UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency.

The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited 'closed' state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active 'open' state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants (i.e. poorly processive, slow, and mechanochemically uncoupled) can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower RNA binding kinetics and enhanced ATP-stimulated RNA dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2 and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.

UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency.

The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited "closed" state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active "open" state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.

A network of DZF proteins controls alternative splicing regulation and fidelity.

Proteins containing DZF (domain associated with zinc fingers) modules play important roles throughout gene expression, from transcription to translation. Derived from nucleotidyltransferases but lacking catalytic residues, DZF domains serve as heterodimerization surfaces between DZF protein pairs. Three DZF proteins are widely expressed in mammalian tissues, ILF2, ILF3 and ZFR, which form mutually exclusive ILF2-ILF3 and ILF2-ZFR heterodimers. Using eCLIP-Seq, we find that ZFR binds across broad intronic regions to regulate the alternative splicing of cassette and mutually exclusive exons. ZFR preferentially binds dsRNA in vitro and is enriched on introns containing conserved dsRNA elements in cells. Many splicing events are similarly altered upon depletion of any of the three DZF proteins; however, we also identify independent and opposing roles for ZFR and ILF3 in alternative splicing regulation. Along with widespread involvement in cassette exon splicing, the DZF proteins control the fidelity and regulation of over a dozen highly validated mutually exclusive splicing events. Our findings indicate that the DZF proteins form a complex regulatory network that leverages dsRNA binding by ILF3 and ZFR to modulate splicing regulation and fidelity.

UPF1 mutants with intact ATPase but deficient helicase activities promote efficient nonsense-mediated mRNA decay.

The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machinery and the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (<200 nt) in physiological contexts and undergoes ATPase-stimulated dissociation from RNA. We combine an in silico screen with these assays to identify and characterize known and novel UPF1 mutants with altered helicase, ATPase, and RNA binding properties. We find that UPF1 mutants with substantially impaired processivity (E797R, G619K/A546H), faster (G619K) or slower (K547P, E797R, G619K/A546H) unwinding rates, and/or reduced mechanochemical coupling (i.e. the ability to harness ATP hydrolysis for work; K547P, R549S, G619K, G619K/A546H) can still support efficient NMD of well-characterized targets in human cells. These data are consistent with a central role for UPF1 ATPase activity in driving cycles of RNA binding and dissociation to ensure accurate NMD target selection.

Temozolomide-induced guanine mutations create exploitable vulnerabilities of guanine-rich DNA and RNA regions in drug-resistant gliomas.

Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive brain cancer glioblastoma (GBM) since 2005. Although initially beneficial, TMZ resistance is universal and second-line interventions are an unmet clinical need. Here, we took advantage of the known mechanism of action of TMZ to target guanines (G) and investigated G-rich G-quadruplex (G4) and splice site changes that occur upon TMZ resistance. We report that TMZ-resistant GBM has guanine mutations that disrupt the G-rich DNA G4s and splice sites that lead to deregulated alternative splicing. These alterations create vulnerabilities, which are selectively targeted by either the G4-stabilizing drug TMPyP4 or a novel splicing kinase inhibitor of cdc2-like kinase. Last, we show that the G4 and RNA binding protein EWSR1 aggregates in the cytoplasm in TMZ-resistant GBM cells and patient samples. Together, our findings provide insight into targetable vulnerabilities of TMZ-resistant GBM and present cytoplasmic EWSR1 as a putative biomarker.

An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination in order to degrade transcripts with premature stop codons and regulate thousands of human genes. Here, we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL , enables condition-dependent remodeling of NMD specificity. Previous studies indicate that the extension of a conserved regulatory loop in the UPF1LL helicase core confers a decreased propensity to dissociate from RNA upon ATP hydrolysis relative to UPF1SL , the major UPF1 isoform. Using biochemical and transcriptome-wide approaches, we find that UPF1LL can circumvent the protective RNA binding proteins PTBP1 and hnRNP L to preferentially bind and down-regulate transcripts with long 3'UTRs normally shielded from NMD. Unexpectedly, UPF1LL supports induction of NMD on new populations of substrate mRNAs in response to activation of the integrated stress response and impaired translation efficiency. Thus, while canonical NMD is abolished by moderate translational repression, UPF1LL activity is enhanced, offering the possibility to rapidly rewire NMD specificity in response to cellular stress.

A systematic review of cellular therapies for the treatment of limbal stem cell deficiency affecting one or both eyes.

PURPOSE: This systematic review (SR) assessed the efficacy, safety and cost-effectiveness of cell-based therapy to manage limbal stem cell deficiency (LSCD), a sight-threatening orphan condition most frequently associated with severe chemical or thermal burns. LSCD has historically been treated by transplanting limbal tissue. In 1997, a new treatment, cultured limbal epithelial autografts, was described for unilateral LSCD. In cases of bilateral disease cultured autologous oral mucosa stem cells have been used. The relative efficacy of different cultured tissue procedures is unknown. METHODS: A protocol was registered with PROSPERO (CRD42017081117). Searches were conducted in 14 databases and 6 conference websites. Two reviewers independently selected studies, conducted data extraction and assessed risk of bias. One reviewer extracted individual patient data (IPD); a second checked extracted data. Data were assessed to determine the feasibility of statistical analysis, with Bayesian synthesis used to estimate improvement achieved by different treatments. RESULTS: Fifty-two studies were eligible for inclusion (1113 eyes); 41 studies (716 eyes) reported IPD. No evidence was identified on cost-effectiveness. This SR was unable to confirm that any of the types of ex vivo cultured stem cell transplants identified for LSCD treatment were statistically superior when assessed against the outcomes of interest. CONCLUSIONS: We believe this SR is the first to include IPD analysis of LSCD data. There is no evidence for the superiority of any method of limbal stem cell transplant. Confirmation of the safety and efficacy of this treatment modality is challenging due to heterogeneity within and between the studies identified. Therefore, recommendations for future research are proposed.

The RNA-binding protein PTBP1 promotes ATPase-dependent dissociation of the RNA helicase UPF1 to protect transcripts from nonsense-mediated mRNA decay.

The sequence-specific RNA-binding proteins PTBP1 (polypyrimidine tract-binding protein 1) and HNRNP L (heterogeneous nuclear ribonucleoprotein L) protect mRNAs from nonsense-mediated decay (NMD) by preventing the UPF1 RNA helicase from associating with potential decay targets. Here, by analyzing in vitro helicase activity, dissociation of UPF1 from purified mRNPs, and transcriptome-wide UPF1 RNA binding, we present the mechanistic basis for inhibition of NMD by PTBP1. Unlike mechanisms of RNA stabilization that depend on direct competition for binding sites among protective RNA-binding proteins and decay factors, PTBP1 promotes displacement of UPF1 already bound to potential substrates. Our results show that PTBP1 directly exploits the tendency of UPF1 to release RNA upon ATP binding and hydrolysis. We further find that UPF1 sensitivity to PTBP1 is coordinated by a regulatory loop in domain 1B of UPF1. We propose that the UPF1 regulatory loop and protective proteins control kinetic proofreading of potential NMD substrates, presenting a new model for RNA helicase regulation and target selection in the NMD pathway.

Activation and inhibition of nonsense-mediated mRNA decay control the abundance of alternative polyadenylation products.

Alternative polyadenylation (APA) produces transcript 3' untranslated regions (3'UTRs) with distinct sequences, lengths, stabilities and functions. We show here that APA products include a class of cryptic nonsense-mediated mRNA decay (NMD) substrates with extended 3'UTRs that gene- or transcript-level analyses of NMD often fail to detect. Transcriptome-wide, the core NMD factor UPF1 preferentially recognizes long 3'UTR products of APA, leading to their systematic downregulation. Counteracting this mechanism, the multifunctional RNA-binding protein PTBP1 regulates the balance of short and long 3'UTR isoforms by inhibiting NMD, in addition to its previously described modulation of co-transcriptional polyadenylation (polyA) site choice. Further, we find that many transcripts with altered APA isoform abundance across multiple tumor types are controlled by NMD. Together, our findings reveal a widespread role for NMD in shaping the outcomes of APA.

EWS-FLI1 modulated alternative splicing of ARID1A reveals novel oncogenic function through the BAF complex.

Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS-FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS-FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS-FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS-FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. We further report a novel feed-forward cycle in which EWS-FLI1 leads to preferential splicing of ARID1A-L, promoting ES growth, and ARID1A-L reciprocally promotes EWS-FLI1 protein stability. Dissecting this interaction may lead to improved cancer-specific drug targeting.

Nonsense-mediated mRNA decay: The challenge of telling right from wrong in a complex transcriptome.

The nonsense-mediated mRNA decay pathway selects and degrades its targets using a dense network of RNA-protein and protein-protein interactions. Together, these interactions allow the pathway to collect copious information about the translating mRNA, including translation termination status, splice junction positions, mRNP composition, and 3'UTR length and structure. The core NMD machinery, centered on the RNA helicase UPF1, integrates this information to determine the efficiency of decay. A picture of NMD is emerging in which many factors contribute to the dynamics of decay complex assembly and disassembly, thereby influencing the probability of decay. The ability of the NMD pathway to recognize mRNP features of diverse potential substrates allows it to simultaneously perform quality control and regulatory functions. In vertebrates, increased transcriptome complexity requires balance between these two functions since high NMD efficiency is desirable for maintenance of quality control fidelity but may impair expression of normal mRNAs. NMD has adapted to this challenge by employing mechanisms to enhance identification of certain potential substrates, while using sequence-specific RNA-binding proteins to shield others from detection. These elaborations on the conserved NMD mechanism permit more sensitive post-transcriptional gene regulation but can have severe deleterious consequences, including the failure to degrade pathogenic aberrant mRNAs in many B cell lymphomas. This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms.

Hedgehog signaling in the airway epithelium of patients with chronic obstructive pulmonary disease.

Genome-wide association studies have linked gene variants of the receptor patched homolog 1 (PTCH1) with chronic obstructive pulmonary disease (COPD). However, its biological role in the disease is unclear. Our objective was to determine the expression pattern and biological role of PTCH1 in the lungs of patients with COPD. Airway epithelial-specific PTCH1 protein expression and epithelial morphology were assessed in lung tissues of control and COPD patients. PTCH1 mRNA expression was measured in bronchial epithelial cells obtained from individuals with and without COPD. The effects of PTCH1 siRNA knockdown on epithelial repair and mucous expression were evaluated using human epithelial cell lines. Ptch1+/- mice were used to assess the effect of decreased PTCH1 on mucous expression and airway epithelial phenotypes. Airway epithelial-specific PTCH1 protein expression was significantly increased in subjects with COPD compared to controls, and its expression was associated with total airway epithelial cell count and thickness. PTCH1 knockdown attenuated wound closure and mucous expression in airway epithelial cell lines. Ptch1+/- mice had reduced mucous expression compared to wildtype mice following mucous induction. PTCH1 protein is up-regulated in COPD airway epithelium and may upregulate mucous expression. PTCH1 provides a novel target to reduce chronic bronchitis in COPD patients.

hnRNP L-dependent protection of normal mRNAs from NMD subverts quality control in B cell lymphoma.

The human nonsense-mediated mRNA decay pathway (NMD) performs quality control and regulatory functions within complex post-transcriptional regulatory networks. In addition to degradation-promoting factors, efficient and accurate detection of NMD substrates involves proteins that safeguard normal mRNAs. Here, we identify hnRNP L as a factor that protects mRNAs with NMD-inducing features including long 3'UTRs. Using biochemical and transcriptome-wide approaches, we provide evidence that the susceptibility of a given transcript to NMD can be modulated by its 3'UTR length and ability to recruit hnRNP L. Integrating these findings with the previously defined role of polypyrimidine tract binding protein 1 in NMD evasion enables enhanced prediction of transcript susceptibility to NMD. Unexpectedly, this system is subverted in B cell lymphomas harboring translocations that produce BCL2:IGH fusion mRNAs. CRISPR/Cas9 deletion of hnRNP L binding sites near the BCL2 stop codon reduces expression of the fusion mRNAs and induces apoptosis. Together, our data indicate that protection by hnRNP L overrides the presence of multiple 3'UTR introns, allowing these aberrant mRNAs to evade NMD and promoting BCL2 overexpression and neoplasia.

Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes.

Cell-free systems are widely used to study mechanisms and regulation of translation, but the use of in vitro transcribed (IVT) mRNAs as translation substrates limits their efficiency and utility. Here, we present an approach for in vitro translation of messenger ribonucleoprotein (mRNP) complexes affinity purified in association with tagged mRNAs expressed in mammalian cells. We show that in vitro translation of purified mRNPs is much more efficient than that achieved using standard IVT mRNA substrates and is compatible with physiological ionic conditions. The high efficiency of affinity-purified mRNP in vitro translation is attributable to both copurified protein components and proper mRNA processing and modification. Further, we use translation inhibitors to show that translation of purified mRNPs consists of separable phases of run-off elongation by copurified ribosomes and de novo initiation by ribosomes present in the translation extracts. We expect that this in vitro system will enhance mechanistic studies of eukaryotic translation and translation-associated processes by allowing the use of endogenous mRNPs as translation substrates under physiological buffer conditions.

ZFR coordinates crosstalk between RNA decay and transcription in innate immunity.

Control of type I interferon production is crucial to combat infection while preventing deleterious inflammatory responses, but the extent of the contribution of post-transcriptional mechanisms to innate immune regulation is unclear. Here, we show that human zinc finger RNA-binding protein (ZFR) represses the interferon response by regulating alternative pre-mRNA splicing. ZFR expression is tightly controlled during macrophage development; monocytes express truncated ZFR isoforms, while macrophages induce full-length ZFR to modulate macrophage-specific alternative splicing. Interferon-stimulated genes are constitutively activated by ZFR depletion, and immunostimulation results in hyper-induction of interferon ß (IFNß/IFNB1). Through whole-genome analyses, we show that ZFR controls interferon signaling by preventing aberrant splicing and nonsense-mediated decay of histone variant macroH2A1/H2AFY mRNAs. Together, our data suggest that regulation of ZFR in macrophage differentiation guards against aberrant interferon responses and reveal a network of mRNA processing and decay that shapes the transcriptional response to infection.

ICE1 promotes the link between splicing and nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway detects aberrant transcripts containing premature termination codons (PTCs) and regulates expression of 5-10% of non-aberrant human mRNAs. To date, most proteins involved in NMD have been identified by genetic screens in model organisms; however, the increased complexity of gene expression regulation in human cells suggests that additional proteins may participate in the human NMD pathway. To identify proteins required for NMD, we performed a genome-wide RNAi screen against >21,000 genes. Canonical members of the NMD pathway were highly enriched as top hits in the siRNA screen, along with numerous candidate NMD factors, including the conserved ICE1/KIAA0947 protein. RNAseq studies reveal that depletion of ICE1 globally enhances accumulation and stability of NMD-target mRNAs. Further, our data suggest that ICE1 uses a putative MIF4G domain to interact with exon junction complex (EJC) proteins and promotes the association of the NMD protein UPF3B with the EJC.

Using Tet-Off Cells and RNAi Knockdown to Assay mRNA Decay.

Cellular mRNA levels are determined by the competing forces of transcription and decay. A wide array of cellular mRNA decay pathways carry out RNA turnover either on a constitutive basis or in response to changing cellular conditions. Here, we outline a method to investigate mRNA decay that employs RNAi knockdown of known or putative decay factors in commercially available Tet-off cell systems. Reporter mRNAs of interest are expressed under the control of a tetracycline-regulated promoter, allowing pulse-chase mRNA decay assays to be conducted. Levels of reporter and constitutively expressed control RNAs throughout the decay assay time course are detected by traditional northern blot analysis and used to calculate mRNA half-lives. We describe the utility of this approach to study nonsense-mediated mRNA decay substrates and factors, but it can be readily adapted to investigate key mechanistic features that dictate the specificity and functions of any mRNA decay pathway.

A system for coordinated analysis of translational readthrough and nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing premature termination codons, limiting the expression of potentially deleterious truncated proteins. This activity positions the pathway as a regulator of the severity of genetic diseases caused by nonsense mutations. Because many genetic diseases result from nonsense alleles, therapeutics inducing readthrough of premature termination codons and/or inhibition of NMD have been of great interest. Several means of enhancing translational readthrough have been reported to concomitantly inhibit NMD efficiency, but tools for systematic analysis of mammalian NMD inhibition by translational readthrough are lacking. Here, we introduce a system that allows concurrent analysis of translational readthrough and mRNA decay. We use this system to show that diverse readthrough-promoting RNA elements have similar capacities to inhibit NMD. Further, we provide evidence that the level of translational readthrough required for protection from NMD depends on the distance of the suppressed termination codon from the end of the mRNA.

Intra-patient variability in adalimumab drug levels within and between cycles in Crohn's disease.

BACKGROUND: Whether therapeutic drug monitoring for adalimumab needs to be performed at trough has not been defined. AIM: To determine intra-patient adalimumab drug-level variation and to identify modulating patient and disease factors. METHODS: In this prospective observational study, adult patients with Crohn's disease established on maintenance adalimumab had drug levels measured repeatedly according to pre-defined schedules (visit 1: day 4-6, visit 2: day 7-9, trough: day 13-14) across two consecutive fortnightly cycles. Disease activity was assessed using Harvey-Bradshaw Index, C-reactive protein and faecal calprotectin. For this analysis, trough levels ≥4.9 µg/mL were considered therapeutic. RESULTS: Nineteen patients underwent 111 evaluations. Mean intra-patient drug levels from paired visits between cycles did not differ (visit1 cycle1: 4.81, cycle2: 5.21 µg/mL, P = 0.24, visit2 cycle1: 4.86, cycle2: 4.82, P = 0.91 and trough cycle1: 3.95, cycle2: 3.95, P = 0.99), irrespective of disease activity. Drug levels were stable over the first 9 days (visit 1-2), but declined to trough by a mean 1.06 and 0.89 µg/mL between visit 1 or 2, respectively (P < 0.001). Models using nontemporal factors (smoking, syringe delivery device) and levels at earlier visits accounted for 66-80% of the variance in trough levels. On receiver-operating curve analysis, thresholds identified in the first 9 days that predicted a therapeutic trough level were similar to the trough threshold itself, with high sensitivity but modest specificity. CONCLUSION: While therapeutic drug monitoring should be performed at trough, a drug level ≥4.9 µg/mL obtained during the first 9 days predicts a therapeutic trough drug level with reasonable confidence.

The RUDY study platform - a novel approach to patient driven research in rare musculoskeletal diseases.

BACKGROUND: Research into rare diseases is becoming more common, with recognition of the significant diagnostic and therapeutic care gaps. Registries are considered a key research methodology to address rare diseases. This report describes the structure of the Rare UK Diseases Study (RUDY) platform that aims to improve research processes and address many of the challenges of carrying out rare musculoskeletal disease research. RUDY is an internet-based platform with online registration, initial verbal consent, online capture of patient reported outcome measures and events within a dynamic consent framework. The database structure, security and governance framework are described. RESULTS: There have been 380 participants recruited into RUDY with completed questionnaire rates in excess of 50 %. There has been one withdrawal and two participants have amended their consent options. CONCLUSIONS: The strengths of RUDY include low burden for the clinical team, low research administration costs with high participant recruitment and ease of data collection and access. This platform has the potential to be used as the model for other rare diseases globally.