Small nuclear RNA genes transcribed by either RNA polymerase II or RNA polymerase III in monocot plants share three promoter elements and use a strategy to regulate gene expression different from that used by their dicot plant counterparts.

RNA polymerase (Pol) II- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes of dicotyledonous plants contain two essential upstream promoter elements, the USE and TATA. The USE is a highly conserved plant snRNA gene-specific element, and its distance from the -30 TATA box, corresponding to approximately three and four helical DNA turns in Pol III and Pol II genes, respectively, is crucial for determining RNA Pol specificity of transcription. Sequences upstream of the USE play no role in snRNA gene transcription in dicot plants. Here we show that for expression of snRNA genes in maize, a monocotyledonous plant, the USE and TATA elements are essential, but not sufficient, for transcription. Efficient expression of both Pol II- and Pol III-specific snRNA genes in transfected maize protoplasts requires an additional element(s) positioned upstream of the USE. This element, named MSP (for monocot-specific promoter; consensus, RGCCCR), is present in one to three copies in monocot snRNA genes and is interchangeable between Pol II- and Pol III-specific genes. The efficiency of snRNA gene expression in maize protoplast is determined primarily by the strength of the MSP element(s); this contrasts with the situation in protoplasts of a dicot plant, Nicotiana plumbaginifolia, where promoter strength is a function of the quality of the USE element. Interestingly, the organization of monocot Pol III-specific snRNA gene promoters closely resembles those of equivalent vertebrate promoters. The data are discussed in the context of the coevolution of Pol II- and Pol III-specific snRNA gene promoters within many eukaryotic organisms.

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Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.2019 ◽

1995 ◽

Vol 15 (4) ◽

pp. 2019-2027 ◽

Cited By ~ 73

Author(s):

J B Yoon ◽

S Murphy ◽

L Bai ◽

Z Wang ◽

R G Roeder

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Small Nuclear Rna ◽

Class Iii ◽

Sequence Element ◽

Pol Ii ◽

Proximal Sequence Element ◽

Nuclear Rna ◽

Class Iii Genes ◽

Pol Iii

The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP-TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific as gene-specific factors for PSE-and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the from used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

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RNA polymerase III-mediated transcription of the trypanosome U2 small nuclear RNA gene is controlled by both intragenic and extragenic regulatory elements.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.2021 ◽

1994 ◽

Vol 14 (3) ◽

pp. 2021-2028 ◽

Cited By ~ 41

Author(s):

A Fantoni ◽

A O Dare ◽

C Tschudi

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Iii ◽

Regulatory Elements ◽

Trna Genes ◽

Regulatory Sequences ◽

Small Nuclear Rna ◽

U2 Snrna ◽

Snrna Gene ◽

Polymerase Iii ◽

Nuclear Rna

Transcription of U2 small nuclear RNA (snRNA) genes in eukaryotes is executed by RNA polymerase II and is dependent on extragenic cis-acting regulatory sequences which are not found in other genes. Here we have mapped promoter elements of the Trypanosoma brucei U2 snRNA gene by transient DNA expression of mutant constructs in insect form trypanosomes. Unlike other eukaryotic U2 snRNA genes, the T. brucei homolog is transcribed by an RNA polymerase III-like enzyme on the basis of its sensitivity to the inhibitors alpha-amanitin and tagetitoxin. Thus, the trypanosome U2 snRNA provides a unique example of an RNA polymerase III transcript carrying a trimethylated cap structure. The promoter of this gene consists of three distinct elements: an intragenic sequence close to the 5' end of the coding region, which is probably required to position the polymerase at the correct transcription start site; and two extragenic elements, located 110 and 160 nucleotides upstream, which are essential for U2 snRNA gene expression. These two elements closely resemble both in sequence and in distance from each other the A and B box consensus sequences of the internal control regions of tRNA genes.

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RNA polymerase III-mediated transcription of the trypanosome U2 small nuclear RNA gene is controlled by both intragenic and extragenic regulatory elements

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.2021-2028.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 2021-2028

Author(s):

A Fantoni ◽

A O Dare ◽

C Tschudi

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Iii ◽

Regulatory Elements ◽

Trna Genes ◽

Regulatory Sequences ◽

Small Nuclear Rna ◽

U2 Snrna ◽

Snrna Gene ◽

Polymerase Iii ◽

Nuclear Rna

Transcription of U2 small nuclear RNA (snRNA) genes in eukaryotes is executed by RNA polymerase II and is dependent on extragenic cis-acting regulatory sequences which are not found in other genes. Here we have mapped promoter elements of the Trypanosoma brucei U2 snRNA gene by transient DNA expression of mutant constructs in insect form trypanosomes. Unlike other eukaryotic U2 snRNA genes, the T. brucei homolog is transcribed by an RNA polymerase III-like enzyme on the basis of its sensitivity to the inhibitors alpha-amanitin and tagetitoxin. Thus, the trypanosome U2 snRNA provides a unique example of an RNA polymerase III transcript carrying a trimethylated cap structure. The promoter of this gene consists of three distinct elements: an intragenic sequence close to the 5' end of the coding region, which is probably required to position the polymerase at the correct transcription start site; and two extragenic elements, located 110 and 160 nucleotides upstream, which are essential for U2 snRNA gene expression. These two elements closely resemble both in sequence and in distance from each other the A and B box consensus sequences of the internal control regions of tRNA genes.

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TATA-binding protein is limiting for both TATA-containing and TATA-lacking RNA polymerase III promoters in Drosophila cells.

Molecular and Cellular Biology ◽

10.1128/mcb.16.12.6909 ◽

1996 ◽

Vol 16 (12) ◽

pp. 6909-6916 ◽

Cited By ~ 27

Author(s):

A Trivedi ◽

A Vilalta ◽

S Gopalan ◽

D L Johnson

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Tata Binding Protein ◽

Gene Promoters ◽

Rna Pol Ii ◽

Pol Ii ◽

Pol Iii ◽

Carboxy Terminal ◽

Drosophila Cells ◽

U6 Rna

We have investigated the role of the TATA-binding protein (TBP) in modulating RNA polymerase (Pol) III gene activity. Epitope-tagged TBP (e-TBP) was both transiently and stably transfected in Drosophila Schneider S-2 cells to increase the total cellular level of TBP. Analysis of the transcripts synthesized from cotransfected tRNA and U6 RNA genes revealed that both types of RNA Pol III promoters were substantially stimulated by an increase in e-TBP in a dose-dependent manner. Furthermore, a TBP-dependent increase in the levels of endogenous tRNA transcripts was produced in the stable line induced to express the e-TBP. We further determined whether the ability of increased TBP to induce RNA Pol III gene expression was due to a direct effect of increased TBP complexes on RNA Pol III gene promoters or an indirect consequence of enhanced expression of RNA Pol II genes. A TBP expression plasmid (e-TBP332), containing a mutation within the highly conserved carboxy-terminal domain, was both transiently and stably transfected into S-2 cells. e-TBP332 augmented the transcription from two RNA Pol II gene promoters indistinguishably from that observed when e-TBP was expressed. In contrast, e-TBP332 was completely defective in its ability to stimulate either the tRNA or U6 RNA gene promoters. In addition, increasing levels of a truncated TBP protein containing only the carboxy-terminal region failed to induce either the tRNA or U6 RNA gene promoter, whereas it retained its ability to stimulate an RNA Pol II promoter. Thus, the TBP-dependent increase in RNA Pol II gene activity is not sufficient for enhanced RNA Pol III gene transcription; rather, a direct effect on RNA Pol III promoters is required. Furthermore, these results provide the first direct evidence that the amino-terminal region of TBP is important for the formation or function of TBP-containing complexes utilized by TATA-less and TATA-containing RNA Pol III promoters. Together, these studies demonstrate that TBP is limiting for the expression of both classes of RNA Pol III promoters in Drosophila cells and implicate an important role for TBP in regulating RNA Pol III gene expression.

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The capped U6 small nuclear RNA is transcribed by RNA polymerase III.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)75890-6 ◽

1987 ◽

Vol 262 (1) ◽

pp. 75-81

Author(s):

R Reddy ◽

D Henning ◽

G Das ◽

M Harless ◽

D Wright

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Iii ◽

Small Nuclear Rna ◽

Polymerase Iii ◽

Nuclear Rna

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Manipulation of RNA Polymerase III by Herpes Simplex Virus-1

10.1101/2021.07.21.453214 ◽

2021 ◽

Author(s):

Sarah E Dremel ◽

Frances L Sivrich ◽

Jessica M Tucker ◽

Britt A Glaunsinger ◽

Neal A DeLuca

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Rna Polymerase ◽

Viral Infection ◽

Rna Polymerase Iii ◽

Herpes Simplex Virus 1 ◽

Pol Ii ◽

Simplex Virus ◽

Pol Iii ◽

Hsv 1

RNA Polymerase III (Pol III) transcribes noncoding RNA, including transfer RNA (tRNA), and acts as a pathogen sensor during the innate immune response. To promote enhanced proliferation, the Pol III machinery is commonly targeted during cancer and viral infection. Herein we employ DM-RNA-Seq, 4SU-Seq, ChIP-Seq, and ATAC-Seq to characterize how Herpes Simplex Virus-1 (HSV-1) perturbs the Pol III landscape. We find that HSV-1 stimulates tRNA expression 10-fold, with mature tRNAs exhibiting a 2-fold increase within 12 hours of infection. Perturbation of host tRNA synthesis requires nuclear viral entry, but not synthesis of specific viral transcripts, nascent viral genomes, or viral progeny. Host tRNA with a specific codon bias were not targeted, rather increased transcription was observed from euchromatic, actively transcribed loci. tRNA upregulation is linked to unique crosstalk between the Pol II and III transcriptional machinery. While viral infection is known to mediate host transcriptional shut off and lead to a depletion of Pol II on host mRNA promoters, we find that Pol II binding to tRNA loci increases. Finally, we report Pol III and associated factors bind the HSV genome, which suggests a previously unrecognized role in HSV-1 gene expression. These data provide insight into novel mechanisms by which HSV-1 alters the host nuclear environment, shifting key processes in favor of the pathogen.

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Engineered mini H1 promoters with dedicated RNA Polymerase II or III activity

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015386 ◽

2020 ◽

pp. jbc.RA120.015386

Author(s):

Zongliang Gao ◽

Yme Ubeles van der Velden ◽

Minghui Fan ◽

Cynthia Alyssa van der Linden ◽

Monique Vink ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Polymerase Iii ◽

Polymerase Activity ◽

Attractive Alternative ◽

Pol Ii ◽

Guide Rnas ◽

Non Coding Rna ◽

Pol Iii ◽

Short Hairpin Rnas

RNA polymerase III promoters such as 7SK, U6 and H1 are widely used for the expression of small non-coding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a non-coding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting the Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly-used H1 promoter in terms of activity and small promoter size, but also concerning safety by exclusive expression of the desired therapeutic transcript (either Pol II or Pol III, but not both).

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Insect small nuclear RNA gene promoters evolve rapidly yet retain conserved features involved in determining promoter activity and RNA polymerase specificity

Nucleic Acids Research ◽

10.1093/nar/gkl982 ◽

2006 ◽

Vol 35 (1) ◽

pp. 21-34 ◽

Cited By ~ 32

Author(s):

Genaro Hernandez ◽

Faramarz Valafar ◽

William E. Stumph

Keyword(s):

Rna Polymerase ◽

Promoter Activity ◽

Gene Promoters ◽

Small Nuclear Rna ◽

Nuclear Rna

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Activity of chimeric U small nuclear RNA (snRNA)/mRNA genes in transfected protoplasts of Nicotiana plumbaginifolia: U snRNA 3'-end formation and transcription initiation can occur independently in plants.

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6403 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6403-6415 ◽

Cited By ~ 7

Author(s):

S Connelly ◽

W Filipowicz

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Gene Promoter ◽

Nicotiana Plumbaginifolia ◽

Gene Promoters ◽

Coding Region ◽

Pol Ii ◽

U2 Snrna ◽

Snrna Gene

Formation of the 3' ends of RNA polymerase II (Pol II)-specific U small nuclear RNAs (U snRNAs) in vertebrate cells is dependent upon transcription initiation from the U snRNA gene promoter. Moreover, U snRNA promoters are unable to direct the synthesis of functional polyadenylated mRNAs. In this work, we have investigated whether U snRNA 3'-end formation and transcription initiation are also coupled in plants. We have first characterized the requirements for 3'-end formation of an Arabidopsis U2 snRNA expressed in transfected protoplasts of Nicotiana plumbaginifolia. We found that the 3'-end-adjacent sequence CA (N)3-10AGTNNAA, conserved in plant Pol II-specific U snRNA genes, is essential for the 3'-end formation of U2 transcripts and, similar to the vertebrate 3' box, is highly tolerant to mutation. The 3'-flanking regions of an Arabidopsis U5 and a maize U2 snRNA gene can effectively substitute for the Arabidopsis U2 3'-end formation signal, indicating that these signals are functionally equivalent among different Pol II-transcribed snRNA genes. The plant U snRNA 3'-end formation signal can be recognized irrespective of whether transcription initiation occurs at U snRNA or mRNA gene promoters, although efficiency of 3' box utilization is higher when transcription initiation occurs at the U snRNA promoter. Moreover, transcripts initiated from the U2 gene promoter can be spliced and polyadenylated. Transcription from a Pol III-specific plant U snRNA gene promoter is not compatible with polyadenylation. Finally, we reveal that initiation at a Pol II-specific plant U snRNA gene promoter can occur in the absence of the snRNA coding region and a functional snRNA 3'-end formation signal, demonstrating that these sequences play no role in determining the RNA polymerase specificity of plant U snRNA genes.

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