Control of endothelial cell function and arteriogenesis by MEG3:EZH2 epigenetic regulation of integrin expression.

Epigenetic processes involving long non-coding RNAs regulate endothelial gene expression. However, the underlying regulatory mechanisms causing endothelial dysfunction remain to be elucidated. Enhancer of zeste homolog 2 (EZH2) is an important rheostat of histone H3K27 trimethylation (H3K27me3) that represses endothelial targets, but EZH2 RNA binding capacity and EZH2:RNA functional interactions have not been explored in post-ischemic angiogenesis. We used formaldehyde/UV-assisted crosslinking ligation and sequencing of hybrids and identified a new role for maternally expressed gene 3 (MEG3). MEG3 formed the predominant RNA:RNA hybrid structures in endothelial cells. Moreover, MEG3:EZH2 assists recruitment onto chromatin. By EZH2-chromatin immunoprecipitation, following MEG3 depletion, we demonstrated that MEG3 controls recruitment of EZH2/H3K27me3 onto integrin subunit alpha4 (ITGA4) promoter. Both MEG3 knockdown or EZH2 inhibition (A-395) promoted ITGA4 expression and improved endothelial cell migration and adhesion to fibronectin in vitro. The A-395 inhibitor re-directed MEG3-assisted chromatin remodeling, offering a direct therapeutic benefit by increasing endothelial function and resilience. This approach subsequently increased the expression of ITGA4 in arterioles following ischemic injury in mice, thus promoting arteriogenesis. Our findings show a context-specific role for MEG3 in guiding EZH2 to repress ITGA4. Novel therapeutic strategies could antagonize MEG3:EZH2 interaction for pre-clinical studies.

Improved detection and consistency of RNA-interacting proteomes using DIA SILAC.

The RNA-interacting proteome is commonly characterized by UV-crosslinking followed by RNA purification, with protein recovery quantified using SILAC labeling followed by data-dependent acquisition (DDA) of proteomic data. However, the low efficiency of UV-crosslinking, combined with limited sensitivity of the DDA approach often restricts detection to relatively abundant proteins, necessitating multiple mass spec injections of fractionated peptides for each biological sample. Here we report an application of data-independent acquisition (DIA) with SILAC in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment. This gave 15% greater protein detection and lower inter-replicate variation relative to the same biological materials analyzed using DDA, while allowing single-shot analysis of the sample. As proof of concept, we determined the effects of arsenite treatment on the RNA-bound proteome of HEK293T cells. The DIA dataset yielded similar GO term enrichment for RNA-binding proteins involved in cellular stress responses to the DDA dataset while detecting extra proteins unseen by DDA. Overall, the DIA SILAC approach improved detection of proteins over conventional DDA SILAC for generating RNA-interactome datasets, at a lower cost due to reduced machine time. Analyses are described for TRAPP data, but the approach is suitable for proteomic analyses following essentially any RNA-binding protein enrichment technique.

The conserved RNA-binding protein Seb1 promotes cotranscriptional ribosomal RNA processing by controlling RNA polymerase I progression.

Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. We show here that the conserved fission yeast RNA-binding protein Seb1 associates with the RNAPI transcription machinery and promotes RNAPI pausing states along the rDNA. The overall faster progression of RNAPI at the rDNA in Seb1-deficient cells impaired cotranscriptional pre-rRNA processing and the production of mature rRNAs. Given that Seb1 also influences pre-mRNA processing by modulating RNAPII progression, our findings unveil Seb1 as a pause-promoting factor for RNA polymerases I and II to control cotranscriptional RNA processing.

A posttranscriptional pathway regulates cell wall mRNA expression in budding yeast.

The fungal cell wall provides protection and structure and is an important target for antifungal compounds. A mitogen-activated protein (MAP) kinase cascade termed the cell wall integrity (CWI) pathway regulates transcriptional responses to cell wall damage. Here, we describe a posttranscriptional pathway that plays an important complementary role. We report that the RNA-binding proteins (RBPs) Mrn1 and Nab6 specifically target the 3' UTRs of a largely overlapping set of cell wall-related mRNAs. These mRNAs are downregulated in the absence of Nab6, indicating a function in target mRNA stabilization. Nab6 acts in parallel to CWI signaling to maintain appropriate expression of cell wall genes during stress. Cells lacking both pathways are hypersensitive to antifungal compounds targeting the cell wall. Deletion of MRN1 partially alleviates growth defects associated with Δnab6, and Mrn1 has an opposing function in mRNA destabilization. Our results uncover a posttranscriptional pathway that mediates cellular resistance to antifungal compounds.

The dual nature of the nucleolus.

The nucleolus is best known for housing the highly ordered assembly line that produces ribosomal subunits. The >100 ribosome assembly factors in the nucleolus are thought to cycle between two states: an operative state (when integrated into subunit assembly intermediates) and a latent state (upon release from intermediates). Although it has become commonplace to refer to the nucleolus as "being a multilayered condensate," and this may be accurate for latent factors, there is little reason to think that such assertions pertain to the operative state of assembly factors.

The RNA-Binding Landscape of HAX1 Protein Indicates Its Involvement in Translation and Ribosome Assembly.

HAX1 is a human protein with no known homologues or structural domains. Mutations in the HAX1 gene cause severe congenital neutropenia through mechanisms that are poorly understood. Previous studies reported the RNA-binding capacity of HAX1, but the role of this binding in physiology and pathology remains unexplained. Here, we report the transcriptome-wide characterization of HAX1 RNA targets using RIP-seq and CRAC, indicating that HAX1 binds transcripts involved in translation, ribosome biogenesis, and rRNA processing. Using CRISPR knockouts, we find that HAX1 RNA targets partially overlap with transcripts downregulated in HAX1 KO, implying a role in mRNA stabilization. Gene ontology analysis demonstrated that genes differentially expressed in HAX1 KO (including genes involved in ribosome biogenesis and translation) are also enriched in a subset of genes whose expression correlates with HAX1 expression in four analyzed neoplasms. The functional connection to ribosome biogenesis was also demonstrated by gradient sedimentation ribosome profiles, which revealed differences in the small subunit:monosome ratio in HAX1 WT/KO. We speculate that changes in HAX1 expression may be important for the etiology of HAX1-linked diseases through dysregulation of translation.

The SPARC complex defines RNAPII promoters in Trypanosoma brucei.

Kinetoplastids are a highly divergent lineage of eukaryotes with unusual mechanisms for regulating gene expression. We previously surveyed 65 putative chromatin factors in the kinetoplastid Trypanosoma brucei. Our analyses revealed that the predicted histone methyltransferase SET27 and the Chromodomain protein CRD1 are tightly concentrated at RNAPII transcription start regions (TSRs). Here, we report that SET27 and CRD1, together with four previously uncharacterized constituents, form the SET27 promoter-associated regulatory complex (SPARC), which is specifically enriched at TSRs. SET27 loss leads to aberrant RNAPII recruitment to promoter sites, accumulation of polyadenylated transcripts upstream of normal transcription start sites, and conversion of some normally unidirectional promoters to bidirectional promoters. Transcriptome analysis in the absence of SET27 revealed upregulated mRNA expression in the vicinity of SPARC peaks within the main body of chromosomes in addition to derepression of genes encoding variant surface glycoproteins (VSGs) located in subtelomeric regions. These analyses uncover a novel chromatin-associated complex required to establish accurate promoter position and directionality.

The promises and pitfalls of specialized ribosomes.

The concept of specialized ribosomes has garnered equal amounts of interest and skepticism since it was first introduced. We ask researchers in the field to provide their perspective on the topic and weigh in on the evidence (or lack thereof) and what the future may bring.

A disease-linked lncRNA mutation in RNase MRP inhibits ribosome synthesis.

RMRP encodes a non-coding RNA forming the core of the RNase MRP ribonucleoprotein complex. Mutations cause Cartilage Hair Hypoplasia (CHH), characterized by skeletal abnormalities and impaired T cell activation. Yeast RNase MRP cleaves a specific site in the pre-ribosomal RNA (pre-rRNA) during ribosome synthesis. CRISPR-mediated disruption of RMRP in human cells lines caused growth arrest, with pre-rRNA accumulation. Here, we analyzed disease-relevant primary cells, showing that mutations in RMRP impair mouse T cell activation and delay pre-rRNA processing. Patient-derived human fibroblasts with CHH-linked mutations showed similar pre-rRNA processing delay. Human cells engineered with the most common CHH mutation (70AG in RMRP) show specifically impaired pre-rRNA processing, resulting in reduced mature rRNA and a reduced ratio of cytosolic to mitochondrial ribosomes. Moreover, the 70AG mutation caused a reduction in intact RNase MRP complexes. Together, these results indicate that CHH is a ribosomopathy.

Yeast Ssd1 is a non-enzymatic member of the RNase II family with an alternative RNA recognition site.

Ssd1, a conserved fungal RNA-binding protein, is important in stress responses, cell division and virulence. Ssd1 is closely related to Dis3L2 of the RNase II family of nucleases, but lacks catalytic activity and likely suppresses translation of bound mRNAs. Previous studies identified RNA motifs enriched in Ssd1-associated transcripts, yet the sequence requirements for Ssd1 binding are not defined. Here, we identify precise binding sites of Ssd1 on RNA using in vivo cross-linking and cDNA analysis. These sites are enriched in 5' untranslated regions of a subset of mRNAs encoding cell wall proteins. We identified a conserved bipartite motif that binds Ssd1 with high affinity in vitro. Active RNase II enzymes have a characteristic, internal RNA binding path; the Ssd1 crystal structure at 1.9 Å resolution shows that remnants of regulatory sequences block this path. Instead, RNA binding activity has relocated to a conserved patch on the surface of the protein. Structure-guided mutations of this surface prevent Ssd1 from binding RNA in vitro and phenocopy Ssd1 deletion in vivo. These studies provide a new framework for understanding the function of a pleiotropic post-transcriptional regulator of gene expression and give insights into the evolution of regulatory and binding elements in the RNase II family.

NANOS2 is a sequence-specific mRNA-binding protein that promotes transcript degradation in spermatogonial stem cells.

Spermatogonial stem cells (SSCs) sustain spermatogenesis and fertility throughout adult male life. The conserved RNA-binding protein NANOS2 is essential for the maintenance of SSCs, but its targets and mechanisms of function are not fully understood. Here, we generated a fully functional epitope-tagged Nanos2 mouse allele and applied the highly stringent cross-linking and analysis of cDNAs to define NANOS2 RNA occupancy in SSC lines. NANOS2 recognizes the AUKAAWU consensus motif, mostly found in the 3' untranslated region of defined messenger RNAs (mRNAs). We find that NANOS2 is a regulator of key signaling and metabolic pathways whose dosage or activity are known to be critical for SSC maintenance. NANOS2 interacts with components of CCR4-NOT deadenylase complex in SSC lines, and consequently, NANOS2 binding reduces the half-lives of target transcripts. In summary, NANOS2 contributes to SSC maintenance through the regulation of target mRNA stability and key self-renewal pathways.

Integrative vectors for regulated expression of SARS-CoV-2 proteins implicated in RNA metabolism.

Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His 8, the C-terminus with His 8-Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In T-REx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.

The expression of Rpb10, a small subunit common to RNA polymerases, is modulated by the R3H domain-containing Rbs1 protein and the Upf1 helicase.

The biogenesis of eukaryotic RNA polymerases is poorly understood. The present study used a combination of genetic and molecular approaches to explore the assembly of RNA polymerase III (Pol III) in yeast. We identified a regulatory link between Rbs1, a Pol III assembly factor, and Rpb10, a small subunit that is common to three RNA polymerases. Overexpression of Rbs1 increased the abundance of both RPB10 mRNA and the Rpb10 protein, which correlated with suppression of Pol III assembly defects. Rbs1 is a poly(A)mRNA-binding protein and mutational analysis identified R3H domain to be required for mRNA interactions and genetic enhancement of Pol III biogenesis. Rbs1 also binds to Upf1 protein, a key component in nonsense-mediated mRNA decay (NMD) and levels of RPB10 mRNA were increased in a upf1Δ strain. Genome-wide RNA binding by Rbs1 was characterized by UV cross-linking based approach. We demonstrated that Rbs1 directly binds to the 3' untranslated regions (3'UTRs) of many mRNAs including transcripts encoding Pol III subunits, Rpb10 and Rpc19. We propose that Rbs1 functions by opposing mRNA degradation, at least in part mediated by NMD pathway. Orthologues of Rbs1 protein are present in other eukaryotes, including humans, suggesting that this is a conserved regulatory mechanism.

Stress-Induced Translation Inhibition through Rapid Displacement of Scanning Initiation Factors.

Cellular responses to environmental stress are frequently mediated by RNA-binding proteins (RBPs). Here, we examined global RBP dynamics in Saccharomyces cerevisiae in response to glucose starvation and heat shock. Each stress induced rapid remodeling of the RNA-protein interactome without corresponding changes in RBP abundance. Consistent with general translation shutdown, ribosomal proteins contacting the mRNA showed decreased RNA association. Among translation components, RNA association was most reduced for initiation factors involved in 40S scanning (eukaryotic initiation factor 4A [eIF4A], eIF4B, and Ded1), indicating a common mechanism of translational repression. In unstressed cells, eIF4A, eIF4B, and Ded1 primarily targeted the 5' ends of mRNAs. Following glucose withdrawal, 5' binding was abolished within 30 s, explaining the rapid translation shutdown, but mRNAs remained stable. Heat shock induced progressive loss of 5' RNA binding by initiation factors over ∼16 min and provoked mRNA degradation, particularly for translation-related factors, mediated by Xrn1. Taken together, these results reveal mechanisms underlying translational control of gene expression during stress.

Nascent Transcript Folding Plays a Major Role in Determining RNA Polymerase Elongation Rates.

Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5' enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding.

Substrate specificity of the TRAMP nuclear surveillance complexes.

During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity is apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association shows co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduces Mtr4 recruitment and increases RNA abundance for mRNAs specifically showing high Trf5 binding.

Extended ncRNAs Interfere with Promoter Nucleosome Dynamics.

Eukaryotic genomes generate vast numbers of non-protein-coding RNAs (ncRNAs) that can inhibit mRNA synthesis through transcription interference, but the mechanisms are unclear. Gill et al. show that transcription of antisense ncRNAs induces 'elongation marks' on histones in promoter regions. These inhibit active nucleosome positioning required to maintain open transcription-initiation sites.

Regulation of the RNAPII Pool Is Integral to the DNA Damage Response.

In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.

High density of unrepaired genomic ribonucleotides leads to Topoisomerase 1-mediated severe growth defects in absence of ribonucleotide reductase.

Cellular levels of ribonucleoside triphosphates (rNTPs) are much higher than those of deoxyribonucleoside triphosphates (dNTPs), thereby influencing the frequency of incorporation of ribonucleoside monophosphates (rNMPs) by DNA polymerases (Pol) into DNA. RNase H2-initiated ribonucleotide excision repair (RER) efficiently removes single rNMPs in genomic DNA. However, processing of rNMPs by Topoisomerase 1 (Top1) in absence of RER induces mutations and genome instability. Here, we greatly increased the abundance of genomic rNMPs in Saccharomyces cerevisiae by depleting Rnr1, the major subunit of ribonucleotide reductase, which converts ribonucleotides to deoxyribonucleotides. We found that in strains that are depleted of Rnr1, RER-deficient, and harbor an rNTP-permissive replicative Pol mutant, excessive accumulation of single genomic rNMPs severely compromised growth, but this was reversed in absence of Top1. Thus, under Rnr1 depletion, limited dNTP pools slow DNA synthesis by replicative Pols and provoke the incorporation of high levels of rNMPs in genomic DNA. If a threshold of single genomic rNMPs is exceeded in absence of RER and presence of limited dNTP pools, Top1-mediated genome instability leads to severe growth defects. Finally, we provide evidence showing that accumulation of RNA/DNA hybrids in absence of RNase H1 and RNase H2 leads to cell lethality under Rnr1 depletion.