Herpes simplex virus 1 expressing GFP-tagged virion host shutoff (vhs) protein uncouples the activities of degradation and nuclear retention of the infected cell transcriptome

Virion host shutoff (vhs) protein is an endoribonuclease encoded by herpes simplex virus 1 (HSV1). Vhs causes a number of changes to the infected cell environment that favour translation of late (L) virus proteins: cellular mRNAs are degraded, immediate-early (IE) and early (E) viral transcripts are sequestered in the nucleus with polyA binding protein (PABPC1), and dsRNA is degraded to help dampen the PKR-dependent stress response. To further our understanding of the cell biology of vhs, we constructed a virus expressing vhs tagged at its C-terminus with GFP. When first expressed, vhs-GFP localised to juxtanuclear clusters, and later it colocalised and interacted with its binding partner VP16, and was packaged into virions. Despite vhs-GFP maintaining activity when expressed in isolation, it failed to degrade mRNA or relocalise PABPC1 during infection, while viral transcript levels were similar to those seen for a vhs knockout virus. PKR phosphorylation was also enhanced in vhs-GFP infected cells, in line with a failure to degrade dsRNA. Nonetheless, mRNA FISH revealed that as in Wt but not Dvhs infection, IE and E, but not L transcripts were retained in the nucleus of vhs-GFP infected cells at late times. Moreover, a representative cellular transcript which is ordinarily highly susceptible to vhs degradation, was also retained in the nucleus. These results reveal that the vhs-induced nuclear retention of the infected cell transcriptome is dependent on vhs expression but not on its endoribonuclease activity, uncoupling these two functions of vhs.

The CCAAT-binding complex mediates azole susceptibility of Aspergillus fumigatus by suppressing SrbA expression and cleavage

In fungal pathogens, the transcription factor SrbA (a sterol regulatory element-binding protein, SREBP) and CBC (CCAAT binding complex) have been reported to regulate azole resistance by competitively binding the TR34 region (34 mer) in the promoter of the drug target gene, erg11A. However, current knowledge about how the SrbA and CBC coordinately mediate erg11A expression remains limited. In this study, we uncovered a novel relationship between HapB (a subunit of CBC) and SrbA in which deletion of hapB significantly prolongs the nuclear retention of SrbA by increasing its expression and cleavage under azole treatment conditions, thereby enhancing Erg11A expression for drug resistance. Furthermore, we verified that loss of HapB significantly induces the expression of the rhomboid protease RbdB, Dsc ubiquitin E3 ligase complex, and signal peptide peptidase SppA, which are required for the cleavage of SrbA, suggesting that HapB acts as a repressor for these genes which contribute to the activation of SrbA by proteolytic cleavage. Together, our study reveals that CBC functions not only to compete with SrbA for binding to erg11A promoter region but also to affect SrbA expression, cleavage, and translocation to nuclei for the function, which ultimately regulate Erg11A expression and azole resistance.

Cue-specific remodeling of the neuronal transcriptome through intron retention programs

Sub-cellular compartmentalization through the nuclear envelope has for a long time been primarily considered a physical barrier that separates nuclear and cytosolic contents. More recently, nuclear compartmentalization has emerged to harbor key regulatory functions in gene expression. A sizeable proportion of protein-coding mRNAs is more prevalent in the nucleus than in the cytosol reflecting the existence of mechanisms to control mRNA release into the cytosol. However, the biological relevance of the nuclear retention of mRNAs remains unclear. Here, we provide a comprehensive map of the subcellular localization of mRNAs in mature neurons and reveal that transcripts stably retaining introns are broadly targeted for nuclear retention. We systematically probed these transcripts upon neuronal stimulation and found that sub-populations of nuclear-retained transcripts are bi-directionally regulated in response to cues: some appear targeted for degradation while others undergo splicing completion to generate fully mature mRNAs which are exported to the cytosol to increase functional gene expression. Remarkably, different forms of stimulation mobilize distinct groups of intron-retaining transcripts and this selectivity arises from the activation of specific signaling pathways. Overall, our findings uncover cue-specific control of intron retention as a major regulator of acute remodeling of the neuronal transcriptome.

Amplification and attenuation of noisy expression by export processes

Inside mammalian cells, single genes are known to be transcribed in stochastic bursts leading to the synthesis of nuclear RNAs that are subsequently exported to the cytoplasm to create mRNAs. We systematically characterize the role of export processes in shaping the extent of random fluctuations (i.e. noise) in the mRNA level of a given gene. Using the method of Partitioning of Poisson arrivals, we derive an exact analytical expression for the noise in mRNA level assuming that the nuclear retention time of each RNA is an independent and identically distributed random variable following an arbitrary distribution. These results confirm recent experimental/theoretical findings that decreasing the nuclear export rate buffers the noise in mRNA level, and counterintuitively, decreasing the noise in the nuclear retention time enhances the noise in the mRNA level. Next, we further generalize the model to consider a dynamic extrinsic disturbance that affects the nuclear-to-cytoplasm export. Our results show that noise in the mRNA level varies non-monotonically with the disturbance timescale. More specifically, high- and low-frequency external disturbances have little impact on the mRNA noise level, while noise is amplified at intermediate frequencies. In summary, our results systematically uncover how the coupling of bursty transcription with nuclear export can both attenuate or amplify noise in mRNA levels depending on the nuclear retention time distribution and the presence of extrinsic fluctuations.

Intermittent High Glucose Elevates Nuclear Localization of EZH2 to Cause H3K27me3-Dependent Repression of KLF2 Leading to Endothelial Inflammation

Epigenetic mechanisms have emerged as one of the key pathways promoting diabetes-associated complications. Herein, we explored the role of enhancer of zeste homolog 2 (EZH2) and its product histone 3 lysine 27 trimethylation (H3K27me3) in high glucose-mediated endothelial inflammation. To examine this, we treated cultured primary endothelial cells (EC) with different treatment conditions—namely, constant or intermittent or transient high glucose. Intermittent high glucose maximally induced endothelial inflammation by upregulating transcript and/or protein-level expression of ICAM1 and P-selectin and downregulating eNOS, KLF2, and KLF4 protein levels. We next investigated the underlining epigenetic mechanisms responsible for intermittent hyperglycemia-dependent endothelial inflammation. Compared with other high glucose treatment groups, intermittent high glucose-exposed EC exhibited an increased level of H3K27me3 caused by reduction in EZH2 threonine 367 phosphorylation and nuclear retention of EZH2. Intermittent high glucose also promoted polycomb repressive complex-2 (PRC2) assembly and EZH2′s recruitment to histone H3. Abrupt enrichment of H3K27me3 on KLF2 and KLF4 gene promoters caused repression of these genes, further supporting endothelial inflammation. In contrast, reducing H3K27me3 through small molecule and/or siRNA-mediated inhibition of EZH2 rescued KLF2 level and inhibited endothelial inflammation in intermittent high glucose-challenged cultured EC and isolated rat aorta. These findings indicate that abrupt chromatin modifications cause high glucose-dependent inflammatory switch of EC.

Stress-induced inhibition of mRNA export triggers RNase III-mediated decay of the BDF2 mRNA

The expression of bromodomain-containing proteins that regulate chromatin structure and accessibility must be tightly controlled to ensure the appropriate regulation of gene expression. In the yeast S. cerevisiae, Bromodomain Factor 2 (BDF2) expression is extensively regulated post-transcriptionally during stress by RNase III-mediated decay (RMD), which is triggered by cleavage of the BDF2 mRNA in the nucleus by the RNase III homologue Rnt1p. Previous studies have shown that RMD-mediated down-regulation of BDF2 is hyper-activated in osmotic stress conditions, yet the mechanisms driving the enhanced nuclear cleavage of BDF2 RNA under these conditions remain unknown. Here, we show that RMD hyper-activation can be detected in multiple stress conditions that inhibit mRNA export, and that Rnt1p remains primarily localized in the nucleus during salt stress. We show that globally inhibiting mRNA nuclear export by anchoring away mRNA biogenesis or export factors out of the nucleus can recapitulate RMD hyper-activation in the absence of stress. RMD hyperactivation requires Rnt1p nuclear localization but does not depend on the BDF2 gene endogenous promoter, and its efficiency is affected by the structure of the stem-loop cleaved by Rnt1p. Because multiple stress conditions have been shown to mediate global inhibition of mRNA export, our results suggest that the hyperactivation of RMD is primarily the result of the increased nuclear retention of the BDF2 mRNA during stress.

Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export

Nuclear clearance of the DNA/RNA-binding protein TDP-43 is a pathologic hallmark of amyotrophic lateral sclerosis and frontotemporal dementia that remains unexplained. Moreover, our current understanding of TDP-43 nucleocytoplasmic shuttling does not fully explain the predominantly nuclear localization of TDP-43 in healthy cells. Here, we used permeabilized and live-cell models to investigate TDP-43 nuclear export and the role of RNA in TDP-43 localization. We show that TDP-43 nuclear efflux occurs in low-ATP conditions and independent of active mRNA export, consistent with export by passive diffusion through nuclear pore channels. TDP-43 nuclear residence requires binding to GU-rich nuclear intronic pre-mRNAs, based on the induction of TDP-43 nuclear efflux by RNase and GU-rich oligomers and TDP-43 nuclear retention conferred by pre-mRNA splicing inhibitors. Mutation of TDP-43 RNA recognition motifs disrupts TDP-43 nuclear accumulation and abolishes transcriptional blockade-induced TDP-43 nuclear efflux, demonstrating strict dependence of TDP-43 nuclear localization on RNA binding. Thus, the nuclear abundance of GU-rich intronic pre-mRNAs, as dictated by the balance of transcription and pre-mRNA processing, regulates TDP-43 nuclear sequestration and availability for passive nuclear export.

Membrane-bound SCF and VCAM-1 synergistically regulate the morphology of hematopoietic stem cells

Membrane-bound factors expressed by niche stromal cells constitute a unique class of localized cues and regulate the long-term functions of adult stem cells, yet little is known about the underlying mechanisms. Here, we used a supported lipid bilayer (SLB) to recapitulate the membrane-bound interactions between hematopoietic stem cells (HSCs) and niche stromal cells. HSCs cluster membrane-bound stem cell factor (mSCF) at the HSC-SLB interface. They further form a polarized morphology with aggregated mSCF under a large protrusion through a synergy with VCAM-1 on the bilayer, which drastically enhances HSC adhesion. These features are unique to mSCF and HSCs among the factors and hematopoietic populations we examined. The mSCF–VCAM-1 synergy and the polarized HSC morphology require PI3K signaling and cytoskeletal reorganization. The synergy also enhances nuclear retention of FOXO3a, a crucial factor for HSC maintenance, and minimizes its loss induced by soluble SCF. Our work thus reveals a unique role and signaling mechanism of membrane-bound factors in regulating stem cell morphology and function.