New roles for elongation factors in RNA polymerase II ubiquitylation and degradation.

RNA polymerase II (RNAPII) encounters numerous impediments on its way to completing mRNA synthesis across a gene. Paused and arrested RNAPII are reactivated or rescued by elongation factors that travel with polymerase as it transcribes DNA. However, when RNAPII fails to resume transcription, such as when it encounters an unrepairable bulky DNA lesion, it is removed by the targeting of its largest subunit, Rpb1, for degradation by the ubiquitin-proteasome system (UPS). We are starting to understand this process better and how the UPS marks Rbp1 for degradation. This review will focus on the latest developments and describe new functions for elongation factors that were once thought to only promote elongation in unstressed conditions in the removal and degradation of RNAPII. I propose that in addition to changes in RNAPII structure, the composition and modification of elongation factors in the elongation complex determine whether to rescue or degrade RNAPII.

Examining the influence of irritability and ADHD on domains of parenting stress.

Parents of children with ADHD typically report higher levels of parenting stress than parents of typically developing children. Children with ADHD display developmentally inappropriate levels of hyperactivity, impulsivity, and inattention. Some children with ADHD are also prone to particularly high levels of tonic irritability that may explain some of the impairments typically found in ADHD. The present study sought to determine the unique impact of ADHD and tonic irritability on child-related parenting stress domains (e.g., difficult child, parent-child dysfunctional interactions). 145 mothers of children with and without ADHD aged 7-12 years participated in the current study. Mothers completed self-report measures of parenting stress as well as a diagnostic structured interview. Ecological momentary assessment (EMA) was used to assess tonic irritability in an ecological environment. Indirect effects models were specified using PROCESS Model 4. For the parent-child dysfunctional interaction domain, the data were best fit by a model specifying a significant total effect of ADHD that was fully accounted for by an indirect effect through irritability. For the difficult child domain, model testing indicated a significant total effect of ADHD that was partially accounted for by an indirect effect through irritability. The current study adds support to the growing body of literature acknowledging the role of tonic irritability in children with ADHD. Furthermore, the results provide novel insight in the complex relation of irritability, child ADHD, and domains of parenting stress.

DEF1: Much more than an RNA polymerase degradation factor.

Degradation Factor 1 was discovered 20 years ago as a yeast protein copurifying with Rad26, a helicase involved in transcription-coupled DNA repair. It was subsequently shown to control the ubiquitylation and destruction of the large subunit of DNA damage-arrested RNA Polymerase II. Since that time, much has been learned about Def1's role in polymerase destruction and new functions of the protein have been revealed. We now understand that Def1 is involved in more than just RNA polymerase II regulation. Most of its known functions are associated with maintaining chromosome and genomic integrity, but other exciting activities outside this realm have been suggested. Here we review this fascinating protein, describe its regulation and present a hypothesis that Def1 is a central coordinator of ubiquitin signaling pathways in cells.

Nucleosome Dynamics during Transcription Elongation.

The nucleosome is the basic packing unit of the eukaryotic genome. Dynamic interactions between DNA and histones in the nucleosome are the molecular basis of gene accessibility regulation that governs the kinetics of various DNA-templated processes such as transcription elongation by RNA Polymerase II (Pol II). On the basis of single-molecule FRET measurements with chemically modified histones, we investigated the nucleosome dynamics during transcription elongation and how it is affected by histone acetylation at H3 K56 and the histone chaperone Nap1, both of which can affect DNA-histone interactions. We observed that H3K56 acetylation dramatically shortens the pause duration of Pol II near the entry region of the nucleosome, while Nap1 induces no noticeable difference. We also found that the elongation rate of Pol II through the nucleosome is unaffected by the acetylation or Nap1. These results indicate that H3K56 acetylation facilitates Pol II translocation through the nucleosome by assisting paused Pol II to resume and that Nap1 does not affect Pol II progression. Following transcription, only a small fraction of nucleosomes remain intact, which is unaffected by H3K56 acetylation or Nap1. These results suggest that (i) spontaneous nucleosome opening enables Pol II progression, (ii) Pol II mediates nucleosome reassembly very inefficiently, and (iii) Nap1 in the absence of other factors does not promote nucleosome disassembly or reassembly during transcription.

Ccr4-Not maintains genomic integrity by controlling the ubiquitylation and degradation of arrested RNAPII.

The Ccr4-Not complex regulates essentially every aspect of gene expression, from mRNA synthesis to protein destruction. The Not4 subunit of the complex contains an E3 RING domain and targets proteins for ubiquitin-dependent proteolysis. Ccr4-Not associates with elongating RNA polymerase II (RNAPII), which raises the possibility that it controls the degradation of elongation complex components. Here, we demonstrate that Ccr4-Not controls the ubiquitylation and turnover of Rpb1, the largest subunit of RNAPII, during transcription arrest. Deleting NOT4 or mutating its RING domain strongly reduced the DNA damage-dependent ubiquitylation and destruction of Rpb1. Surprisingly, in vitro ubiquitylation assays indicate that Ccr4-Not does not directly ubiquitylate Rpb1 but instead promotes Rpb1 ubiquitylation by the HECT domain-containing ligase Rsp5. Genetic analyses suggest that Ccr4-Not acts upstream of RSP5, where it acts to initiate the destruction process. Ccr4-Not binds Rsp5 and forms a ternary complex with it and the RNAPII elongation complex. Analysis of mutant Ccr4-Not lacking the RING domain of Not4 suggests that it both recruits Rsp5 and delivers the E2 Ubc4/5 to RNAPII. Our work reveals a previously unknown function of Ccr4-Not and identifies an essential new regulator of RNAPII turnover during genotoxic stress.

Temperamental negative affect, emotion-specific regulation, and concurrent internalizing and externalizing pathology among children with ADHD.

Children with attention-deficit/hyperactivity disorder (ADHD) experience high rates of temperamental negative affect and comorbid internalizing and externalizing pathology. The current study explored the role of emotion-specific regulation in accounting for the link between temperamental negative affect and psychopathology among children with ADHD. Forty parents of children ages 8-11 (N =29 males, N =11 females) completed measures of child temperament, emotion-specific dysregulation (i.e., anger dysregulation, sadness dysregulation), and psychopathology. Children completed a measure of emotion-specific dysregulation. Results revealed that anger dysregulation fully statistically accounted for the relationship between temperamental negative affect and concurrent externalizing problems. Sadness dysregulation did not account for the relationship between temperamental negative affect and internalizing problems. These novel findings implicate the robust role of anger dysregulation in explaining the link between temperamental negative affect and concurrent externalizing pathology. The results of this study have significant implications for the treatment of emotionally driven externalizing behavior among children with ADHD.

Biochemical methods to characterize RNA polymerase II elongation complexes.

Transcription of DNA into RNA is critical for all life, and RNA polymerases are enzymes tasked with this activity. In eukaryotes, RNA Polymerase II (RNAPII) is responsible for transcription of all protein coding genes and many non-coding RNAs. RNAPII carries out the remarkable feat of unwinding the stable double-stranded DNA template, synthesizing the transcript and re-forming the double helix behind it with great precision and speed. In vitro, RNAPII is capable of carrying out templated RNA chain elongation in the absence of any accessory proteins. However, in cells, the transcription of genes is influenced by several factors, including DNA structure, chromatin, co-transcriptional processes, and DNA binding proteins, which impede the smooth progression of RNAPII down the template. Many transcription elongation proteins have evolved to mitigate the complications and barriers encountered by polymerase during transcription. Many of these elongation factors physically interact with components of the RNAPII elongation complex, including the growing RNA transcript and the DNA template entering and exiting RNAPII. To better understand how transcription elongation factors (EFs) regulate RNAPII, elegant methods are required to probe the structure of the elongation complex. Here, we describe a collection of biochemical assays to interrogate the structure of the RNAPII elongation complex of Saccharomyces cerevisiae that are capable of providing insights into the function of EFs and the elongation process.

Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II.

Transcription elongation through the nucleosome is a precisely coordinated activity to ensure timely production of RNA and accurate regulation of co-transcriptional histone modifications. Nucleosomes actively participate in transcription regulation at various levels and impose physical barriers to RNA polymerase II (RNAPII) during transcription elongation. Despite its high significance, the detailed dynamics of how RNAPII translocates along nucleosomal DNA during transcription elongation and how the nucleosome structure dynamically conforms to the changes necessary for RNAPII progression remain poorly understood. Transcription elongation through the nucleosome is a complex process and investigating the changes of the nucleosome structure during this process by ensemble measurements is daunting. This is because it is nearly impossible to synchronize elongation complexes within a nucleosome or a sub-nucleosome to a designated location at a high enough efficiency for desired sample homogeneity. Here we review our recently developed single-molecule FRET experimental system and method that has fulfilled this deficiency. With our method, one can follow the changes in the structure of individual nucleosomes during transcription elongation. We demonstrated that this method enables the detailed measurements of the kinetics of transcription elongation through the nucleosome and its regulation by a transcription factor, which can be easily extended to investigations of the roles of environmental variables and histone post-translational modifications in regulating transcription elongation.

Genome-Wide Mapping of Decay Factor-mRNA Interactions in Yeast Identifies Nutrient-Responsive Transcripts as Targets of the Deadenylase Ccr4.

The Ccr4 (carbon catabolite repression 4)-Not complex is a major regulator of stress responses that controls gene expression at multiple levels, from transcription to mRNA decay. Ccr4, a "core" subunit of the complex, is the main cytoplasmic deadenylase in Saccharomyces cerevisiae; however, its mRNA targets have not been mapped on a genome-wide scale. Here, we describe a genome-wide approach, RNA immunoprecipitation (RIP) high-throughput sequencing (RIP-seq), to identify the RNAs bound to Ccr4, and two proteins that associate with it, Dhh1 and Puf5 All three proteins were preferentially bound to lowly abundant mRNAs, most often at the 3' end of the transcript. Furthermore, Ccr4, Dhh1, and Puf5 are recruited to mRNAs that are targeted by other RNA-binding proteins that promote decay and mRNA transport, and inhibit translation. Although Ccr4-Not regulates mRNA transcription and decay, Ccr4 recruitment to mRNAs correlates better with decay rates, suggesting it imparts greater control over transcript abundance through decay. Ccr4-enriched mRNAs are refractory to control by the other deadenylase complex in yeast, Pan2/3, suggesting a division of labor between these deadenylation complexes. Finally, Ccr4 and Dhh1 associate with mRNAs whose abundance increases during nutrient starvation, and those that fluctuate during metabolic and oxygen consumption cycles, which explains the known genetic connections between these factors and nutrient utilization and stress pathways.

The elongation factor Spt4/5 regulates RNA polymerase II transcription through the nucleosome.

RNA polymerase II (RNAPII) passes through the nucleosome in a coordinated manner, generating several intermediate nucleosomal states as it breaks and then reforms histone-DNA contacts ahead of and behind it, respectively. Several studies have defined transcription-induced nucleosome intermediates using only RNA Polymerase. However, RNAPII is decorated with elongation factors as it transcribes the genome. One such factor, Spt4/5, becomes an integral component of the elongation complex, making direct contact with the 'jaws' of RNAPII and nucleic acids in the transcription scaffold. We have characterized the effect of incorporating Spt4/5 into the elongation complex on transcription through the 601R nucleosome. Spt4/5 suppressed RNAPII pausing at the major H3/H4-induced arrest point, resulting in downstream re-positioning of RNAPII further into the nucleosome. Using a novel single molecule FRET system, we found that Spt4/5 affected the kinetics of DNA re-wrapping and stabilized a nucleosomal intermediate with partially unwrapped DNA behind RNAPII. Comparison of nucleosomes of different sequence polarities suggest that the strength of the DNA-histone interactions behind RNAPII specifies the Spt4/5 requirement. We propose that Spt4/5 may be important to coordinate the mechanical movement of RNAPII through the nucleosome with co-transcriptional chromatin modifications during transcription, which is affected by the strength of histone-DNA interactions.

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.

Ccr4-Not and TFIIS Function Cooperatively To Rescue Arrested RNA Polymerase II.

Expression of the genome requires RNA polymerase II (RNAPII) to transcribe across many natural and unnatural barriers, and this transcription across barriers is facilitated by protein complexes called elongation factors (EFs). Genetic studies in Saccharomyces cerevisiae yeast suggest that multiple EFs collaborate to assist RNAPII in completing the transcription of genes, but the molecular mechanisms of how they cooperate to promote elongation are not well understood. The Ccr4-Not complex participates in multiple steps of mRNA metabolism and has recently been shown to be an EF. Here we describe how Ccr4-Not and TFIIS cooperate to stimulate elongation. We find that Ccr4-Not and TFIIS mutations show synthetically enhanced phenotypes, and biochemical analyses indicate that Ccr4-Not and TFIIS work synergistically to reactivate arrested RNAPII. Ccr4-Not increases the recruitment of TFIIS into elongation complexes and enhances the cleavage of the displaced transcript in backtracked RNAPII. This is mediated by an interaction between Ccr4-Not and the N terminus of TFIIS. In addition to revealing insights into how these two elongation factors cooperate to promote RNAPII elongation, our study extends the growing body of evidence suggesting that the N terminus of TFIIS acts as a docking/interacting site that allows it to synergize with other EFs to promote RNAPII transcription.

The Rpb4/7 module of RNA polymerase II is required for carbon catabolite repressor protein 4-negative on TATA (Ccr4-not) complex to promote elongation.

Gene expression relies on the balance between mRNA synthesis in the nucleus and decay in the cytoplasm, processes once thought to be separate. We now know that transcription and decay rates are coordinated, but the factors or molecular mechanisms are unclear. The Ccr4-Not complex regulates multiple stages of gene expression, from mRNA synthesis to protein destruction. One of its functions is to promote RNA polymerase II elongation by reactivating arrested elongation complexes. Here we explored the features of polymerase required for Ccr4-Not to promote elongation and found that the Rpb4/7 module is important for Ccr4-Not to associate with elongation complexes and stimulate elongation. Rpb4/7 has also been implicated in coordinating mRNA synthesis and decay, but its role in this process is controversial. The interplay between Ccr4-Not and Rpb4/7 described here suggests a mechanism for how the cell coordinates mRNA synthesis and decay.

Gene expression as a circular process: cross-talk between transcription and mRNA degradation in eukaryotes; International University of Andalusia (UNIA) Baeza, Spain.

Studies on the regulation of gene expression in eukaryotes in the past 20 years have consistently revealed increasing levels of complexity. Thirty years ago it seemed that we had understood the basic principles of gene regulation in eukaryotes. It was thought that regulation of transcription was the first and most important stage at which gene expression was regulated, and transcriptional regulation was considered to be very simple, with DNA-binding activators and repressors talking to the basic transcription machinery. This simple model was overthrown when it became clear that other stages of gene expression are also highly regulated. More recently, other dogmas have started to collapse. In particular, the idea that a linkage between the different steps in gene expression is restricted to processes ongoing in the same compartment has fallen out of favor. It is now evident that functional and physical linkage occurs in eukaryotes. We know that factors contributing to transcription in the nucleus can be found in the cytoplasm, and that RNA binding proteins that contribute to RNA decay in the cytoplasm are present in the nucleus. However, shuttling of such factors between nucleus and cytoplasm has traditionally been thought to serve a simple regulatory purpose, for instance, to avoid untimely activation of a transcription factor in the nucleus. Alternatively, it was thought to be necessary to recruit RNA binding proteins to the relevant RNAs. The notion that is now emerging is that factors thought to have evolved to specialize in regulating a single step of gene regulation in one cellular compartment may be contributing to the regulation of mRNAs at multiple steps along the lifecycle of an mRNA.

A highly conserved region within H2B is important for FACT to act on nucleosomes.

Histone N-terminal tails play crucial roles in chromatin-related processes. The tails of histones H3 and H4 are highly conserved and well characterized, but much less is known about the functions of the tails of histones H2A and H2B and their sequences are more divergent among eukaryotes. Here we characterized the function of the only highly conserved region in the H2B tail, the H2B repression (HBR) domain. Once thought to play a role only in repression, it also has an uncharacterized function in gene activation and DNA damage responses. We report that deletion of the HBR domain impairs the eviction of nucleosomes at the promoters and open reading frames of genes. A closer examination of the HBR domain mutants revealed that they displayed phenotypes similar to those of histone chaperone complex FACT mutants, including an increase in intragenic transcription and the accumulation of free histones in cells. Biochemical characterization of recombinant nucleosomes indicates that deletion of the HBR domain impairs FACT-dependent removal of H2A-H2B from nucleosomes, suggesting that the HBR domain plays an important role in allowing FACT to disrupt dimer-DNA interactions. We have uncovered a previously unappreciated role for the HBR domain in regulating chromatin structure and have provided insight into how FACT acts on nucleosomes.

The control of elongation by the yeast Ccr4-not complex.

The Ccr4-Not complex is a highly conserved nine-subunit protein complex that has been implicated in virtually all aspects of gene control, including transcription, mRNA decay and quality control, RNA export, translational repression and protein ubiquitylation. Understanding its mechanisms of action has been difficult due to the size of the complex and the fact that it regulates mRNAs and proteins at many levels in both the cytoplasm and the nucleus. Recently, biochemical and genetic studies on the yeast Ccr4-Not complex have revealed insights into its role in promoting elongation by RNA polymerase II. This review will describe what is known about the Ccr4-Not complex in regulating transcription elongation and its possible collaboration with other factors traveling with RNAPII across genes. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

Ccr4-Not complex: the control freak of eukaryotic cells.

The purpose of this review is to provide an analysis of the latest developments on the functions of the carbon catabolite-repression 4-Not (Ccr4-Not) complex in regulating eukaryotic gene expression. Ccr4-Not is a nine-subunit protein complex that is conserved in sequence and function throughout the eukaryotic kingdom. Although Ccr4-Not has been studied since the 1980s, our understanding of what it does is constantly evolving. Once thought to solely regulate transcription, it is now clear that it has much broader roles in gene regulation, such as in mRNA decay and quality control, RNA export, translational repression and protein ubiquitylation. The mechanism of actions for each of its functions is still being debated. Some of the difficulty in drawing a clear picture is that it has been implicated in so many processes that regulate mRNAs and proteins in both the cytoplasm and the nucleus. We will describe what is known about the Ccr4-Not complex in yeast and other eukaryotes in an effort to synthesize a unified model for how this complex coordinates multiple steps in gene regulation and provide insights into what questions will be most exciting to answer in the future.