Control of RNA Pol II Speed by PNUTS-PP1 and Spt5 Dephosphorylation Facilitates Termination by a "Sitting Duck Torpedo" Mechanism.

Control of transcription speed, which influences many co-transcriptional processes, is poorly understood. We report that PNUTS-PP1 phosphatase is a negative regulator of RNA polymerase II (Pol II) elongation rate. The PNUTS W401A mutation, which disrupts PP1 binding, causes genome-wide acceleration of transcription associated with hyper-phosphorylation of the Spt5 elongation factor. Immediately downstream of poly(A) sites, Pol II decelerates from >2 kb/min to <1 kb/min, which correlates with Spt5 dephosphorylation. Pol II deceleration and Spt5 dephosphorylation require poly(A) site recognition and the PNUTS-PP1 complex, which is in turn necessary for transcription termination. These results lead to a model for termination, the "sitting duck torpedo" mechanism, where poly(A) site-dependent deceleration caused by PNUTS-PP1 and Spt5 dephosphorylation is required to convert Pol II into a viable target for the Xrn2 terminator exonuclease. Spt5 and its bacterial homolog NusG therefore have related functions controlling kinetic competition between RNA polymerases and the termination factors that pursue them.

DNA damage regulates alternative splicing through inhibition of RNA polymerase II elongation.

DNA damage induces apoptosis and many apoptotic genes are regulated via alternative splicing (AS), but little is known about the control mechanisms. Here we show that ultraviolet irradiation (UV) affects cotranscriptional AS in a p53-independent way, through the hyperphosphorylation of RNA polymerase II carboxy-terminal domain (CTD) and a subsequent inhibition of transcriptional elongation, estimated in vivo and in real time. Phosphomimetic CTD mutants not only display lower elongation but also duplicate the UV effect on AS. Consistently, nonphosphorylatable mutants prevent the UV effect. Apoptosis promoted by UV in cells lacking p53 is prevented when the change in AS of the apoptotic gene bcl-x is reverted, confirming the relevance of this mechanism. Splicing-sensitive microarrays revealed a significant overlap of the subsets of genes that have changed AS with UV and those that have reduced expression, suggesting that transcriptional coupling to AS is a key feature of the DNA-damage response.

mRNA decapping factors and the exonuclease Xrn2 function in widespread premature termination of RNA polymerase II transcription.

We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a, and Dcp2 and the termination factor TTF2 coimmunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease "torpedo" that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2, and TTF2 localize near transcription start sites (TSSs) by ChIP-seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors Xrn2 and TTF2 shifted polymerase away from the TSS toward upstream and downstream distal positions. This redistribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the "torpedo" mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated cotranscriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation.

Integration of the unfolded protein and oxidative stress responses through SKN-1/Nrf.

The Unfolded Protein Response (UPR) maintains homeostasis in the endoplasmic reticulum (ER) and defends against ER stress, an underlying factor in various human diseases. During the UPR, numerous genes are activated that sustain and protect the ER. These responses are known to involve the canonical UPR transcription factors XBP1, ATF4, and ATF6. Here, we show in C. elegans that the conserved stress defense factor SKN-1/Nrf plays a central and essential role in the transcriptional UPR. While SKN-1/Nrf has a well-established function in protection against oxidative and xenobiotic stress, we find that it also mobilizes an overlapping but distinct response to ER stress. SKN-1/Nrf is regulated by the UPR, directly controls UPR signaling and transcription factor genes, binds to common downstream targets with XBP-1 and ATF-6, and is present at the ER. SKN-1/Nrf is also essential for resistance to ER stress, including reductive stress. Remarkably, SKN-1/Nrf-mediated responses to oxidative stress depend upon signaling from the ER. We conclude that SKN-1/Nrf plays a critical role in the UPR, but orchestrates a distinct oxidative stress response that is licensed by ER signaling. Regulatory integration through SKN-1/Nrf may coordinate ER and cytoplasmic homeostasis.

Enhanced oxidative stress resistance through activation of a zinc deficiency transcription factor in Brachypodium distachyon.

Identification of viable strategies to increase stress resistance of crops will become increasingly important for the goal of global food security as our population increases and our climate changes. Considering that resistance to oxidative stress is oftentimes an indicator of health and longevity in animal systems, characterizing conserved pathways known to increase oxidative stress resistance could prove fruitful for crop improvement strategies. This report argues for the usefulness and practicality of the model organism Brachypodium distachyon for identifying and validating stress resistance factors. Specifically, we focus on a zinc deficiency B. distachyon basic leucine zipper transcription factor, BdbZIP10, and its role in oxidative stress in the model organism B. distachyon. When overexpressed, BdbZIP10 protects plants and callus tissue from oxidative stress insults, most likely through distinct and direct activation of protective oxidative stress genes. Increased oxidative stress resistance and cell viability through the overexpression of BdbZIP10 highlight the utility of investigating conserved stress responses between plant and animal systems.

Specific SKN-1/Nrf stress responses to perturbations in translation elongation and proteasome activity.

SKN-1, the Caenorhabditis elegans Nrf1/2/3 ortholog, promotes both oxidative stress resistance and longevity. SKN-1 responds to oxidative stress by upregulating genes that detoxify and defend against free radicals and other reactive molecules, a SKN-1/Nrf function that is both well-known and conserved. Here we show that SKN-1 has a broader and more complex role in maintaining cellular stress defenses. SKN-1 sustains expression and activity of the ubiquitin-proteasome system (UPS) and coordinates specific protective responses to perturbations in protein synthesis or degradation through the UPS. If translation initiation or elongation is impaired, SKN-1 upregulates overlapping sets of cytoprotective genes and increases stress resistance. When proteasome gene expression and activity are blocked, SKN-1 activates multiple classes of proteasome subunit genes in a compensatory response. SKN-1 thereby maintains UPS activity in the intestine in vivo under normal conditions and promotes survival when the proteasome is inhibited. In contrast, when translation elongation is impaired, SKN-1 does not upregulate proteasome genes, and UPS activity is then reduced. This indicates that UPS activity depends upon presence of an intact translation elongation apparatus; and it supports a model, suggested by genetic and biochemical studies in yeast, that protein synthesis and degradation may be coupled processes. SKN-1 therefore has a critical tissue-specific function in increasing proteasome gene expression and UPS activity under normal conditions, as well as when the UPS system is stressed, but mounts distinct responses when protein synthesis is perturbed. The specificity of these SKN-1-mediated stress responses, along with the apparent coordination between UPS and translation elongation activity, may promote protein homeostasis under stress or disease conditions. The data suggest that SKN-1 may increase longevity, not only through its well-documented role in boosting stress resistance, but also through contributing to protein homeostasis.

Identification and characterization of a salt stress-inducible zinc finger protein from Festuca arundinacea.

BACKGROUND: Increased biotic and abiotic plant stresses due to climate change together with an expected global human population of over 9 billion by 2050 intensifies the demand for agricultural production on marginal lands. Soil salinity is one of the major abiotic stresses responsible for reduced crop productivity worldwide and the salinization of arable land has dramatically increased over the last few decades. Consequently, as land becomes less amenable for conventional agriculture, plants grown on marginal soils will be exposed to higher levels of soil salinity. Forage grasses are a critical component of feed used in livestock production worldwide, with many of these same species of grasses being utilized for lawns, erosion prevention, and recreation. Consequently, it is important to develop a better understanding of salt tolerance in forage and related grass species. FINDINGS: A gene encoding a ZnF protein was identified during the analysis of a salt-stress suppression subtractive hybridization (SSH) expression library from the forage grass species Festuca arundinacea. The expression pattern of FaZnF was compared to that of the well characterized gene for delta 1-pyrroline-5-carboxylate synthetase (P5CS), a key enzyme in proline biosynthesis, which was also identified in the salt-stress SSH library. The FaZnF and P5CS genes were both up-regulated in response to salt and drought stresses suggesting a role in dehydration stress. FaZnF was also up-regulated in response to heat and wounding, suggesting that it might have a more general function in multiple abiotic stress responses. Additionally, potential downstream targets of FaZnF (a MAPK [Mitogen-Activated Protein Kinase], GST [Glutathione-S-Transferase] and lipoxygenase L2) were found to be up-regulated in calli overexpressing FaZnF when compared to control cell lines. CONCLUSIONS: This work provides evidence that FaZnF is an AN1/A20 zinc finger protein that is involved in the regulation of at least two pathways initiated by the salt stress response, thus furthering our understanding of the mechanisms of cellular action during a stress that is applicable to commercial crops worldwide.

Mitochondrial SKN-1/Nrf mediates a conserved starvation response.

SKN-1/Nrf plays multiple essential roles in development and cellular homeostasis. We demonstrate that SKN-1 executes a specific and appropriate transcriptional response to changes in available nutrients, leading to metabolic adaptation. We isolated gain-of-function (gf) alleles of skn-1, affecting a domain of SKN-1 that binds the transcription factor MXL-3 and the mitochondrial outer membrane protein PGAM-5. These skn-1(gf) mutants perceive a state of starvation even in the presence of plentiful food. The aberrant monitoring of cellular nutritional status leads to an altered survival response in which skn-1(gf) mutants transcriptionally activate genes associated with metabolism, adaptation to starvation, aging, and survival. The triggered starvation response is conserved in mice with constitutively activated Nrf and may contribute to the tumorgenicity associated with activating Nrf mutations in mammalian somatic cells. Our findings delineate an evolutionarily conserved metabolic axis of SKN-1/Nrf, further establishing the complexity of this pathway.

Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II.

Promoter-proximal pausing by RNA polymerase II (Pol II) ensures gene-specific regulation and RNA quality control. Structural considerations suggested a requirement for initiation-factor eviction in elongation-factor engagement and pausing of transcription complexes. Here we show that selective inhibition of Cdk7--part of TFIIH--increases TFIIE retention, prevents DRB sensitivity-inducing factor (DSIF) recruitment and attenuates pausing in human cells. Pause release depends on Cdk9-cyclin T1 (P-TEFb); Cdk7 is also required for Cdk9-activating phosphorylation and Cdk9-dependent downstream events--Pol II C-terminal domain Ser2 phosphorylation and histone H2B ubiquitylation--in vivo. Cdk7 inhibition, moreover, impairs Pol II transcript 3'-end formation. Cdk7 thus acts through TFIIE and DSIF to establish, and through P-TEFb to relieve, barriers to elongation: incoherent feedforward that might create a window to recruit RNA-processing machinery. Therefore, cyclin-dependent kinases govern Pol II handoff from initiation to elongation factors and cotranscriptional RNA maturation.

TOR signaling and rapamycin influence longevity by regulating SKN-1/Nrf and DAF-16/FoxO.

The TOR kinase, which is present in the functionally distinct complexes TORC1 and TORC2, is essential for growth but associated with disease and aging. Elucidation of how TOR influences life span will identify mechanisms of fundamental importance in aging and TOR functions. Here we show that when TORC1 is inhibited genetically in C. elegans, SKN-1/Nrf, and DAF-16/FoxO activate protective genes, and increase stress resistance and longevity. SKN-1 also upregulates TORC1 pathway gene expression in a feedback loop. Rapamycin triggers a similar protective response in C. elegans and mice, but increases worm life span dependent upon SKN-1 and not DAF-16, apparently by interfering with TORC2 along with TORC1. TORC1, TORC2, and insulin/IGF-1-like signaling regulate SKN-1 activity through different mechanisms. We conclude that modulation of SKN-1/Nrf and DAF-16/FoxO may be generally important in the effects of TOR signaling in vivo and that these transcription factors mediate an opposing relationship between growth signals and longevity.

TFIIH-associated Cdk7 kinase functions in phosphorylation of C-terminal domain Ser7 residues, promoter-proximal pausing, and termination by RNA polymerase II.

The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7(as/as) mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5' ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase "leaking" into the gene. Consistent with a 5' pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36-two marks of elongation-within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3' ends, it was detected within genes and 3'-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination.

RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes.

We investigated co-transcriptional recruitment of pre-mRNA processing factors to human genes. Capping factors associate with paused RNA polymerase II (pol II) at the 5' ends of quiescent genes. They also track throughout actively transcribed genes and accumulate with paused polymerase in the 3' flanking region. The 3' processing factors cleavage stimulation factor and cleavage polyadenylation specificity factor are maximally recruited 0.5-1.5 kilobases downstream of poly(A) sites where they coincide with capping factors, Spt5, and Ser2-hyperphosphorylated, paused pol II. 3' end processing factors also localize at transcription start sites, and this early recruitment is enhanced after polymerase arrest with the elongation factor DRB. These results suggest that promoters may help specify recruitment of 3' end processing factors. We propose a dual-pausing model wherein elongation arrests near the transcription start site and in the 3' flank to allow co-transcriptional processing by factors recruited to the pol II ternary complex.