structure. These results show that the chromatin structure directly participates in regulation of a Pol III-transcribed gene under different states of its activity in vivo.

scholarly journals Mutations in the RNA Polymerase III Subunit Rpc11p That Decrease RNA 3′ Cleavage Activity Increase 3′-Terminal Oligo(U) Length and La-Dependent tRNA Processing

2005 ◽  
Vol 25 (2) ◽  
pp. 621-636 ◽  
Author(s):  
Ying Huang ◽  
Robert V. Intine ◽  
Amy Mozlin ◽  
Samuel Hasson ◽  
Richard J. Maraia
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Protein A ◽  
Activity Increase ◽  
Cleavage Activity ◽  
Polymerase Iii ◽  
La Protein ◽  
Pol Iii ◽  
And Function ◽  
Zinc Ribbon

ABSTRACT Termination by RNA polymerase III (Pol III) produces RNAs whose 3′ oligo(U) termini are bound by La protein, a chaperone that protects RNAs from 3′ exonucleases and promotes their maturation. Multiple reports indicate that yeasts use La-dependent and -independent pathways for tRNA maturation, with defective pre-tRNAs being most sensitive to decay and most dependent on La for maturation and function. The Rpc11p subunit of Pol III shows homology with the zinc ribbon of TFIIS and is known to mediate RNA 3′ cleavage and to be important for termination. We used a La-dependent opal suppressor, tRNASerUGAM, which suppresses ade6-704 and the accumulation of red pigment, to screen Schizosaccaromyces pombe for rpc11 mutants that increase tRNA-mediated suppression. Analyses of two zinc ribbon mutants indicate that they are deficient in Pol III RNA 3′ cleavage activity and produce pre-tRNASerUGAM transcripts with elongated 3′-oligo(U) tracts that are better substrates for La. A substantial fraction of pre-tRNASerUGAM contains too few 3′ Us for efficient La binding and appears to decay in wild-type cells but has elongated oligo(U) tracts and matures along the La-dependent pathway in the mutants. The data indicate that Rpc11p limits RNA 3′-U length and that this significantly restricts pre-tRNAs to a La-independent pathway of maturation in fission yeast.

scholarly journals Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 in Drosophila

2016 ◽  
Author(s):  
Shrivani Sriskanthadevan-Pirahas ◽  
Rujuta Deshpande ◽  
Byoungchun Lee ◽  
Savraj S. Grewal
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Tissue Growth ◽  
Polymerase Iii ◽  
Kinase Cascade ◽  
Important Challenge ◽  
Pol Iii ◽  
And Function ◽  
Stimulation Of

ABSTRACTThe small G-protein Ras is a conserved regulator of cell and tissue growth. These effects of Ras are mediated largely through activation of a canonical RAF-MEK-ERK kinase cascade. An important challenge is to identify how this Ras/ERK pathway alters cellular metabolism to drive growth. Here we report on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling in Drosophila. We find that activation of Ras/ERK signalling promotes tRNA synthesis both in vivo and in cultured Drosophila S2 cells. We also show that Pol III function is required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells in Drosophila tissues. We find that the transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead we show that the main way that Ras promotes Pol III function and tRNA synthesis is by inhibiting the nuclear localization and function of the Pol III repressor Maf1. We propose that inhibition of Maf1 and stimulation of tRNA synthesis is one way by which Ras signalling enhances protein synthesis to promote cell and tissue growth.

scholarly journals CHD8 Associates with Human Staf and Contributes to Efficient U6 RNA Polymerase III Transcription

2007 ◽  
Vol 27 (24) ◽  
pp. 8729-8738 ◽  
Author(s):  
Chih-Chi Yuan ◽  
Xinyang Zhao ◽  
Laurence Florens ◽  
Selene K. Swanson ◽  
Michael P. Washburn ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Chromatin Remodeling ◽  
Rna Polymerase Iii ◽  
Chromatin Modification ◽  
Small Nuclear Rna ◽  
Polymerase Iii ◽  
Chromatin Template ◽  
Pol Iii

ABSTRACT Chromatin remodeling and histone modification are essential for eukaryotic transcription regulation, but little is known about chromatin-modifying activities acting on RNA polymerase III (Pol III)-transcribed genes. The human U6 small nuclear RNA promoter, located 5′ of the transcription start site, consists of a core region directing basal transcription and an activating region that recruits the transcription factors Oct-1 and Staf (ZNF143). Oct-1 activates transcription in part by helping recruit core binding factors, but nothing is known about the mechanisms of transcription activation by Staf. We show that Staf activates U6 transcription from a preassembled chromatin template in vitro and associates with several proteins linked to chromatin modification, among them chromodomain-helicase-DNA binding protein 8 (CHD8). CHD8 binds to histone H3 di- and trimethylated on lysine 4. It resides on the human U6 promoter as well as the mRNA IRF3 promoter in vivo and contributes to efficient transcription from both these promoters. Thus, Pol III transcription from type 3 promoters uses some of the same factors used for chromatin remodeling at Pol II promoters.

scholarly journals RNA Polymerase III Transcription Factor IIIB Is a Target for Repression by Pocket Proteins p107 and p130

1999 ◽  
Vol 19 (6) ◽  
pp. 4255-4261 ◽  
Author(s):  
Josephine E. Sutcliffe ◽  
Carol A. Cairns ◽  
Angela McLees ◽  
Simon J. Allison ◽  
Kerrie Tosh ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Direct Interaction ◽  
Double Knockout ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Pol Iii ◽  
Related Proteins

ABSTRACT RNA polymerase III (Pol III) transcription is subject to repression by the retinoblastoma protein RB, both in vitro and in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88–90, 1996). This is achieved through a direct interaction between RB and TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061–2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755–14761, 1997). p107 and p130 are two closely related proteins that display 30 to 35% identity with the RB polypeptide and share some of its functions. We show that p107 and p130 can both repress Pol III transcription in transient transfection assays or when added to cell extracts. Pull-down assays and immunoprecipitations using recombinant components demonstrate that a subunit of TFIIIB interacts physically with p107 and p130. In addition, endogenous TFIIIB is shown by cofractionation and coimmunoprecipitation to associate stably with both p107 and p130. Disruption of this interaction in vivo by using the E7 oncoprotein of human papillomavirus results in a marked increase in Pol III transcription. Pol III activity is also deregulated in fibroblasts derived from p107 p130 double knockout mice. We conclude that TFIIIB is targeted for repression not only by RB but also by its relatives p107 and p130.

scholarly journals Functions of paralogous RNA polymerase III subunits POLR3G and POLR3GL in mouse development

2020 ◽  
Vol 117 (27) ◽  
pp. 15702-15711
Author(s):  
Xiaoling Wang ◽  
Alan Gerber ◽  
Wei-Yi Chen ◽  
Robert G. Roeder
Keyword(s):  
Rna Polymerase ◽  
Knockout Mice ◽  
Mammalian Cells ◽  
Target Genes ◽  
Rna Polymerase Iii ◽  
Embryonic Stem ◽  
Mouse Development ◽  
Polymerase Iii ◽  
Pol Iii

Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol IIIα) and the related POLR3GL in the other form (Pol IIIβ). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.

scholarly journals Maf1p, a Negative Effector of RNA Polymerase III inSaccharomyces cerevisiae

2001 ◽  
Vol 21 (15) ◽  
pp. 5031-5040 ◽  
Author(s):  
Krzysztof Pluta ◽  
Olivier Lefebvre ◽  
Nancy C. Martin ◽  
Wieslaw J. Smagowicz ◽  
David R. Stanford ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Growth Defect ◽  
Class Iii ◽  
Gene Encoding ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Pol Iii

ABSTRACT Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

scholarly journals Involvement of RNA Polymerase III in Immune Responses

2015 ◽  
Vol 35 (10) ◽  
pp. 1848-1859 ◽  
Author(s):  
Damian Graczyk ◽  
Robert J. White ◽  
Kevin M. Ryan
Keyword(s):  
Transcription Factor ◽  
Immune Responses ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Trna Genes ◽  
Malignant Cells ◽  
Polymerase Iii ◽  
Mouse Macrophages ◽  
Tightly Coupled ◽  
Pol Iii

Inflammation in the tumor microenvironment has many tumor-promoting effects. In particular, tumor-associated macrophages (TAMs) produce many cytokines which can support tumor growth by promoting survival of malignant cells, angiogenesis, and metastasis. Enhanced cytokine production by TAMs is tightly coupled with protein synthesis. In turn, translation of proteins depends on tRNAs, short abundant transcripts that are made by RNA polymerase III (Pol III). Here, we connect these facts by showing that stimulation of mouse macrophages with lipopolysaccharides (LPS) from the bacterial cell wall causes transcriptional upregulation of tRNA genes. The transcription factor NF-κB is a key transcription factor mediating inflammatory signals, and we report that LPS treatment causes an increased association of the NF-κB subunit p65 with tRNA genes. In addition, we show that p65 can directly associate with the Pol III transcription factor TFIIIB and that overexpression of p65 induces Pol III-dependent transcription. As a consequence of these effects, we show that inhibition of Pol III activity in macrophages restrains cytokine secretion and suppresses phagocytosis, two key functional characteristics of these cells. These findings therefore identify a radical new function for Pol III in the regulation of macrophage function which may be important for the immune responses associated with both normal and malignant cells.

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