Thiolutin has complex effects in vivo but is a direct inhibitor of RNA polymerase II in vitro.

Thiolutin is a natural product transcription inhibitor with an unresolved mode of action. Thiolutin and the related dithiolopyrrolone holomycin chelate Zn2+ and previous studies have concluded that RNA Polymerase II (Pol II) inhibition in vivo is indirect. Here, we present chemicogenetic and biochemical approaches to investigate thiolutin's mode of action in Saccharomyces cerevisiae. We identify mutants that alter sensitivity to thiolutin. We provide genetic evidence that thiolutin causes oxidation of thioredoxins in vivo and that thiolutin both induces oxidative stress and interacts functionally with multiple metals including Mn2+ and Cu2+, and not just Zn2+. Finally, we show direct inhibition of RNA polymerase II (Pol II) transcription initiation by thiolutin in vitro in support of classical studies that thiolutin can directly inhibit transcription in vitro. Inhibition requires both Mn2+ and appropriate reduction of thiolutin as excess DTT abrogates its effects. Pause prone, defective elongation can be observed in vitro if inhibition is bypassed. Thiolutin effects on Pol II occupancy in vivo are widespread but major effects are consistent with prior observations for Tor pathway inhibition and stress induction, suggesting that thiolutin use in vivo should be restricted to studies on its modes of action and not as an experimental tool.

Dissecting the Pol II transcription cycle and derailing cancer with CDK inhibitors.

Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription. Here we review the molecular mechanisms by which specific CDKs are thought to act at discrete steps in the transcription cycle and describe the recent emergence of transcriptional CDKs as promising drug targets in cancer. We emphasize recent advances in understanding the transcriptional CDK network that were facilitated by development and deployment of small-molecule inhibitors with increased selectivity for individual CDKs. Unexpectedly, several of these compounds have also shown selectivity in killing cancer cells, despite the seemingly universal involvement of their target CDKs during transcription in all cells. Finally, we describe remaining and emerging challenges in defining functions of individual CDKs in transcription and co-transcriptional processes and in leveraging CDK inhibition for therapeutic purposes.

TP53 loss creates therapeutic vulnerability in colorectal cancer.

TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

Remodeling of the enhancer landscape during macrophage activation is coupled to enhancer transcription.

Recent studies suggest a hierarchical model in which lineage-determining factors act in a collaborative manner to select and prime cell-specific enhancers, thereby enabling signal-dependent transcription factors to bind and function in a cell-type-specific manner. Consistent with this model, TLR4 signaling primarily regulates macrophage gene expression through a pre-existing enhancer landscape. However, TLR4 signaling also induces priming of ∼3,000 enhancer-like regions de novo, enabling visualization of intermediates in enhancer selection and activation. Unexpectedly, we find that enhancer transcription precedes local mono- and dimethylation of histone H3 lysine 4 (H3K4me1/2). H3K4 methylation at de novo enhancers is primarily dependent on the histone methyltransferases Mll1, Mll2/4, and Mll3 and is significantly reduced by inhibition of RNA polymerase II elongation. Collectively, these findings suggest an essential role of enhancer transcription in H3K4me1/2 deposition at de novo enhancers that is independent of potential functions of the resulting eRNA transcripts.

The CDK7 inhibitor THZ1 alters RNA polymerase dynamics at the 5' and 3' ends of genes.

The t(8;21) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). We found that t(8;21) AML were extremely sensitive to THZ1, which triggered apoptosis after only 4 h. We used precision nuclear run-on transcription sequencing (PROseq) to define the global effects of THZ1 and other CDK inhibitors on RNA polymerase II dynamics. Inhibition of CDK7 using THZ1 caused wide-spread loss of promoter-proximal paused RNA polymerase. This loss of 5' pausing was associated with accumulation of polymerases in the body of a large number of genes. However, there were modest effects on genes regulated by 'super-enhancers'. At the 3' ends of genes, treatment with THZ1 suppressed RNA polymerase 'read through' at the end of the last exon, which resembled a phenotype associated with a mutant RNA polymerase with slower elongation rates. Consistent with this hypothesis, polyA site-sequencing (PolyA-seq) did not detect differences in poly A sites after THZ1 treatment. PROseq analysis after short treatments with THZ1 suggested that these 3' effects were due to altered CDK7 activity at the 5' end of long genes, and were likely to be due to slower rates of elongation.

Tyrosine kinase inhibitors induce alternative spliced BCR-ABLIns35bp variant via inhibition of RNA polymerase II on genomic BCR-ABL.

To elucidate dynamic changes in native BCR-ABL and alternatively spliced tyrosine kinase inhibitor (TKI)-resistant but function-dead BCR-ABLIns35bp variant, following commencement or discontinuation of TKI therapy, each transcript was serially quantified in patients with chronic myeloid leukemia (CML) by deep sequencing. Because both transcripts were amplified together using conventional PCR system for measuring International Scale (IS), deep sequencing method was used for quantifying such BCR-ABL variants. At the initial diagnosis, 7 of 9 patients presented a small fraction of cells possessing BCR-ABLIns35bp , accounting for 0.8% of the total IS BCR-ABL, corresponding to actual BCR-ABLIns35bp value of 1.1539% IS. TKI rapidly decreased native BCR-ABL but not BCR-ABLIns35bp , leading to the initial increase in the proportion of BCR-ABLIns35bp . Thereafter, both native BCR-ABL and BCR-ABLIns35bp gradually decreased in the course of TKI treatment, whereas small populations positive for TKI-resistant BCR-ABLIns35bp continued fluctuating at low levels, possibly underestimating the molecular response (MR). Following TKI discontinuation, sequencing analysis of 54 patients revealed a rapid relapse, apparently derived from native BCR-ABL+ clones. However, IS fluctuating at low levels around MR4.0 marked a predominant persistence of cells expressing function-dead BCR-ABLIns35bp , suggesting that TKI resumption was unnecessary. We clarified the possible mechanism underlying mis-splicing BCR-ABLIns35bp , occurring at the particular pseudo-splice site within intron8, which can be augmented by TKI treatment through inhibition of RNA polymerase II phosphorylation. No mutations were found in spliceosomal genes. Therefore, monitoring IS functional BCR-ABL extracting BCR-ABLIns35bp would lead us to a correct evaluation of MR status, thus determining the adequate therapeutic intervention.

Low-dose triptolide enhances antitumor effect of JQ1 on acute myeloid leukemia through inhibiting RNA polymerase II in vitro and in vivo.

The bromodomain and extra-terminal (BET) domain inhibitor JQ1 exerts potent anticancer activity in various cancer cells. However, the resistance to BET inhibitors in leukemia stem cells limits its implication in acute myeloid leukemia (AML). High concentration of triptolide (TPL) presents anticancer activities but with adverse effects. Here, we investigated whether the combination of low-dose TPL with JQ1 could help to circumvent the dilemma of drug resistance and side effect in treating AML. AML cell lines, primary cells from 10 AML patients with different status, as well as AML mice model were subjected to different treatments and apoptotic related protein expression were evaluated. Data showed that low-dose TPL combined with JQ1 effectively killed AML cell lines and primary cells from AML patients without exerting significantly greater lethal activity against normal cells. Mechanism study revealed that low-dose TPL combined with JQ1 triggered reactive oxygen species production and induced mitochondrial-mediated apoptosis in AML cells, in which the inhibition of RNA polymerase II to downregulate c-Myc was mainly responsible for the enhanced activity of TPL in combination with JQ1. In vivo study presented that cotreatment with low-dose TPL and JQ1 significantly reduced tumor burden of the NOD/SCID mice engrafted with MOLM-13 cells. In conclusion, low-dose TPL enhanced the antitumor effect of JQ1 on AML without increasing the side effects, supporting a potential option for AML treatment.

Analysis of RNA polymerase II ubiquitylation and proteasomal degradation.

Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called "last resort pathway", which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation. Different approaches have been described to study RNAPII poly-ubiquitylation and degradation, each method with its own advantages and caveats. Here, we describe optimised strategies for detecting ubiquitylated RNAPII and studying its degradation, but these protocols are suitable for studying other ubiquitylated proteins as well.

A review on kinases phosphorylating the carboxyl-terminal domain of RNA polymerase II-Biological functions and inhibitors.

RNA polymerase II (RNA Pol II) plays a major role in gene transcription for eukaryote. One of the major modes of regulation in eukaryotes is the phosphorylation of the carboxyl-terminal domain (CTD) of RNA Pol II. The current study found that the phosphorylation of Ser2, Ser5, Ser7, Thr4 and Tyr1 among the heptapeptide repeats of CTD plays a key role in the transcription process. We therefore review the biological functions and inhibitors of kinases that phosphorylate these amino acid residues including transcriptional cyclin-dependent protein kinases (CDKs), bromodomain-containing protein 4 (BRD4), Polo-like kinases 3 (Plk3) and Abelson murine leukemia viral oncogene 1 and 2 (c-Abl1/2).

Mechanism of RNA polymerase II stalling by DNA alkylation.

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

Cryo-EM structure of a mammalian RNA polymerase II elongation complex inhibited by α-amanitin.

RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, we present the high-resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II but forms additional contacts with metazoan-specific residues, which explains why its affinity to mammalian Pol II is ∼3000 times higher than for yeast Pol II. Our work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.

Alu element-containing RNAs maintain nucleolar structure and function.

Non-coding RNAs play a key role in organizing the nucleus into functional subcompartments. By combining fluorescence microscopy and RNA deep-sequencing-based analysis, we found that RNA polymerase II transcripts originating from intronic Alu elements (aluRNAs) were enriched in the nucleolus. Antisense-oligo-mediated depletion of aluRNAs or drug-induced inhibition of RNA polymerase II activity disrupted nucleolar structure and impaired RNA polymerase I-dependent transcription of rRNA genes. In contrast, overexpression of a prototypic aluRNA sequence increased both nucleolus size and levels of pre-rRNA, suggesting a functional link between aluRNA, nucleolus integrity and pre-rRNA synthesis. Furthermore, we show that aluRNAs interact with nucleolin and target ectopic genomic loci to the nucleolus. Our study suggests an aluRNA-based mechanism that links RNA polymerase I and II activities and modulates nucleolar structure and rRNA production.

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription.

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

Protein network construction using reverse phase protein array data.

In this paper, we introduce a novel computational method for constructing protein networks based on reverse phase protein array (RPPA) data to identify complex patterns in protein signaling. The method is applied to phosphoproteomic profiles of basal expression and activation/phosphorylation of 76 key signaling proteins in three breast cancer cell lines (MCF7, LCC1, and LCC9). Temporal RPPA data are acquired at 48h, 96h, and 144h after knocking down four genes in separate experiments. These genes are selected from a previous study as important determinants for breast cancer survival. Interaction networks are constructed by analyzing the expression levels of protein pairs using a multivariate analysis of variance model. A new scoring criterion is introduced to determine relevant protein pairs. Through a network topology based analysis, we search for wiring patterns to identify key proteins that are associated with significant changes in expression levels across various experimental conditions.

The Nonbulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in Vitro.

The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impedes transcription, which could make each a viable substrate for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II. These constructs were incubated in HeLa nuclear extracts that contained active RNA polymerase II, and the resulting transcripts were resolved by denaturing polyacrylamide gel electrophoresis. The structurally rigid Sp strongly blocks transcription elongation, permitting 1.6 ± 0.5% nominal lesion bypass. In contrast, the conformationally flexible Gh poses less of a block to human RNAPII, allowing 9 ± 2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is minimally affected by glycosylase activity found in the HeLa nuclear extract. These data specifically suggest that both Sp and Gh may well be susceptible to TCR because each poses a significant block to human RNA polymerase II progression. A more general principle is also proposed: Conformational flexibility may be an important structural feature of DNA lesions that enhances their transcriptional bypass.

Biochemical Analysis of Yeast Suppressor of Ty 4/5 (Spt4/5) Reveals the Importance of Nucleic Acid Interactions in the Prevention of RNA Polymerase II Arrest.

RNA polymerase II (RNAPII) undergoes structural changes during the transitions from initiation, elongation, and termination, which are aided by a collection of proteins called elongation factors. NusG/Spt5 is the only elongation factor conserved in all domains of life. Although much information exists about the interactions between NusG/Spt5 and RNA polymerase in prokaryotes, little is known about how the binding of eukaryotic Spt4/5 affects the biochemical activities of RNAPII. We characterized the activities of Spt4/5 and interrogated the structural features of Spt5 required for it to interact with elongation complexes, bind nucleic acids, and promote transcription elongation. The eukaryotic specific regions of Spt5 containing the Kyrpides, Ouzounis, Woese domains are involved in stabilizing the association with the RNAPII elongation complex, which also requires the presence of the nascent transcript. Interestingly, we identify a region within the conserved NusG N-terminal (NGN) domain of Spt5 that contacts the non-template strand of DNA both upstream of RNAPII and in the transcription bubble. Mutating charged residues in this region of Spt5 did not prevent Spt4/5 binding to elongation complexes, but abrogated the cross-linking of Spt5 to DNA and the anti-arrest properties of Spt4/5, thus suggesting that contact between Spt5 (NGN) and DNA is required for Spt4/5 to promote elongation. We propose that the mechanism of how Spt5/NGN promotes elongation is fundamentally conserved; however, the eukaryotic specific regions of the protein evolved so that it can serve as a platform for other elongation factors and maintain its association with RNAPII as it navigates genomes packaged into chromatin.

High-throughput single-molecule screen for small-molecule perturbation of splicing and transcription kinetics.

In eukaryotes, mRNA synthesis is catalyzed by RNA polymerase II and involves several distinct steps, including transcript initiation, elongation, cleavage, and transcript release. Splicing of RNA can occur during (co-transcriptional) or after (post-transcriptional) RNA synthesis. Thus, RNA synthesis and processing occurs through the concerted activity of dozens of enzymes, each of which is potentially susceptible to perturbation by small molecules. However, there are few, if any, high-throughput screening strategies for identifying drugs which perturb a specific step in RNA synthesis and processing. Here we have developed a high-throughput fluorescence microscopy approach in single cells to screen for inhibitors of specific enzymatic steps in RNA synthesis and processing. By utilizing the high affinity interaction between bacteriophage capsid proteins (MS2, PP7) and RNA stem loops, we are able to fluorescently label the intron and exon of a ß-globin reporter gene in human cells. This approach allows one to measure the kinetics of transcription, splicing and release in both fixed and living cells using a tractable, genetically encoded assay in a stable cell line. We tested this reagent in a targeted screen of molecules that target chromatin readers and writers and identified three compounds that slow transcription elongation without changing transcription initiation.

RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template.

Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

Small regulatory RNAs inhibit RNA polymerase II during the elongation phase of transcription.

Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defence against parasitic nucleic acids and genome rearrangement. Many small regulatory RNAs are thought to function in nuclei. For instance, in plants and fungi, short interfering RNA (siRNAs) associate with nascent transcripts and direct chromatin and/or DNA modifications. To understand further the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in Caenorhabditis elegans nuclei. Here we show that the gene nuclear RNAi defective-2 (nrde-2) encodes an evolutionarily conserved protein that is required for siRNA-mediated silencing in nuclei. NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi. We find that nuclear-localized siRNAs direct an NRDE-2-dependent silencing of pre-messenger RNAs (pre-mRNAs) 3' to sites of RNAi, an NRDE-2-dependent accumulation of RNA polymerase (RNAP) II at genomic loci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 3' to sites of RNAi. These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally. In addition, these results establish a novel mode of RNAP II regulation: siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.

Modular RNA heats up.

In this issue of Molecular Cell, Mariner et al. (2008) demonstrate that Alu RNA from a human SINE represses RNA polymerase II transcription during heat shock. This noncoding RNA is the first example of a "protein-like" transcription factor with a distinct modular architecture.