RNA Pol III: Historical Experiments and Epigenetics

Gina ViavatteneApril 10, 2015

PART ONE:

Historical Experiments

Finding Three Distinct RNA Polymerases

Prior to 1969, regulation of RNA synthesis in bacteria was believed to be regulated by a single RNA polymerase

Several considerations support the idea that multiple RNA pols exist in eukaryotic cells: (ex: when considering the possibility that a specific enzyme responsible for rRNA synthesis)

Ribosomal RNA has distinct base composition Ribosomal genes localized in nucleoli Synthesis of ribosomal RNA is regulated

independently of other RNA

Finding Three Distinct RNA Polymerases

Previous studies of isolated nuclei show:In the presence of Mg++, ribosomal-like RNA is produced (very GC rich), and activity is localized in nucleolus

In presence of Mn++, more DNA-like RNA is produced (nucleotide ratios similar to that of template DNA), and activity is more dispersed through nucleoplasm

Suggests that the more than one RNA polymerase may exist

However, this effect could also be explained by differential effects of ions, ultimately altering the transcriptional efficiency of a single enzyme

Finding Three Distinct RNA Polymerases

Model Organisms: Sea urchin embryo:

- Other studies have shown independent regulation of rRNA and DNA-like RNA early in development

Adult rat liver- Techniques for isolation of nuclei and nucleoli

already exist

This study developed procedures to solubilize RNA pol activity, and resolve each species using chromatography.

Finding Three Distinct RNA Polymerases

Each species is readily separated from the others by chromatographic procedures

Maintain their distinct properties even after freezing, thawing, etc.

R. G. Roeder, et al., Nature, 1969

Finding Three Distinct RNA Polymerases

Each species is readily separated from the others by chromatographic procedures

Maintain their distinct properties even after freezing, thawing, etc.

R. G. Roeder, et al., Nature, 1969

Finding Three Distinct RNA Polymerases

Each species has its own metal ion profile

I and II ratios are very similar in both organisms

R. G. Roeder, et al., Nature, 1969

Finding Three Distinct RNA Polymerases

Each species has its own salt activity profile

I and II similar in both organisms

R. G. Roeder, et al., Nature, 1969

Finding Three Distinct RNA Polymerases

Relative activity with native and denatured templates is different for each species

R. G. Roeder, et al., Nature, 1969

RNA Pol III TranscriptionFast forward to 2002: Now know that RNA pol III is responsible for transcribing genes encoding structural and catalytic RNAs, all less than 400 bp in length

Three types of promoters (1-3)

Transcription factors responsible for recognizing gene internal and extragenic promoter elements in order to recruit RNA pol III and initiate transcription

The Three Promoter Types

Schramm, L., & Hernandez, N. Genes Dev. (2002)

Type 1 Promoters

First characterized were Xenopus laevis 5S RNA genes

Bogenhagen et al./ Sakonju, et al. described a series of experiments in which they ultimately determine the existence of the intragenic elements necessary for initiation and termination of transcription in the 5S RNA genes.

Created 5’ and 3’ deletion mutants on this gene and provided transcriptional analyses of these mutants which suggested the coding region of the gene was essential for transcription initiation and termination

Type 2 Promoters

Sharp et al. describe how they created 5’ and 3’ deletion mutants (pictured below) within the Drosophila tRNA Arg gene.

Transcriptional analysis of the 5’ clones and 3’ clones ultimately led them to define the outside borders of the internal transcriptional control regions required for tRNA gene transcription.

Type 3 Promoters

Das et al. describe that while the coding regions of genes were sufficient to induce transcription from type 1 and type 2 promoters, the coding sequence of the U6 RNA gene is not necessary to induce transcription.

PART TWO:

RNA Pol III and Epigenetics

RNA Pol III and Epigenetics

• Epigenetics is the study of heritable changes in the genome that occur without altering the DNA sequence

• Epigenetic mechanisms include: ATP-dependent chromatin remodeling, Covalent histone modifications, and DNA methylation

• Selvakumar, et al. report analysis of the human U6 snRNA family implies relationship between gene potency and DNA methylation

DNMTs Regulate Human RNA Pol III Transcription

Red dots = CpG sites(can be methylated)U6-3-4-5 = non-functional (lacking promoter elements)

Methylation of actively transcribed U6 snRNA may regulate gene expression

Selvakumar, et al., J Biol Chem, 2012

DNMTs Regulate Human RNA Pol III Transcription

dAzaC = DNMT inhibitor (no methylation)

After incubation for 5 days, transcription activity significantly increased

Selvakumar, et al., J Biol Chem, 2012

DNMTs Regulate Human RNA Pol III Transcription

In vitro transcription analysis using premethylated U6 reporter plasmid

Premethylated plasmid inhibited U6 snRNA transcription

Selvakumar, et al., J Biol Chem, 2012

Tumor Suppressor RB Enables DNA Methylation

Selvakumar, et al., J Biol Chem, 2012

Western Blot showing RB expression in untreated HeLa cells, or cells transiently transfected with the RB expression vector, as well as cells transfected with empty vector.

In summaryIn the late 1960s and early 1970s, three distinct RNA polymerases were identified in eukaryotic organisms.

In the 1980s, more intricate details about RNA pol III transcription were discovered, including the various promoter structures, characterization of the transcription factors, and the mechanisms by which transcription initiated.

More recently, studies linking RNA pol III transcription and epigenetic modifications have begun to surface.

One such modification is DNA methylation, and the Selvakumar, et al. study is a fine example showing probable regulation of RNA pol III transcription by DNMTs.

This can also be expanded to possibly explain how tumor suppressors like RB function to regulate proper cell growth

• Roeder, R. G. & Rutter, W. J. Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms. Nature 224, 234–237 (1969)

• Kedinger, C.,et al. α-amanitin: a specific inhibitor of one of two DNA-dependent RNA polymerase activities from calf thymus. Biochem. Biophys. Res. Commun. 38, 165–171 (1970)

• Pogo, A.O., et al. Modification of Ribonucleic Acid Synthesis in Nuclei Isolated from Normal and Regenerating Liver. PNAS 57 (3), 743-750 (1967)

• Schramm, L., & Hernandez, N. Recruitment of RNA polymerase III to its target promoters. Genes Dev. (2002)

• Bogenhagen, et al. A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3’ Border of the region. Cell 19, 27-35 (1980)

• Sakonju, et al. A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3’ Border of the region. Cell 19, 13-25 (1980)

• Sharp, et al. Internal control regions for transcription of eukaryituc tRBA gebes. PNAS 78, 6657-6661 (1981)

• Das, et al. Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III. EMBO 7, 503-512 (1988)

• Selvakumar, et al. Regulation of Human RNA Polymerase III Transcription by DNMT1 and DNMT3a DNA Methyltransferases. J Biol Chem. 287(10): 7039–7050 (2012)

References