Nab3 nuclear granule accumulation is driven by respiratory capacity.

Numerous biological processes involve proteins capable of transiently assembling into subcellular compartments necessary for cellular functions. One process is the RNA polymerase II transcription cycle which involves initiation, elongation, co-transcriptional modification of nascent RNA, and termination. The essential yeast transcription termination factor Nab3 is required for termination of small non-coding RNAs and accumulates into a compact nuclear granule upon glucose removal.

Variable penetrance of Nab3 granule accumulation quantified by a new tool for high-throughput single-cell granule analysis.

Reorganization of cellular proteins into subcellular compartments, such as the concentration of RNA-binding proteins into cytoplasmic stress granules and P-bodies, is a well-recognized, widely studied physiological process currently under intense investigation. One example of this is the induction of the yeast Nab3 transcription termination factor to rearrange from its pan-nucleoplasmic distribution to a granule at the nuclear periphery in response to nutrient limitation. Recent work in many cell types has shown that protein condensation in the nucleus is functionally important for transcription initiation, RNA processing, and termination.

Recent advances in understanding RNA polymerase II structure and function.

More than 50 years after the identification of RNA polymerase II, the enzyme responsible for the transcription of most eukaryotic genes, studies have continued to reveal fresh aspects of its structure and regulation. New technologies, coupled with years of development of a vast catalog of RNA polymerase II accessory proteins and activities, have led to new revelations about the transcription process. The maturation of cryo-electron microscopy as a tool for unraveling the detailed structure of large molecular machines has provided numerous structures of the enzyme and its accessory factors.

A fluorescent assay for the genetic dissection of the RNA polymerase II termination machinery.

RNA polymerase II is a highly processive enzyme that synthesizes mRNAs and some non-protein coding RNAs. Termination of transcription, which entails release of the transcript and disengagement of the polymerase, requires an active process. In yeast, there are at least two multi-protein complexes needed for termination of transcription, depending upon which class of RNAs are being acted upon.

Nab3's localization to a nuclear granule in response to nutrient deprivation is determined by its essential prion-like domain.

Ribonucleoprotein (RNP) granules are higher order assemblies of RNA, RNA-binding proteins, and other proteins, that regulate the transcriptome and protect RNAs from environmental challenge. There is a diverse range of RNP granules, many cytoplasmic, which provide various levels of regulation of RNA metabolism. Here we present evidence that the yeast transcription termination factor, Nab3, is targeted to intranuclear granules in response to glucose starvation by Nab3's proline/glutamine-rich, prion-like domain (PrLD) which can assemble into amyloid in vitro.

Compositional complexity of rods and rings.

Rods and rings (RRs) are large linear- or circular-shaped structures typically described as polymers of IMPDH (inosine monophosphate dehydrogenase). They have been observed across a wide variety of cell types and species and can be induced to form by inhibitors of IMPDH. RRs are thought to play a role in the regulation of de novo guanine nucleotide synthesis; however, the function and regulation of RRs is poorly understood.

The hnRNP-like Nab3 termination factor can employ heterologous prion-like domains in place of its own essential low complexity domain.

Many RNA-binding proteins possess domains with a biased amino acid content. A common property of these low complexity domains (LCDs) is that they assemble into an ordered amyloid form, juxtaposing RNA recognition motifs in a subcellular compartment in which RNA metabolism is focused. Yeast Nab3 is one such protein that contains RNA-binding domains and a low complexity, glutamine/proline-rich, prion-like domain that can self-assemble.

Recent advances in understanding transcription termination by RNA polymerase II.

Transcription termination is a fundamental process in which RNA polymerase ceases RNA chain extension and dissociates from the chromatin template, thereby defining the end of the transcription unit. Our understanding of the biological role and functional importance of termination by RNA polymerase II and the range of processes in which it is involved has grown significantly in recent years. A large set of nucleic acid-binding proteins and enzymes have been identified as part of the termination machinery.

Determinants of Amyloid Formation for the Yeast Termination Factor Nab3.

Low complexity protein sequences are often intrinsically unstructured and many have the potential to polymerize into amyloid aggregates including filaments and hydrogels. RNA-binding proteins are unusually enriched in such sequences raising the question as to what function these domains serve in RNA metabolism. One such yeast protein, Nab3, is an 802 amino acid termination factor that contains an RNA recognition motif and a glutamine/proline rich domain adjacent to a region with structural similarity to a human hnRNP.

Termination of Transcription of Short Noncoding RNAs by RNA Polymerase II.

The RNA polymerase II transcription cycle is often divided into three major stages: initiation, elongation, and termination. Research over the last decade has blurred these divisions and emphasized the tightly regulated transitions that occur as RNA polymerase II synthesizes a transcript from start to finish. Transcription termination, the process that marks the end of transcription elongation, is regulated by proteins that interact with the polymerase, nascent transcript, and/or chromatin template.

Amyloid-like assembly of the low complexity domain of yeast Nab3.

Termination of transcription of short non-coding RNAs is carried out in yeast by the Nab3-Nrd1-Sen1 complex. Nab3 and Nrd1 are hnRNP-like proteins that dimerize and bind RNA with sequence specificity. We show here that an essential region of Nab3 that is predicted to be prion-like based upon its sequence bias, formed amyloid-like filaments.

A network of interdependent molecular interactions describes a higher order Nrd1-Nab3 complex involved in yeast transcription termination.

Nab3 and Nrd1 are yeast heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins that heterodimerize and bind RNA. Genetic and biochemical evidence reveals that they are integral to the termination of transcription of short non-coding RNAs by RNA polymerase II. Here we define a Nab3 mutation (nab3Δ134) that removes an essential part of the protein's C terminus but nevertheless can rescue, in trans, the phenotype resulting from a mutation in the RNA recognition motif of Nab3.

Yeast Nab3 protein contains a self-assembly domain found in human heterogeneous nuclear ribonucleoprotein-C (hnRNP-C) that is necessary for transcription termination.

Nab3 is an RNA-binding protein whose function is important for terminating transcription by RNA polymerase II. It co-assembles with Nrd1, and the resulting heterodimer of these heterogeneous nuclear ribonucleoprotein-C (hnRNP)-like proteins interacts with the nascent transcript and RNA polymerase II. Previous genetic analysis showed that a short carboxyl-terminal region of Nab3 is functionally important for termination and is located far from the Nab3 RNA recognition domain in the primary sequence.

Decapping goes nuclear.

In this issue of Molecular Cell, Brannan et al. (2012) propose a novel function for RNA-decapping and transcription termination in aborting "divergent" promoter-proximal elongation complexes.

A genetic screen for terminator function in yeast identifies a role for a new functional domain in termination factor Nab3.

The yeast IMD2 gene encodes an enzyme involved in GTP synthesis. Its expression is controlled by guanine nucleotides through a set of alternate start sites and an intervening transcriptional terminator. In the off state, transcription results in a short non-coding RNA that starts upstream of the gene.

Properties of an intergenic terminator and start site switch that regulate IMD2 transcription in yeast.

The IMD2 gene in Saccharomyces cerevisiae is regulated by intracellular guanine nucleotides. Regulation is exerted through the choice of alternative transcription start sites that results in synthesis of either an unstable short transcript terminating upstream of the start codon or a full-length productive IMD2 mRNA. Start site selection is dictated by the intracellular guanine nucleotide levels.

Metabolic regulation of IMD2 transcription and an unusual DNA element that generates short transcripts.

Transcriptional regulation of IMD2 in yeast (Saccharomyces cerevisiae) is governed by the concentration of intracellular guanine nucleotide pools. The mechanism by which pool size is measured and transduced to the transcriptional apparatus is unknown. Here we show that DNA sequences surrounding the IMD2 initiation site constitute a repressive element (RE) involved in guanine regulation that contains a novel transcription-blocking activity.

The gene encoding the fragile X RNA-binding protein is controlled by nuclear respiratory factor 2 and the CREB family of transcription factors.

FMR1 encodes an RNA-binding protein whose absence results in fragile X mental retardation. In most patients, the FMR1 gene is cytosine-methylated and transcriptionally inactive. NRF-1 and Sp1 are known to bind and stimulate the active, but not the methylated/silenced, FMR1 promoter.

Dissection of the molecular basis of mycophenolate resistance in Saccharomyces cerevisiae.

IMP dehydrogenase (IMPDH) is required for the de novo synthesis of guanine nucleotides. While most invertebrates have one IMPDH gene and humans and mice have two, Saccharomyces cerevisiae contains four, IMD1-IMD4. Although Imd2 is 92% identical to Imd3, it is the only S.

Detection of the mycophenolate-inhibited form of IMP dehydrogenase in vivo.

IMP dehydrogenase (IMPDH) is the rate-limiting enzyme for de novo GMP synthesis. Its activity is correlated with cell growth, and it is the target of a number of proven and experimental drug therapies including mycophenolic acid (MPA). MPA inhibits the enzyme by trapping a covalent nucleotide-enzyme intermediate.

Occupancy and synergistic activation of the FMR1 promoter by Nrf-1 and Sp1 in vivo.

Fragile X syndrome is due to mutation of the FMR1 gene. The most common mutation is an expansion of a CGG repeat in the 5' UTR that triggers dense DNA methylation and formation of a heterochromatin-like structure which lead to transcriptional silencing. In vitro experiments have identified several transcription factors, including Sp1, Nrf-1 and USF1/2, as potential regulators of normal FMR1 promoter activity.

Large-scale screening of yeast mutants for sensitivity to the IMP dehydrogenase inhibitor 6-azauracil.

Mutations in several genes encoding components of the RNA polymerase II elongation machinery render S. cerevisiae cells sensitive to the drug 6-azauracil (6AU), an inhibitor of IMP dehydrogenase and orotidylate decarboxylase. It is thought that a reduction in nucleotide levels following drug treatment causes transcriptional elongation to be more dependent on a fully functional RNA polymerase.

Functional distinctions between IMP dehydrogenase genes in providing mycophenolate resistance and guanine prototrophy to yeast.

IMP dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo synthesis of GTP. Yeast with mutations in the transcription elongation machinery are sensitive to inhibitors of this enzyme such as 6-azauracil and mycophenolic acid, at least partly because of their inability to transcriptionally induce IMPDH. To understand the molecular basis of this drug-sensitive phenotype, we have dissected the expression and function of a four-gene family in yeast called IMD1 through IMD4.

Histone modifications depict an aberrantly heterochromatinized FMR1 gene in fragile x syndrome.

Fragile X syndrome is caused by an expansion of a polymorphic CGG triplet repeat that results in silencing of FMR1 expression. This expansion triggers methylation of FMR1's CpG island, hypoacetylation of associated histones, and chromatin condensation, all characteristics of a transcriptionally inactive gene. Here, we show that there is a graded spectrum of histone H4 acetylation that is proportional to CGG repeat length and that correlates with responsiveness of the gene to DNA demethylation but not with chromatin condensation.