Tethering Assays Research Articles

Using the Tether Function Assay to Identify Potential Regulators of mRNA Translation and mRNA Decay.

RNA binding proteins (RBPs) and their associated partners are key factors of posttranscriptional control of gene expression. To study and manipulate the functional consequences of binding of these regulators to their targets, several tethering assays have been developed, in which a protein of interest is brought to a reporter mRNA through heterologous RNA-protein interaction motifs. The effect of such constrained binding is then monitored by measuring the accumulation of the reporter protein and mRNA. This chapter describes a protocol for the λN-BoxB tether system in transiently transfected mammalian cells. Combining the luciferase reporter technology to quantify protein amounts by light measurement and RNA amounts by RT-qPCR, this assay provides a simple and robust way to analyze the consequences of any protein binding in a controlled and defined manner.

Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion

RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we developed tethered function luciferase-based splicing reporters that provide rapid, scalable and robust readouts of exon inclusion changes and used these to evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing and affinity purification–mass spectrometry to investigate a subset of candidates with no prior association with splicing. Integrative analysis of these assays indicates surprising roles for TRNAU1AP, SCAF8 and RTCA in the modulation of hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using these identified domains, we engineered programmable fusion proteins that outperform current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. This tethering approach characterizes the ability of RBPs to induce exon inclusion and yields new molecular parts for programmable splicing control.

M6A modification negatively regulates translation by switching mRNA from polysome to P-body via IGF2BP3

In the cytoplasm, mRNAs are dynamically partitioned into translating and non-translating pools, but the mechanism for this regulation has largely remained elusive. Here, we report that m6A regulates mRNA partitioning between polysome and P-body where a pool of non-translating mRNAs resides. By quantifying the m6A level of polysomal and cytoplasmic mRNAs with m6A-LAIC-seq and m6A-LC-MS/MS in HeLa cells, we observed that polysome-associated mRNAs are hypo-m6A-methylated, whereas those enriched in P-body are hyper-m6A-methylated. Downregulation of the m6A writer METTL14 enhances translation by switching originally hyper-m6A-modified mRNAs from P-body to polysome. Conversely, by proteomic analysis, we identify a specific m6A reader IGF2BP3 enriched in P-body, and via knockdown and molecular tethering assays, we demonstrate that IGF2BP3 is both necessary and sufficient to switch target mRNAs from polysome to P-body. These findings suggest a model for the dynamic regulation of mRNA partitioning between the translating and non-translating pools in an m6A-dependent manner.

Silver Ions Inhibit Bacterial Movement and Stall Flagellar Motor.

Silver (Ag) in different forms has been gaining broad attention due to its antimicrobial activities and the increasing resistance of bacteria to commonly prescribed antibiotics. However, various aspects of the antimicrobial mechanism of Ag have not been understood, including how Ag affects bacterial motility, a factor intimately related to bacterial virulence. Here, we report our study on how Ag+ ions affect the motility of E. coli bacteria using swimming, tethering, and rotation assays. We observed that the bacteria slowed down dramatically by >70% when subjected to Ag+ ions, providing direct evidence that Ag+ ions inhibit the motility of bacteria. In addition, through tethering and rotation assays, we monitored the rotation of flagellar motors and observed that the tumbling/pausing frequency of bacteria increased significantly by 77% in the presence of Ag+ ions. Furthermore, we analyzed the results from the tethering assay using the hidden Markov model (HMM) and found that Ag+ ions decreased bacterial tumbling/pausing-to-running transition rate significantly by 75%. The results suggest that the rotation of bacterial flagellar motors was stalled by Ag+ ions. This work provided a new quantitative understanding of the mechanism of Ag-based antimicrobial agents in bacterial motility.

Rapid Gene Evolution in an Ancient Post-transcriptional and Translational Regulatory System Compensates for Meiotic X Chromosomal Inactivation.

It is conventionally assumed that conserved pathways evolve slowly with little participation of gene evolution. Nevertheless, it has been recently observed that young genes can take over fundamental functions in essential biological processes, for example, development and reproduction. It is unclear how newly duplicated genes are integrated into ancestral networks and reshape the conserved pathways of important functions. Here, we investigated origination and function of two autosomal genes that evolved recently in Drosophila: Poseidon and Zeus, which were created by RNA-based duplications from the X-linked CAF40, a subunit of the conserved CCR4–NOT deadenylase complex involved in posttranscriptional and translational regulation. Knockdown and knockout assays show that the two genes quickly evolved critically important functions in viability and male fertility. Moreover, our transcriptome analysis demonstrates that the three genes have a broad and distinct effect in the expression of hundreds of genes, with almost half of the differentially expressed genes being perturbed exclusively by one paralog, but not the others. Co-immunoprecipitation and tethering assays show that the CAF40 paralog Poseidon maintains the ability to interact with the CCR4–NOT deadenylase complex and might act in posttranscriptional mRNA regulation. The rapid gene evolution in the ancient posttranscriptional and translational regulatory system may be driven by evolution of sex chromosomes to compensate for the meiotic X chromosomal inactivation (MXCI) in Drosophila.

Spatial and Temporal Patterns in Macroherbivore Grazing in a Multi-Species Tropical Seagrass Meadow of the Great Barrier Reef

Macroherbivory is an important process in seagrass meadows worldwide; however, the impact of macroherbivores on seagrasses in the Great Barrier Reef (GBR) has received little attention. We used exclusion cages and seagrass tethering assays to understand how the intensity of macroherbivory varies over space and time in the seagrass meadows around Green Island (Queensland), and what impact this has on overall meadow structure. Rates of macroherbivory were comparatively low, between 0.25–44% of daily seagrass productivity; however, rates were highly variable over a one-year period, and among sites. Loss of seagrass material to macroherbivory was predominantly due to fish; however, urchin herbivory was also taking place. Macroherbivory rates were of insufficient intensity to impact overall meadow structure. No macroherbivory events were identified on video cameras that filmed in the day, indicating that feeding may be occurring infrequently in large shoals, or at night. While relatively low compared to some meadows, seagrass macroherbivory was still an important process at this site. We suggest that in this highly protected area of the GBR, where the ecosystem and food webs remain largely intact, macroherbivory was maintained at a low level and was unlikely to cause the large-scale meadow structuring influence that can be seen in more modified seagrass systems.

Membrane Tethering Potency of Rab-Family Small GTPases Is Defined by the C-Terminal Hypervariable Regions.

Membrane tethering is a crucial step to determine the spatiotemporal specificity of secretory and endocytic trafficking pathways in all eukaryotic endomembrane systems. Recent biochemical studies by a chemically-defined reconstitution approach reveal that, in addition to the structurally-diverse classic tethering factors such as coiled-coil tethering proteins and multisubunit tethering complexes, Rab-family small GTPases also retain the inherent membrane tethering functions to directly and physically bridge two distinct lipid bilayers by themselves. Although Rab-mediated membrane tethering reactions are fairly efficient and specific in the physiological context, its mechanistic basis is yet to be understood. Here, to explore whether and how the intrinsic tethering potency of Rab GTPases is controlled by their C-terminal hypervariable region (HVR) domains that link the conserved small GTPase domains (G-domains) to membrane anchors at the C-terminus, we quantitatively compared tethering activities of two representative Rab isoforms in humans (Rab5a, Rab4a) and their HVR-deleted mutant forms. Strikingly, deletion of the HVR linker domains enabled both Rab5a and Rab4a isoforms to enhance their intrinsic tethering potency, exhibiting 5- to 50-fold higher initial velocities of tethering for the HVR-deleted mutants than those for the full-length, wild-type Rabs. Furthermore, we revealed that the tethering activity of full-length Rab5a was significantly reduced by the omission of anionic lipids and cholesterol from membrane lipids and, however, membrane tethering driven by HVR-deleted Rab5a mutant was completely insensitive to the headgroup composition of lipids. Reconstituted membrane tethering assays with the C-terminally-truncated mutants of Rab4a further uncovered that the N-terminal residues in the HVR linker, located adjacent to the G-domain, are critical for regulating the intrinsic tethering activity. In conclusion, our current findings establish that the non-conserved, flexible C-terminal HVR linker domains define membrane tethering potency of Rab-family small GTPases through controlling the close attachment of the globular G-domains to membrane surfaces, which confers the active tethering-competent state of the G-domains on lipid bilayers.

Intragenic recruitment of NF-\u03baB drives splicing modifications upon activation by the oncogene Tax of HTLV-1

Chronic NF-κB activation in inflammation and cancer has long been linked to persistent activation of NF-κB–responsive gene promoters. However, NF-κB factors also massively bind to gene bodies. Here, we demonstrate that recruitment of the NF-κB factor RELA to intragenic regions regulates alternative splicing upon NF-κB activation by the viral oncogene Tax of HTLV-1. Integrative analyses of RNA splicing and chromatin occupancy, combined with chromatin tethering assays, demonstrate that DNA-bound RELA interacts with and recruits the splicing regulator DDX17, in an NF-κB activation-dependent manner. This leads to alternative splicing of target exons due to the RNA helicase activity of DDX17. Similar results were obtained upon Tax-independent NF-κB activation, indicating that Tax likely exacerbates a physiological process where RELA provides splice target specificity. Collectively, our results demonstrate a physical and direct involvement of NF-κB in alternative splicing regulation, which significantly revisits our knowledge of HTLV-1 pathogenesis and other NF-κB-related diseases.

Predation patterns across states of landscape fragmentation can shift with seasonal transitions.

Nested scales of habitat heterogeneity may independently or synergistically influence faunal interactions. Fragmentation effects (i.e., the breaking apart of landscapes) and edge effects (i.e., ecological differences between edges and interiors of patches, nested within landscapes) are distinct yet related ecological concepts, linked mathematically by the habitat edge-to-area ratio. Our study quantified the separate and interactive effects of fragmentation and edge on predation using temperate seagrass. To assess how predation and generalized consumption were influenced by fragmentation state (i.e., continuous, fragmented), and proximity to edge (i.e., edges, interiors), we used tethering assays with two prey-items: juvenile crabs, Callinectes sapidus, and "squidpops" (dried squid mantle). We also investigated whether faunal densities (a proxy for consumption potential) and temperature (a proxy for a broad suite of seasonal changes) correlated with predation across landscapes. Results showed fragmentation state affected predation (i.e., crab) mortality, yet edge effects did not. Moreover, the directionality of fragmentation effects shifted across a temperature/seasonal gradient. Predation mortality more than doubled in continuous landscapes amidst temperature increases, surpassing initially higher mortality in fragmented landscapes, which did not systematically vary with temperature. This mortality magnitude "flip" matched spatiotemporal trends in faunal densities between continuous and fragmented meadows. Consumption rates of both prey-items increased alongside temperature and neither demonstrated edge effects. However, crabs showed fragmentation effects not seen with squidpops, suggesting differing foraging strategies used by consumers of these prey-items. We conclude that fragmentation and edge effects have dynamic influences on temperate predator-prey interactions, as faunal favorability of habitat heterogeneity can "flip" temporally.

Genomic Clustering Facilitates Nuclear Processing of Suboptimal Pri-miRNA Loci

Nuclear processing of most miRNAs is mediated by Microprocessor, comprised of RNase III enzyme Drosha and its cofactor DGCR8. Here, we uncover a hidden layer of Microprocessor regulation via studies of Dicer-independent mir-451, which is clustered with canonical mir-144. Although mir-451 is fully dependent on Drosha/DGCR8, its short stem and small terminal loop render it an intrinsically weak Microprocessor substrate. Thus, it must reside within a cluster for normal biogenesis, although the identity and orientation of its neighbor are flexible. We use DGCR8 tethering assays and operon structure-function assays to demonstrate that local recruitment and transfer of Microprocessor enhances suboptimal substrate processing. This principle applies more broadly since genomic analysis indicates suboptimal canonical miRNAs are enriched in operons, and we validate several of these experimentally. Proximity-based enhancement of suboptimal hairpin processing provides a rationale for genomic retention of certain miRNA operons and may explain preferential evolutionary emergence of miRNA operons.

Variation in consumer pressure along 2500 km in a major upwelling system: crab predators are more important at higher latitudes

Consumer pressure in benthic communities is predicted to be higher at low than at high latitudes, but support for this pattern has been ambiguous, especially for herbivory. To understand large-scale variation in biotic interactions, we quantify consumption (predation and herbivory) along 2500 km of the Chilean coast (19°S–42°S). We deployed tethering assays at ten sites with three different baits: the crab Petrolisthes laevigatus as living prey for predators, dried squid as dead prey for predators/scavengers, and the kelp Lessonia spp. for herbivores. Underwater videos were used to characterize the consumer community and identify those species consuming baits. The species composition of consumers, frequency of occurrence, and maximum abundance (MaxN) of crustaceans and the blenniid fish Scartichthys spp. varied across sites. Consumption of P. laevigatus and kelp did not vary with latitude, while squid baits were consumed more quickly at mid and high latitudes. This is likely explained by the increased occurrence of predatory crabs, which was positively correlated with consumption of squidpops after 2 h. Crabs, rather than fish, were the principal consumers of squid baits (91% of all recorded predation events) at sites south of 30°S. Fish and crustaceans preyed in similar proportion on P. laevigatus, with most fish predation events at northern sites. The absence of any strong latitudinal patterns in consumption rate of tethered prey is likely due to redundancy among consumers across the latitudinal range, with crustaceans gaining in importance with increasing latitude, possibly replacing fish as key predators.

Dissecting the functions of SMG5, SMG7, and PNRC2 in nonsense-mediated mRNA decay of human cells.

The term “nonsense-mediated mRNA decay” (NMD) originally described the degradation of mRNAs with premature translation–termination codons (PTCs), but its meaning has recently been extended to be a translation-dependent post-transcriptional regulator of gene expression affecting 3%–10% of all mRNAs. The degradation of NMD target mRNAs involves both exonucleolytic and endonucleolytic pathways in mammalian cells. While the latter is mediated by the endonuclease SMG6, the former pathway has been reported to require a complex of SMG5–SMG7 or SMG5–PNRC2 binding to UPF1. However, the existence, dominance, and mechanistic details of these exonucleolytic pathways are divisive. Therefore, we have investigated the possible exonucleolytic modes of mRNA decay in NMD by examining the roles of UPF1, SMG5, SMG7, and PNRC2 using a combination of functional assays and interaction mapping. Confirming previous work, we detected an interaction between SMG5 and SMG7 and also a functional need for this complex in NMD. In contrast, we found no evidence for the existence of a physical or functional interaction between SMG5 and PNRC2. Instead, we show that UPF1 interacts with PNRC2 and that it triggers 5′–3′ exonucleolytic decay of reporter transcripts in tethering assays. PNRC2 interacts mainly with decapping factors and its knockdown does not affect the RNA levels of NMD reporters. We conclude that PNRC2 is probably an important mRNA decapping factor but that it does not appear to be required for NMD.

Abstract LB-A13: Targeting poly-ADP ribose glycohydrolase: pursuing a synthetic lethal therapeutic strategy to treat all pancreatic cancers

Abstract This study lays the groundwork to target poly-ADP ribose glycohydrolase (PARG), a DNA repair enzyme that counteracts PARP-1 activity and is upregulated in response to PARP inhibitor (PARPi) treatment. The goal of this study is to optimize PARPi-based therapies for all pancreatic cancer patients, irrespective of their DNA repair status (i.e., homologous repair (HR) deficiency). Metastatic pancreatic ductal adenocarcinoma (PDA) is a lethal disease with an average survival of less than one year, which highlights the need for novel treatment regimens. Though PARPi therapy offers a personalized strategy in a subset of tumors with HR defects, initially responsive patients typically become resistant to therapy. Therefore, identifying and combating PARPi resistance mechanisms is a rising concern. We have previously shown that PARG is a critical player in mediating resistance; therefore we evaluated PARG as a candidate target in PDA. Importantly, we demonstrated that the pro-oncogenic protein, HuR (ELAVL1), upregulates PARG specifically in tumor microenvironment niches (e.g., drug exposure), thus providing a unique, cancer-specific therapeutic window for targeting PARG. We evaluated a series of potent first-in-class, cell-active small molecule PARG inhibitors; PDDX-01, PDDX02 and PDDX-04 in pre-clinical models of PDA. These PARGi effectively inhibited PARG activity, caused persistence of PAR polymers, increased accumulation of γH2AX foci and inhibited short and long-term growth of PDA cell lines. Similar to PARPi agents, PARG inhibition was also shown to be synthetic lethal; DNA damage repair (DDR)- deficient cell lines were more sensitive than the DDR- proficient PDA cell lines. This was further validated in isogenic colorectal cell lines with varying DNA repair functionality; DLD1 lines with BRCA2 (+/+, +/-, -/-) and RKO lines with FANCC (+/+, +/-, -/-) modulation. Using chromatin tethering assays, these PARGis effectively trapped PARP1 on DNA, alone as well as combination with the PARPi olaparib, and a DNA damaging agent oxaliplatin. We also developed and characterized a PARG inhibition model in vivo in a DNA- repair proficient PDA line, MIA PaCa2. The doxycycline-inducible shPARG knockdown was validated for PARG inhibition, PAR accumulation and increased PARPi sensitivity in vitro. Complementing xenograft experiments were performed wherein 3 million cells were injected per flank in nude female athymic mice. At an average tumor volume of 50mm3, respective groups were fed DOX- chow to induce PARG knockdown and treated with olaparib intraperitoneally at 50mg/kg five times a week. PARG inhibition by doxycycline induction significantly decreased tumor volumes (50% decrease, p-value 0.0165), which was further enhanced with olaparib treatment (70% decrease, p- value 0.0004), when compared with control arms. Taken together, these studies help define and break a potent PARPi resistance mechanism and also validate pursuing PARG as a valuable “druggable” target in both HR-proficient and deficient PDA cells. Citation Format: Saswati N. Chand, AnnJosette Ramirez, Aditi Jain, Avinoam Nevlar, Cinthya Yabar-Lowder, Joseph A. Cozzitorto, Dominic I. James, Ian D. Waddell, Charles J. Yeo, Michael J. Pishvaian, Jordan M. Winter, Jonathan R. Brody. Targeting poly-ADP ribose glycohydrolase: pursuing a synthetic lethal therapeutic strategy to treat all pancreatic cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A13.

Refining the tethering of American oysters (Crassostrea virginica) to measure the effects of two environmental stressors

Tethering assays, or the physical restraint of test organisms, has been used in the past to measure selected organisms' response to stressors while removing the observer from the experimental setting. Although informative for monitoring and hypothesis testing, these assays often used microfilaments that have been found to be too invasive or prone to biases given their effects on test organisms' behavior. Here, we describe a new variation of tethering using American oysters (Crassostrea virginica) and illustrate its use in the study of their mortality rates as a result of two stressors: siltation and predation by a non-indigenous species. Our protocol identified a resistant (non-toxic) glue that could be used to attach oysters to stone slabs, thus partially mimicking the natural cementation of the shell to natural substrates. This variation of tethering was harmless and maintained oysters' body position and natural ability to filter feed. Using tethered oysters in separate two-week field cage experiments, we also show how siltation and predation by a non-indigenous species (the European green crab, Carcinus maenas), caused a gradual, easily measurable increase in oyster mortality rates. We argue that this variation of tethering is a cost-effective and advantageous way to monitor or test the effects of these and other stressors on oysters and similar species.

Trinucleotide repeat containing 6c (TNRC6c) is essential for microvascular maturation during distal airspace sacculation in the developing lung

GW182 (also known asTNRC6) family members are critically involved in the final effector phase of miRNA-mediated mRNA repression. The three mammalian paralogs, TNRC6a, b and c, are thought to be redundant based on Argonaute (Ago) binding, tethering assays, and RNAi silencing of individual members in cell lines. To test this idea, we generated TNRC6a, b and c knockout mice. TNRC6a mutants die at mid-gestation, while b- and c- deleted mice are born at a Mendelian ratio. However, the majority of TNRC6b and all TNRC6c mutants die within 24h after birth, the latter with respiratory failure. Necropsy of TNRC6c mutants revealed normal-appearing airways that give rise to abnormally thick-walled distal gas exchange sacs. Immunohistological analysis of mutant lungs demonstrated a normal distribution of bronchiolar and alveolar cells, indicating that loss of TNRC6c did not abrogate epithelial cell differentiation. The cellular kinetics and relative proportions of endothelial, epithelial, and mesenchymal cells were also not altered. However, the underlying capillary network was simplified and endothelial cells had failed to become tightly apposed to the surface epithelium in TNRC6c mutants, presumably causing the observed respiratory failure. TGFβ family mutant mice exhibit a similar lung phenotype of thick-walled air sacs and neonatal lethality, and qRT-PCR confirmed dynamic downregulation of TGFβ1 and TGFβR2 in TNRC6c mutant lungs during sacculation. VEGFR, but not VEGF-A ligand, was also lower, likely reflecting the overall reduced capillary density in TNRC6c mutants. Together, these results demonstrate that GW182 paralogs are not functionally redundant in vivo. Surprisingly, despite regulating a general cellular process, TNRC6c is selectively required only in the distal lung and not until late in gestation for proper expression of the TGFβ family genes that drive sacculation. These results imply a complex and indirect mode of regulation of sacculation by TNRC6c, mediated in part by dynamic transcriptional repression of an inhibitor of TGFβ family gene expression.

Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA-guided gene silencing invivo.

Argonaute proteins associate with microRNAs and are key components of gene silencing pathways. With such a pivotal role, these proteins represent ideal targets for regulatory post-translational modifications. Using quantitative mass spectrometry, we find that a C-terminal serine/threonine cluster is phosphorylated at five different residues in human and Caenorhabditis elegans In human, hyper-phosphorylation does not affect microRNA binding, localization, or cleavage activity of Ago2. However, mRNA binding is strongly affected. Strikingly, on Ago2 mutants that cannot bind microRNAs or mRNAs, the cluster remains unphosphorylated indicating a role at late stages of gene silencing. In C.elegans, the phosphorylation of the conserved cluster of ALG-1 is essential for microRNA function invivo Furthermore, a single point mutation within the cluster is sufficient to phenocopy the loss of its complete phosphorylation. Interestingly, this mutant retains its capacity to produce and bind microRNAs and represses expression when artificially tethered to an mRNA Altogether, our data suggest that the phosphorylation state of the serine/threonine cluster is important for Argonaute-mRNA interactions.

Mitochondrial Fusion Proteins: A Tale of Two Membranes

Dynamins are a class of GTPase enzymes responsible for the fusion, fission, and vesiculation of cellular lipid membranes throughout the cell. The dynamin-like proteins Optic Atrophy 1 (Opa1) and Mitofusin (Mfn) 1 and 2 are responsible for the fusion of the mitochondrial inner and outer membranes, respectively. Mutations in any of these proteins can lead to neuropathies including blindness and Charcot-Marie-Tooth, a disease characterized by progressive loss of distal muscle tissue. Currently, little is known structurally or biochemically about any of these proteins. We have developed a protocol for expressing and purifying biologically relevant and biochemically active shortened isoforms (OpaGG and Mfn1GG) in sufficient quantity to begin crystallographic studies. Both have comparable GTPase activity compared to full-length, unstimulated Dynamin 1 when assayed at room temperature and interestingly OpaGG exists as a tetramer when assayed by size exclusion chromatography. In addition, the long, membrane-bound isoforms of Opa1 and Mfn1 have been expressed and purified in large quantities. To date, we have shown full length Mfn1 can be incorporated into proteoliposomes and in the presence of GTP forms dense tethers as seen by cryo-EM. This tethering is reversible as shown by confocal microscopy. Currently we are developing tethering assays for Opa1 and fusion assays for both Opa1 and Mfn1.

Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences.

The turnover of messenger RNAs (mRNAs) is a key regulatory step of gene expression in eukaryotic cells. Due to the complexity of the mammalian degradation machinery, the contribution of decay factors to the directionality of mRNA decay is poorly understood. Here we characterize a molecular tool to interrogate mRNA turnover via the detection of XRN1-resistant decay fragments (xrFrag). Using nonsense-mediated mRNA decay (NMD) as a model pathway, we establish xrFrag analysis as a robust indicator of accelerated 5′–3′ mRNA decay. In tethering assays, monitoring xrFrag accumulation allows to distinguish decapping and endocleavage activities from deadenylation. Moreover, xrFrag analysis of mRNA degradation induced by miRNAs, AU-rich elements (AREs) as well as the 3′ UTRs of cytokine mRNAs reveals the contribution of 5′–3′ decay and endonucleolytic cleavage. Our work uncovers formerly unrecognized modes of mRNA turnover and establishes xrFrag as a powerful tool for RNA decay analyses.

Functional Analysis of BTG1 and BTG2 Variants in Non-Hodgkin Lymphoma

Non-Hodgkin's lymphoma (NHL) represents a highly heterogeneous group of lympho-proliferative disorders and accounts for more than half of the cases of haematological malignancies. These disorders are characterised by multiple steps of accumulating genetic mutations resulting in the selection of a malignant clone. Recently, mutations in the anti-proliferative genes B-cell translocation 1 gene (BTG1) and B-cell translocation 2 gene (BTG2) have been identified in approximately 10-15% of NHL cases, which suggests a direct involvement of BTG1 and BTG2 in malignant transformation.BTG1 and BTG2 are characterised by the conserved amino-terminal BTG domain, which mediates interactions with the Caf1 catalytic subunit of the Ccr4-Not deadenylase complex. In addition, the BTG domain binds to the poly(A)-binding protein PABPC1. This complex plays a critical role in mRNA deadenylation and degradation as well as translational repression.It is currently unclear how, or indeed whether, mutations in BTG1 and BTG2 affect the function of the gene products. Therefore, we used a combination of sequence analysis and molecular modelling to predict the functional consequences of mutations previously identified in NHL. Sorting intolerant from tolerant (SIFT) prediction tool was used to predict amino acidic residues potentially interfering with the protein function. In total we assessed 45 mutations in BTG1 and BTG2 derived from NHL samples, including diffuse large B-cell lymphoma, germinal centre B-cell-like, activated B-cell-like lymphoma, follicular lymphoma and Burkitt'slymphoma. Of the mutations analysed, 25 were predicted to interfere with the function of BTG1 and BTG2. We therefore analysed the ability of these protein variants to interact with known partners and functionally assessed the role of the mutated proteins in cell cycle progression, translational repression and mRNA degradation. Using a yeast two-hybrid system, fourteen mutations were shown to affect the interaction of BTG1 or BTG2 with the Caf1catalytic subunit of the Ccr4-Not deadenylase complex. In addition, when we transfected into mammalian cells, these BTG1 and BTG2 variants, unlike the wild-type proteins, were not able to inhibit cell cycle progression. Interestingly, our findings show that several mutations (M11I, F25C, P58L, I115V) on BTG1 do not require interaction with the Caf1 deadenylase enzyme to reduce reporter activity as established using 3' UTR tethering assays. This suggests that BTG1 and BTG2 may also have a role in regulating cell cycle progression and RNA degradation via Ccr4-Not-independent mechanisms.The data show that mutations in BTG1 and BTG2, commonly found in NHL, are functionally significant and are likely to contribute to malignant transformation and tumour cell grow. DisclosuresAlmasmoum:Umm Al-Qura University: Employment, Research Funding.

Multivalent Rab interactions determine tether-mediated membrane fusion.

Membrane fusion at endomembranes requires cross-talk between Rab GTPases and tethers to drive SNARE-mediated lipid bilayer mixing. Several tethers have multiple Rab-binding sites with largely untested function. Here we dissected the lysosomal HOPS complex as a tethering complex with just two binding sites for the Rab7-like Ypt7 protein to determine their relevance for fusion. Using tethering and fusion assays combined with HOPS mutants, we show that HOPS-dependent fusion requires both Rab-binding sites, with Vps39 being the stronger Ypt7 interactor than Vps41. The intrinsic amphipathic lipid packaging sensor (ALPS) motif within HOPS Vps41, a target of the vacuolar kinase Yck3, is dispensable for tethering and fusion but can affect tethering if phosphorylated. In combination, our data demonstrate that a multivalent tethering complex uses its two Rab bindings to determine the place of SNARE assembly and thus fusion at endomembranes.