Catalytic RNAs, or ribozymes, are involved in a number of essential biological processes, such as replication of RNA genomes and mobile genetic elements, RNA splicing, translation, and RNA degradation. The function of ribozymes requires the formation of active sites decorated with RNA functional groups within defined three-dimensional (3D) structures. The genotype (sequence) of RNAs ultimately determines what 3D structures they adopt . These 3D structures, in turn, give rise to biochemical activity, which can further elaborate them by catalytic rearrangements or association with other molecules. The fitness landscape of a non-periodic linear polymer, such as RNA, relates its primary structure to a phenotype. Two major challenges in the analysis of ribozymes is to map all possible genotypes to their corresponding catalytic activity , and to understand whether their genotypes and three-dimensional structures can support multiple different catalytic functions. Recently, the combined results of experiments that employ in vitro evolution methods, high-throughput sequencing and crystallographic structure determination have hinted at answers to these two questions: while the fitness landscape of ribozymes is rugged, meaning that their catalytic activity cannot be optimized by a smooth trajectory in sequence space, once an RNA achieves a stable three-dimensional fold, it can be endowed with distinctly different biochemical activities through small changes in genotype. This functional plasticity of highly structured RNAs may be particularly advantageous for the adaptation of organisms to drastic changes in selective pressure, or for the development of new biotechnological tools. RNA enzNA level ,6. In adNA level ,8,9. Mann + 1-dimensional function relating all possible alleles of the n loci of a genotype to the phenotypic fitness [n residues [nX, where X is the number of residue types, for instance 4 for RNA and 21 for proteins, and n is the length of the polymer), and the evaluation of the phenotypic fitness corresponding to each genotype. Advances in high-throughput sequencing methodology, combined with experimental designs in which the abundance of a sequence serves as a proxy for phenotypic fitness, have made it possible to experimentally examine the fitness landscapes of DNAs, RNAs and peptides in the past decade.In the most general sense, the relationship between genotype and phenotype can be described by a fitness landscape, and adaptation is an optimizing walk on this high-dimensional surface. In its original formulation, the fitness landscape was envisioned as an  fitness . The conresidues . FitnessEven with the most efficient sequencing methods, the total sequence space of an RNA can only be examined exhaustively for short sequences . The bin13 sequence variants of the parental ribozyme, allowing it to self-ligate to a biotin-tagged substrate RNA. They used this tag to isolate reactive species at different time-points, and subjected these to high-throughput sequencing, thereby producing a sequencing-based apparent ligation rate for each sequence [For longer RNA molecules, experimentally constructed fitness landscapes will necessarily be sparsely sampled. Pitt and Ferré-D’Amaré analyzed the fitness landscape of a previously in vitro selected 54-nt RNA ligase ribozyme by constructing a library containing 6 × 10sequence . These asequence . Thus, wThe ruggedness of RNA fitness landscapes is also reflected in the presence of multiple phenotypic peaks, which are not connected by genotypes that do not decrease fitness, i.e., the absence of neutral networks. Jiménez et al., through a combination of in vitro selection and high-throughput sequencing, generated a fitness landscape describing the ability of 24-nt RNAs to bind to guanosine triphosphate (GTP), finding a near-random distribution of disjoint fitness . This stOne possible solution to the conundrum posed by the rugged fitness landscapes inhabited by functional RNAs is that their evolution is not through optimization of one biochemical activity, but by the adoption of new capabilities. In a pioneering study, Schultes and Bartel examined such a scenario by constructing, in a step-wise manner, a mutational pathway that interconverted between an RNA ligase (the in vitro selected Class III ligase ribozyme) and an RNA cleaving ribozyme  . Through4SU synthase), is a 124-nt in vitro selected ribozyme that catalyzes formation of the glycosidic bond between an activated ribose and 4-thiouracil [4SU synthase, an in vitro selection was performed to examine if closely related variants of the 4SU synthase that catalyze the synthesis of the purine nucleotide 6-thioguanosine  exist [6SG synthase ribozymes that were isolated did not share any detectable structural similarity with the 4SU synthase. Structural probing and site-directed mutagenesis suggests that the 6SG synthases are structurally less complex than the 4SU synthases, being comprised of just two simple stem-loops. This work is consistent with the notion that, at least in some cases, evolution of new biochemical activities goes hand-in-hand with evolution of a new RNA structure.Acquisition of a new catalytic function through stepwise mutation of an RNA may be challenging, however, even if the two chemistries are very similar. The pyrimidine nucleotide synthase ribozyme  exist . Despite6SG synthase, and can catalyze glycosidic bond formation between 6-thioguanine and either an activated or non-activated ribose , with comparable efficiency. This is a case of a ribozyme which, by using a promiscuous active site that can accommodate different substrates, can catalyze two reactions using the same overall architecture.Is a new structure necessary for each different ribozyme? Depending on how they are functionalized, many conserved and abundant protein folds  are shared by enzymes that catalyze disparate reactions. Although the ligase-HDV and glycosidic bond synthase studies reviewed above may suggest that one RNA structure can only support one reaction, early ribozyme engineering studies using the Tetrahymena ribozyme, which is a considerably larger and more structurally complex RNA, argue otherwise. In addition to self-splicing, variants of this RNA have been reported to catalyze, to varying extents, a number of different reactions. Examples include RNA cleavage, RNA polymerization, and peptide bond hydrolysis ,21,22,23glmS riboswitch-ribozyme [The ribozyme  is uniquribozyme . In its ribozyme . As the ribozyme  as well glmS ribozyme is a relatively efficient catalyst, attaining a maximal cleavage rate of ~100 min−1 in the presence of GlcN6P, comparable to the rates of some of the fastest known ribozymes, such as the Class I ligase (maximum rate of 800 min−1) [Kd ~ 300 mM) [l-serine and Tris (tris(hydroxymethyl)aminomethane), which all share vicinal amine and hydroxyl groups in equivalent stereochemical relationships, can activate the ribozyme to various extents (d-serine is not an activator) [The 0 min−1) . Consisttivator) . In pringlmS ribozyme provided the first glimpse of the catalytic mechanism of this ~145-nt RNA [Ka of the amine group of GlcN6P and other non-physiological activators [glmS ribozyme does not change conformation upon GlcN6P binding, cleavage, or release of GlcN6P post-reaction [The crystal structure of the 5-nt RNA ,33. The 5-nt RNA , and thetivators ,34. Biocreaction ,35,36. TglmS ribozyme is, thus far, the only known natural ribozyme that has an absolute requirement for an exogenous coenzyme. Since many other ribozymes that catalyze the same sequence-specific RNA cleavage reaction through internal transesterification  are known, the discovery of the general acid catalyst function for GlcN6P in the glmS ribozyme raised a fundamental question about its evolutionary origin. Did the glmS ribozyme evolve from a GlcN6P-binding RNA  by becoming catalytic, or did it evolve from a coenzyme-independent ribozyme that became dependent on GlcN6P? Because of the exceptional structural stability of the glmS ribozyme fold, experiments addressing this question would also illuminate the broader question of whether a stable RNA fold can be decorated with different residues to support multiple biochemical activities.The glmS ribozyme [14glmS ribozyme variants in which residues in proximity to the binding site were mutagenized, without affecting any of the base pairing interactions that define the secondary structure or the three pseudoknots that confer on the RNA its exceptional stability. We then challenged the RNA sequences to catalyze internal RNA cleavage in the absence of GlcN6P, and only those sequences that were active were iteratively amplified and enriched. The selection resulted in the isolation of a glmS ribozyme variant bearing three adenosine mutations (glmSAAA) that can catalyze the same site-specific internal transesterification reaction as the wild-type in the absence of GlcN6P. This mutant can cleave RNA at rates of up to 2.5 min−1 in the complete absence of GlcN6P, requiring only divalent cations for activity (and its activity is not enhanced by GlcN6P). Crystallographic and small-angle X-ray scattering (SAXS) analyses demonstrated that glmSAAA retains the overall fold of the wild-type ribozyme  connects the metalloribozyme glmSAAA with the strictly dependent wild-type glmS ribozyme. Moreover, at least for this highly stable RNA fold, one overall architecture can support two different catalytic activities. This suggests that despite the rugged fitness landscapes that appear to characterize ribozymes, because different biochemical activities can arise through minimal  mutations, these catalysts may evolve by the rapid acquisition of new biochemical functions [To analyze the possible evolutionary pathway between a GlcN6P-independent ancestral ribozyme, such as unctions .Advances in the study of RNA structure and function in the past decade have not only increased knowledge of RNA biology, but also encouraged the engineering of RNAs as biotechnological tools. The characterization of cellular components involved in small RNAs regulatory pathways, for example, have led to the development of silencing RNA (siRNA) therapeutics for targeting specific cancer and disease related genes . The clucis, which leads to the degradation of the mRNA and silences the expression of the downstream genes.Riboswitches and ribozymes have also been expanded into synthetic biology and engineered to perform complex functions in cells that have also been explored as possible players in the development of RNA therapeutics. The Breaker group, for example, engineered an allosteric ribozyme by fusing an ATP-binding RNA motif (aptamer) with the self-cleaving hammerhead ribozyme. Binding of either adenosine or ATP drastically reduces the ribozyme activity, demonstrating how RNAs can be subjected to allosteric regulation using an exogenous small molecule. Using a similar RNA fusion approach, the Breaker lab, along with other research groups, have since engineered synthetic RNAs using the aptamer region as a genetic switch to control the expression of specific proteins . These RglmSAAA ribozyme described above [glmS ribozyme with strict dependence on GlcN6P to a metalloribozyme requiring only divalent metal ions for catalysis. Further ribozyme engineering by in vitro selection or mutational studies based on known RNA structures can therefore be a fruitful approach to rationally design and engineer new ribozymes with novel biological activities.In vitro evolution of ribozymes with pre-existing functions, as in the studies of the ed above , offers


RNA aptamers that bind non-fluorescent dyes and activate their fluorescence are highly sensitive, nonperturbing, and convenient probes in the field of synthetic biology. These RNA molecules, referred to as light-up aptamers, operate as molecular nanoswitches that alter folding and fluorescence function in response to ligand binding, which is important in biosensing and molecular computing. Herein, we demonstrate a conceptually new generation of smart RNA nano-devices based on malachite green (MG)-binding RNA aptamer, which fluorescence output controlled by addition of short DNA oligonucleotides inputs. Four types of RNA switches possessing AND, OR, NAND, and NOR Boolean logic functions were created in modular form, allowing MG dye binding affinity to be changed by altering 3D conformation of the RNA aptamer. It is essential to develop higher-level logic circuits for the production of multi-task nanodevices for data processing, typically requiring combinatorial logic gates. Therefore, we further designed and synthetized higher-level half adder logic circuit by “in parallel” integration of two logic gates XOR and AND within a single RNA nanoparticle. The design utilizes fluorescence emissions from two different RNA aptamers: MG-binding RNA aptamer (AND gate) and Broccoli RNA aptamer that binds DFHBI dye (XOR gate). All computationally designed RNA devices were synthesized and experimentally tested in vitro. The ability to design smart nanodevices based on RNA binding aptamers offers a new route to engineer “label-free” ligand-sensing regulatory circuits, nucleic acid detection systems, and gene control elements. The progression in the field of RNA nanotechnology makes RNA molecules the most promising candidate to fabricate bio-computers due to their variable folding properties as well as their catalytic functions ,2. NumerIn RNA nanotechnology, the development and implementation of RNA-based nanodevices that respond to biomolecular inputs by generating output signals in accordance with logic gate behavior has attracted considerable attention ,34,35,36DNA has been routinely used for the development of biochemical circuits and all basic logic operations, including INHIBIT, IMPLICATION, and XOR have been mimicked with DNA as a template ,48,49,50Herein, we report the design and assembly of a conceptually new generation of molecular logic gates that possess simple AND, OR, NAND, and NOR logic operations implementing the light-up MG-binding RNA aptamer. Single stranded DNA (ssDNA) oligonucleotides were used as inputs to trigger conformational changes in the RNA aptamer. The corresponding output values of OFF (0) and ON (1) are obtained by low and high fluorescence emissions, respectively A.Furthermore, we developed a basic half adder computing platform based on the RNA light-up aptamer strategy B. The deThe function of the half adder is triggered by the same two ssDNA inputs. The ssDNA inputs alter the conformation of the RNA aptamers in such a manner as to either permit or deny fluorogen dye binding to the aptamer, resulting in fluorescent (ON) or non-fluorescent (OFF) states within one nanostructure. This RNA logic gate system demonstrates the great potential of light-up RNA aptamers as an arithmetic tool for molecular programming and will open a way to further development concerning well-regulated molecular electronic devices and biosensors.RNA molecules featuring aptamers that bind a fluorogenic dye and activate its fluorescence have the potential to be highly sensitive and convenient probes in the field of synthetic biology. The initial system is comprised of an RNA hairpin molecule containing the MG RNA binding aptamer sequence and the MG fluorogen dye . The fluIndividual gates employed light-up RNA aptamers consisting of two extended sequences localized at both the 5′- and 3′-ends highlighted in In a similar manner, initial structures for the NAND and NOR gates were chosen to share identical nucleotides as the ON state is a requirement for both structures at the no inputs condition. For each logic gate, the fluorescence intensities were normalized throughout the experiments. The threshold value was determined to be 60% where an intensity greater than this value yields an output = 1, while an intensity below this value yields an output = 0.m), the temperature at which 50% of double stranded nucleic acid is converted to single-standard form. The desired Tm values for inputs hybridization were chosen to be slightly greater than 22 °C, the temperature at which the experiment was performed. Such input selection relies on the equilibrium between duplex (formed by ssDNA and hairpin nts) and hairpin structures where equilibrium shifts slightly in favor of the hairpin structure  was designed using interfering ends. The purpose of this was to form a complementary base pairing with the RNA aptamer MG-binding region as illustrated in The OR construct, which yields output = 0 for scenario 0-0 and output = 1 for scenarios 1-0, 0-1, and 1-1, shares the identical RNA sequence as the AND gate. However, the ssDNA inputs were designed to have several extra nucleotides to achieve an equilibrium state where inclusion of at least one input will trigger conformational change in favor of the correctly folded MG-binding RNA aptamer. The design criterion is based on a strand displacement reaction. Upon binding to its corresponding hairpin, input A or B will create a stable hybrid RNA/DNA duplex. The DNA inputs were selected to contain longer sequences with much higher Tm values as compared to the AND gate, so that disrupted hairpin nts initiate a conformational change of the whole construct favoring formation of the ON aptamer state , OR GATEIn contrast to the AND and OR gates, the NAND and NOR gates were designed so the default structures possess correctly folded light-up MG RNA aptamers and the extensions at the 5′- and 3′-ends do not interfere with the core structure. To produce the corresponding NAND operation, the non-interfering ends must be able to bind input A or input B without sacrificing the conformation of the aptamer. However, when both A and B are presented, the conformation of the aptamer needs to be sufficiently distorted to register an output = 0. The NOR logic gate was constructed utilizing the NAND RNA molecule. max = 650 nm, respectively, in response to DNA oligonucleotide inputs. A threshold value of fluorescence enhancement of 60% was chosen to distinguish the OFF  and ON  states. Various concentrations of RNA molecular gates, inputs, and MG dye in solution were explored, with those yielding the greatest difference in fluorescence between output = 0 and output = 1 reported here. The extent to which these modular RNA logic gates can be used to probe three or more inputs simultaneously will depend on their reliability in tandem.Collectively, the modular approach to the fabrication of RNA Boolean logic gates based on the light-up RNA aptamer was demonstrated. All designed gates produced the expected OFF or ON values corresponding to low or high fluorescence intensity at λThe production of multi-functional nanodevices for data analysis or processing is extremely important and yet challenging due to requirements of multiple coordinated logic gates operations within a single unit. Based on the aforementioned results, we next integrated two different fluorogen-binding RNA aptamers: (i) MG RNA aptamer and (ii) the recently developed Broccoli RNA aptamer that binds DFHBI dye within one RNA complex. As a key building block, the half adder is used to construct more advanced computational circuits and is in high demand in information technology . The repex/em = 465/510 nm (corresponding to the XOR gate) and λex/em = 615/650 nm (corresponding to the AND gate). These gates were rationally designed to use two ssDNA inputs to output two fluorescence signals: SUM (λem = 510 nm) and CARRY (λem = 650 nm) generated by the AND and XOR gates, respectively  gate based on the DFHBI-binding Broccoli light-up RNA aptamer . The fluectively . Both thectively . The conectively B. Atomicectively .To perform the XOR and AND logic operations using the same two ssDNA inputs, additional DNA inhibitor strands were introduced to bind complementary to the light-up RNA aptamers and interfere with their ON states or correctly folded structures. This tetragonal shaped half-adder RNA complex is designed according to the competitive hybridization and displacement principle of the DNA strands. The assembly experiments shown in The Broccoli RNA aptamer was designed to act as an XOR gate. To maintain the proper XOR gate function, an inhibitor DNA strand was designed (XOR_DNA inhibitor). The inhibitor bound to the aptamer, disrupting the binding of DFHBI and thus diminishing fluorescence in the presence of neither input. This XOR_DNA inhibitor contains two loop regions on either side of the aptamer. These internal loops contain eight unpaired nts and are designed to complement the ssDNA inputs. The addition of either ssDNA input destabilizes the RNA aptamer-inhibitor complex, separating the strands enough to reform the functional RNA aptamer shape allowing (1-0) or (0-1) truth values in fluorescence with an output of “1” A.However, in the presence of both inputs, fluorescence is once again inhibited as the hybridization of inputs A and B is favored over hybridization with XOR_DNA inhibitor. To achieve this, inputs A and B were designed to bind more competitively to one another than to the XOR_DNA inhibitor through 17 “sticky” nts at the 5′ end of input A and 3′ end of input B . TherefoThe AND logic operation of the half adder was designed utilizing the fluorescent properties of the MG-binding RNA aptamer system as the output signal. Similar to the XOR gate approach, a DNA inhibitor (AND_DNA) was used to disrupt the RNA aptamer conformation. The AND gate has an output of OFF or (0) in the absence of inputs (0-0) or in the presence of only one of the inputs (1-0 or 0-1). As the inputs are the same as those used for the Broccoli XOR gate, it is critical for the AND_DNA inhibitor to be complementary to the previously designed inputs. For this purpose, the AND_DNA inhibitor was designed to contain “sticky” nts at the 5′- and 3′-ends. These “sticky” nts are complementary to the ssDNA inputs. The addition of either input causes only a partial displacement of the AND_DNA inhibitor from the light-up RNA aptamer resulting in the low output value as demonstrated in Polynucleotide sequence design was carried out using the multi-strand secondary structure prediction programs NUPACK and mfold ,62. To mAll DNA oligonucleotides were purchased from IDT DNA  as desalted products and used without purification. RNA strands corresponding to individual logic gates and to the tetragonal half-adder complex were prepared by in vitro transcription using T7 RNA polymerase . For thi2) buffer and heating the mixture to +80 °C and gradually cooling it down to +4 °C over a period of 1 h on a PCR thermocycler. Once the RNA aptamer self-assembly was achieved, a small amount  of the malachite green oxalate salt  was added to each RNA or RNA/DNA assembly. The mixture was left to incubate for an additional 30 min at 22 °C to reach proper binding equilibrium. DNA inputs oligonucleotides were added to the assembled AND, OR, and NOR gates at the final concentrations of 2 μM (each) making the final stoichiometry of the complexes as follows:The self-assembly of individual MG-based light-up RNA logic gate complexes AND, OR, NAND, and NOR was achieved by mixing equimolar oligonucleotide strands (1 μM) in TMS   dyes were added to the corresponding complexes  to make 5 μM final concentration. The resulting mixture was allowed to incubate for an additional 30 min at 22 °C. After reaching equilibrium, DNA inputs (5 μM each) were added in accordance to the truth table for the half-added RNA systems and fluorescence were recorded after additional 30 min incubation, which was necessary to achieve input driven strand displacement effect Fluorescence was measured on a Fluoromax-3  spectrofluorimeter using a Sub-Micro quartz fluorometer cell . Fluorescence intensities were recorded separately for each dye. For the DFHBI-binding Broccoli RNA aptamer, the excitation wavelength centered at 465 nm and emission was collected in the range of 475–700 nm. For the MG-binding RNA aptamer, the excitation was centered at 615 nm and emission was recorded from the range of 630–750 nm.The fluorescent enhancement was quantified by the ratio of the maximum emission of the fluorogenic dyes bound to its aptamers divided by the emission of the free dyes in solution. The fluorescence enhancement data were normalized after the experiments; a threshold value was chosen to be 60% for the MG based RNA logic gates, an intensity greater than this value yields an output = 1, while an intensity below this value yields an output = 0.Hydrodynamic diameters of assembled half-adder RNA constructs and the control tetragon RNA nanoparticles were measured by a Zetasizer nano-ZS  at 22 °C following previously described protocols .The RNA tetragon and RNA half-adder complexes were imaged with MultiMode AFM NanoScope IV system , following previous methods .Molecular logic gates hold great potential for a wide range of biotechnological applications, including gene expression regulation, biosensors, therapeutic molecule design, metabolic reprogramming, studies of drug-nucleic acid interactions, and tools for elucidating cellular functions. The emergence of RNA nanotechnology offers great opportunities for applications of RNA-based logic gates. In this study, we have used a computational approach to design various oligonucleotide-responsive RNA logic gates  based on the MG-binding RNA aptamer. The structures of four logic gates were designed based on the general 2D architecture depicted in


Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer. Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport.Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis. However, the composition of transported ribonucleoprotein particles (RNPs) and the regulation of their movement are poorly understood. We have reconstituted microtubule minus end-directed transport of mRNAs using purified components. A Bicaudal-D (BicD) adaptor protein and the RNA-binding protein Egalitarian (Egl) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin, thus defining a minimal transport-competent RNP. Unexpectedly, the RNA is required for robust activation of dynein motility. We show that a  In our cells, tiny molecular motors transport the components necessary for life’s biological processes from one location to another. They do so by loading their cargo, and burning up chemical fuel to carry it along pathways made of filaments. For example, one such motor, called dynein, can move molecules of messenger RNA (mRNA) to specific locations within the cell. There, the mRNA will be used as a template to create proteins, which will operate at exactly the right place.Transporting mRNA in this way is critical in processes such as embryonic development and the formation of memories; yet, this mechanism is still poorly understood. Previous work suggested that the mRNA is simply a passenger of the dynein motor, but McClintock et al. asked if this is really the case. Instead, could mRNA regulate its own sorting by controlling the activity of dynein?Studying mRNA trafficking within the complex molecular environment of a cell is challenging, so mRNA transporting machinery was recreated in the laboratory. Only the proteins necessary to build a working system were included in the experiments. In addition to the filaments, the components included dynein and a complex of proteins known as dynactin, which allows the motor to move together with a protein called BICD2. A protein named Egalitarian was used to link the mRNA to BICD2.By filming fluorescently labelled proteins and mRNAs, McClintock et al. discovered that mRNA strongly promotes the movement of the dynein motor. A structured section in the mRNA acts as a docking area for two copies of Egalitarian. This activates BICD2, which then binds to dynein and dynactin, thereby completing the transport machinery. According to these results, the mRNA directs the assembly of the system that will carry it within the cell.Viruses such as HIV and herpesvirus hijack dynein motors to have their genetic information moved around a cell in order to propagate infection. Understanding precisely how mRNA is transported may help to develop new strategies to fight these viruses. Targeting of mRNAs to specific locations within the cytoplasm can confer precise spatial control over protein synthesis and function . By compDrosophila melanogaster and is responsible for localising spatial determinants of embryonic patterning to microtubule minus ends. Transport of these mRNAs is dependent on the Egalitarian (Egl) and Bicaudal-D (BicD) proteins  and part of CC2) can bridge the interaction between dynein and dynactin, forming a mutually dependent triple complex . The bin in vivo , indicatDrosophila BicD from associating with dynein in vivo (Full-length BicD proteins interact poorly with dynein and dynactin and are therefore only weak activators of dynein motility . While m in vivo .Drosophila BicD proteins when it is GTP-bound (Rab6GTP)  , a stateRab6GTP) . These dRab6GTP) . This obWe set out to elucidate molecular mechanisms of dynein-based mRNA transport by Egl and BicD by reconstituting this process in vitro with purified components. Our results define a minimal set of proteins for RNA translocation on microtubules and show that the RNA strongly activates dynein motility. Stimulation of transport by RNA is not dependent on the Egl-LC8 interaction. Rather, our data support a model in which the RNA localisation signal overcomes BicD autoinhibition by augmenting the interaction of Egl with BicD CC3. Our study reveals a pivotal role of an RNA localisation signal in gating the activity of a microtubule motor, and give rise to a model in which cargoes stimulate dynein motility by scaffolding active adaptor protein assemblies.Drosophila dynein and dynactin, we established a system in which Drosophila Egl and an mRNA target are linked to mammalian dynein and dynactin complexes. We took advantage of the strong evolutionary conservation of the Egl/Rab6GTP-binding site of BicD  microscopy in the presence of dynein, dynactin, and Egl/BICD2 . RNAs ana embryo . We obse chamber . Gratifyransport . As obsed system , hairy Rransport  and wereransport . We alsoogenesis . Like haro assay . These eI-factor )  Egl-binding element from the l (ILS)) , which wovements . These dnts . The Egl/DmBicD complex was also produced by co-expression of both proteins in Sf9 insect cells and purification with an affinity tag on Egl. The hairy RNA significantly increased the number of processive movements of dynein in the presence of dynactin and Egl/DmBicD  subunit of dynactin and the heavy chain of dynein (Dhc) by western blotting  (dlc2pt/BICD2 complex supported robust transport of hairy RNA in the presence of dynein and dynactin (The results described above raise the question of how RNA binding stimulates the association of Egl and BicD proteins with dynein and dynactin. We first asked if this involves the binding of Egl to the LC8 dynein light chain. Motility assays were performed with a purified Egl/BICD2 complex in which Egl has two mutations in a consensus LC8-binding site that abolish association with LC8 in vivo and in vitro (Egl+ S969R) . The Egldynactin . Moreovedynactin , as welldynactin . Thus, tGTP-binding site in BICD2 relieves autoinhibition, licensing BICD2N to interact with dynein and dynactin  to evaluate mean molar masses of complexes in the presence and absence of RNA independently of protein conformation. Over a range of protein concentrations, the presence of the  samples .ILS substantially increases the mean molar mass of the Egl/BICD2 sample  – confirmed that the 2 sample . This ef2 sample . Despitelar mass , confirmlar mass . The ILSmponents . Consistmponents . Analysi and Egl . We also protein . CollectILS, Egl and a BicD protein could reach ~400 kDa. The predicted molar masses of the BICD2, DmBicD and Egl polypeptides are 93, 89 and 112 kDa, respectively, while the ILS has a molar mass of 19 kDa. It was previously shown that DmBicD is a dimer  or Cy5. Of the labelled motile RNPs, 14% had signal from both dyes .We have succeeded in reconstituting microtubule-based mRNA transport in vitro using purified proteins and have used this system to define a minimal transport-competent RNP. Although genetic experiments indicate that other proteins can modulate the mRNA transport process in vivo , no otheThere has recently been considerable focus on the regulation of dynein motility, stemming from the discovery that the isolated N-terminal region of BicD proteins can bridge the interaction of dynein and dynactin and thereby activate transport . SubsequPrevious observations suggested that binding of a cargo-associated protein such as Egl to the C-terminal region of BicD is sufficient to overcome the autoinhibited state of the full-length protein, and thereby lead to recruitment of dynein and dynactin. Our study reveals that a purified Egl/BicD complex does not efficiently associate with dynein and dynactin in the absence of an RNA localisation signal. Thus, the availability of the cargo gates robust activation of dynein motility. This mechanism presumably limits unproductive long-range movement of the motor complex in the absence of an RNA consignment.Drosophila does significantly compromise the function of Egl in the maintenance of oocyte fate  to BicD CC3 releases CC1/2. One possibility is that binding of Egl/Rab6GTP competes directly with the interaction of CC3 with the N-terminal sequences. Alternatively, occupancy of the Rab6GTP- and Egl-binding site in CC3 could induce changes in coiled-coil architecture that are propagated along the molecule to release a discrete autoinhibitory interaction  have not been genetically profiled since purchase but were grown in a tissue culture facility dedicated to insect cell expression. The cells were tested for mycoplasma twice a year  and the results were always negative.Drosophila melanogaster Egl isoform B:NM_166623, mouse BICD2:NM_001039179 and Drosophila melanogaster BicD:NM_165220) were synthesised commercially  with codons optimised for expression in Spodoptera frugiperda Sf9 cells, and cloned for use with the MultiBac expression system. Where required, sequences encoding SNAPf tags for fluorescent labelling of protein complexes and ZZ-LTLT tags for IgG-based affinity purification (polh promoter of the pACEBac1 acceptor vector (Drosophila melanogaster BicD (DmBicD) were cloned downstream of the polh promoter of the pIDC donor vector  . The presence of the desired mutations, and no others, was confirmed by sequencing of the entire open-reading frame.The Egl+ S969R)  were geng) using a Beckman Coulter Type 70 Ti fixed-angle rotor in a Beckman Coulter Optima L-100 XP preparative ultracentrifuge.All purification steps were performed at 4°C. Native dynactin was purified from pig brain as described previously . Dynein,−1 TEV protease in a final volume of 15 ml TEV buffer. Eluted protein was recovered by gravity flow through a fresh Econo-column and concentrated to ~1.5 mg ml−1 with a 100 kDa MWCO Amicon Ultra-4 centrifugal filter unit .During centrifugation, IgG Sepharose 6 affinity resin  was applied to a gravity flow Econo-column  and washed twice with five column volumes of lysis buffer . Clarified lysate was added directly to the affinity matrix in the column, which was then sealed and agitated by gentle rolling for 3 hr. After incubation, the lysate was allowed to flow through the column by gravity and the retained affinity matrix washed twice with five column volumes of lysis buffer and twice with five column volumes of TEV buffer . If required, bound SNAP-tagged proteins were fluorescently labelled on-column before proceeding to elution (see below). Bound proteins were eluted by overnight TEV cleavage of the ZZ affinity tag using gentle rolling agitation and ~0.03 mg ml2, 5 mM DTT, 0.1 mM MgATP, 10% glycerol) to remove large aggregates, TEV protease, and other small contaminants. For SEC-MALS and SE-AUC experiments, GF150 was modified to include 5 mM TCEP instead of DTT. For the dynein complex, a TSKgel G4000SWxl with guard column  was used, while a Superose 6 Increase 3.2/300 column  was used for BICD2, Egl/BICD2 and Egl/DmBicD complexes. Fractions containing the dynein complex were pooled and concentrated to ~1 mg ml−1. Fractions containing BICD2, Egl/BICD2 or Egl/DmBicD complexes were pooled without an additional concentration step. All purified proteins were dispensed in aliquots for single use, flash frozen in liquid N2, and stored at −80°C. Protein concentrations were determined using a Coomassie Protein Assay Kit (ThermoFisher Scientific). To assess purity, proteins were resolved by SDS-PAGE using Novex 4–12% Bis-Tris precast gels (ThermoFisher Scientific) and MES-SDS running buffer. Protein bands were visualised using Coomassie Instant Blue protein stain  and imaged with a ChemiDoc XRS + system (Bio-Rad). Protein sizes were evaluated by comparison with Full-Range Rainbow prestained molecular weight markers .The affinity-purified protein complexes were further purified by FPLC-based gel-filtration chromatography  in GF150 buffer  or SNAP-Surface Alexa Fluor 647 (NEB) was performed on-column during affinity capture according to a previously described method that labels >95% of dynein dimers with at least one dye . For thehairy RNA or Cy3-hairy RNA was transcribed in vitro from a gel-purified PCR amplicon template using the MEGAscript T7 Transcription Kit (Ambion). The RNA is a 730-nt region of the 3’UTR containing the RNA localisation signal (hairy RNA was synthesised from the same template using a 1:9 ratio of Alexa488-UTP (ThermoFisher Scientific) to unlabelled UTP. Cy5-I-factor RNA was synthesised from a linearised plasmid template using the MEGAscript SP6 Transcription Kit (Ambion) and a 1:3 ratio of Cy5-UTP to unlabelled UTP. The RNA is 597-nt long and contains the ILS localisation signal . Chambers were then incubated with 2 mg ml−1 streptavidin (Sigma-Aldrich) for 5 min and again washed twice with 20 μl motility buffer. To block any unpassivated surface, chambers were incubated with 20 mg ml−1 α-casein (Sigma-Aldrich) for 5 min and washed twice with 20 μl motility buffer. The prepared chambers were kept in a humidified container until the addition of microtubules and protein/RNA mixtures to prevent desiccation of chamber surfaces.Glass surfaces were prepared as described previously . Motilit2, 0.5 mM EGTA, 1 mM DTT) with 0.5 mM GMPCPP  for 2–4 hr at 37°C. Polymerised microtubules were pelleted in a room temperature table top centrifuge at 18,400 x g for 8.5 min, and washed once with pre-warmed (37°C) BRB80. After pelleting once more, the microtubules were gently resuspended in pre-warmed (37°C) BRB80 containing 40 μM paclitaxel  and used on the same day.Microtubules were polymerised from porcine tubulin  and labelled with fluorophores and biotin by stochastic incorporation of labelled dimers into the microtubule lattice. Mixes of 1.66 μM unlabelled tubulin, 0.15 μM Hilyte488-tubulin, and 0.4 μM biotin-tubulin were incubated in BRB80  that greatly limits photobleaching , and 1 μM RNA. To ensure that all complexes assemble at the same ionic strengths, KCl was supplemented to a final concentration of 50 mM during assembly. Just prior to imaging, stabilised microtubules were immobilised in a prepared motility chamber for 5 min and subsequently washed once with motility buffer that also contained 50 mM KCl, 1 mg mlleaching . Diluted−1) and 100 ms exposure per frame using a Nikon TIRF microscope system controlled with Micro-Manager open-source acquisition software , resulting in pixel dimensions of 105 x 105 nm. Multicolour acquisitions used sequential image capture with switching of emission filters ).For each chamber, a single multicolour acquisition of 500 frames was made at the maximum achievable frame rate (~2 frames ssoftware  and equiP[tub-Egl::GFP] (Sco/CyO P[actin5C-GFP] flies (Bloomington Drosophila Stock Center: RRID:BDSC_4533), which contain genomically-integrated transgenes expressing Egl::GFP or GFP from the ubiquitous α-tubulin or β-actin promoters, respectively. 0–12 hr embryos were dechorionated and flash frozen in liquid N2. 300 μl chilled extraction buffer  was added for each 100 mg of frozen embryos, followed by grinding on ice with a motorised pellet pestle (ThermoFisher Scientific). The material was subjected to 25 passes in a Wheaton dounce tissue grinder (ThermoFisher Scientific) on ice before the addition of 200 μl chilled extraction buffer containing 0.5% Triton-X-100 per 100 mg of embryos. Following gentle mixing, samples were incubated on ice for 5 min and passed through a 23G syringe five times before clarification by two centrifugation steps (each 5 min at 3000 x g). 350 μl aliquots of clarified extract were incubated with 20 units Recombinant RNase Inhibitor  and either 20 μl of a 6.7 μg/μl solution of unlabelled hairy RNA in dH2O or 20 μl dH2O for 30 min at 4°C. Magnetic beads coupled to GFP-binding protein ) were washed twice in PBS, followed by blocking of non-specific interaction sites with 1 mg ml−1 casein in PBS for 30 min at 4°C. After two washes of the beads in extraction buffer, the equivalent of 30 μl of initial bead slurry was mixed with the embryo extracts with or without hairy RNA. Following a 2 hr 30 min incubation at 4°C, beads were washed fives times for 1 min in extraction buffer containing 0.05% Triton-X-100 (three washes in 400 μl of buffer and two washes in 1 ml buffer). Proteins and RNA-protein complexes were eluted from the beads by the addition of 60 μl 1 x lithium dodecyl sulphate (LDS) buffer (ThermoFisher Scientific)/50 mM DTT and incubation at 80°C for 10 min.Extracts were generated from embryos of gl::GFP]  or Sco/CAB_390913); diluted 1:1000); mouse α-Dhc (clone 2C11-C [AB_2091523)  and diluted 1:1000) and rabbit α-p150-C-term  in GF150 buffer (using 5 mM TCEP instead of 5 mM DTT) in the presence or absence of a 10-fold molar excess of ILS RNA were pre-incubated on ice for at least 1 hr and subsequently diluted in GF150 (TCEP) to yield three samples with volumes of 110 µl and protein concentrations of 1, 0.33, and 0.11 mg ml−1. These samples were loaded in 12 mm six-sector cells and subjected to equilibrium sedimentation in an An50Ti rotor using an Optima XL-I analytical ultracentrifuge (Beckmann) at 3200, 5600, and 10,000 rpm until equilibrium was reached at 4˚C. At each speed, comparison of several scans was used to judge whether equilibrium had been reached. Data were processed and analysed using SEDPHAT 13b ([SCR_016254) and plotted with GUSSI ([SCR_014962). The partial-specific volumes (v-bar), solvent density and viscosity were calculated using Sednterp .Duplicate independent preparations of 1 mg mlHAT 13b , GF75 buffer (contains 75 mM KCl with 5 mM DTT), or GF50 buffer (contains 50 mM KCl with 5 mM TCEP). All measurements for BICD2 and Egl/BICD2 were made at room temperature, whereas the relative instability of the Egl/DmBicD complex required measurements be made at 4°C. Where indicated, ILS RNA was added at a 10-fold molar excess over Egl/BICD2 or Egl/DmBicD  and incubated on ice for 1 hr prior to injection on the column. Samples lacking RNA were subjected to the same incubation. Following SEC fractionation, eluted protein was detected on a Wyatt Heleos II 18 angle light scattering instrument coupled to a Wyatt Optilab rEX online refractive index detector in a standard SEC-MALS format. Heleos detector 12 at 99° was replaced with Wyatt’s QELS detector for on-line dynamic light scattering measurements. Protein concentration was determined from the excess differential refractive index based on 0.186 RI increment for 1 g ml−1 protein solution. Concentrations and observed scattered intensities at each point in the chromatograms were used to calculate the absolute molecular mass from the intercept of the Debye plot, using Zimm’s model as implemented in ASTRA software . Fractions were analysed by gel electrophoresis and staining with SYPRO Ruby  or Coomassie Instant Blue according to the manufacturer’s instructions.Samples of BICD2, Egl/BICD2 and Egl/DmBicD were resolved on a Superdex 200 HR10/300 analytical gel filtration column  at 0.5 ml minSCR_002285). Typically, three independent chambers were imaged using protein complexes from at least two independent assembly reactions for each experimental condition. The positions of microtubules were determined by the fluorescent tubulin signal or a projection of RNA/protein signals over the course of the movie. From each of these chambers, 5–10 microtubules were typically selected for analysis with preference given to those that were longer and better isolated from adjacent microtubules. No power analysis was used to determine sample size. Instead the sample size was chosen to allow the identification of a range of effect sizes. To avoid the risk of subconscious bias, microtubules were selected before visualising the motile properties of complexes on them.Kymographs were generated and analysed manually using FIJI  in duration and as processive events if they achieved predominantly minus end displacement >500 nm (five pixels) without significant diffusive behaviour. These parameters were chosen in advance of image acquisition following discussion within the team and all particles that fulfilled the criteria were analysed. As described previously , some moSCR_002798). A two-tailed Student’s t-test or a two-tailed Welch’s t-test was used when comparing two groups where a Gaussian data distribution was expected, with the latter test employed in cases of unequal variance. A Mann-Whitney test was used to compare two groups with non-Gaussian data distributions. An ANOVA test with Dunnett’s correction was used for multiple comparisons.Statistical analyses, curve fitting, and data plotting were performed using Prism 7.0b  and different mRNAs known to be transported in vivo, the authors define a minimal set of components required for RNP motility in vitro. They then systematically dissect the requirements for RNP assembly, which identifies a crucial and unexpected role for the mRNA cargo. A combination of single-molecule motility assays and rigorous biochemical analysis allows the authors to present an insightful model for motile RNP assembly, which postulates that overcoming the autoinhibited state of BicD requires the binding of two Egl proteins to a BicD dimer, and that the Egl-BicD interaction is stabilized by mRNA binding to Egl.The manuscript by McClintock and coworkers reports the establishment of an in vitro system to study dynein-dependent mRNA transport mediated by the adaptor Bicaudal D and its binding partner Egalitarian (Egl), which interacts with a specific subset of mRNAs containing appropriate targeting sequences. Using purified proteins  is not required to recruit (at least some) Egl to BICD2. However, the SDS-PAGE below shows that more Egl is present in the larger complexes when ILS is added. This raises a few questions:• Figure 6B shows that BICD2 and Egl are fully complexed under the conditions used for SEC-MALS (since the Egl/BICD2 peak does not show a BICD2-like shoulder). This suggests that the RNA  How much more Egl is recruited to BICD2 in the presence of ILS is present in a 10-fold molar excess in these experiments, why do the authors think it does not compete in what appears to be an ILS-mediated recruitment of additional Egl to the Egl/BICD2 complex?2) Since the • The data in Figure 7 suggest that a significant fraction of the transport complexes contain two RNAs. Figure 7B-E show that about 40% of complexes contain BICD2s and Egls with different dyes. Since one would expect 50% of the dimers to have two colors, this means that 80% of the complexes have two copies of BICD2, or Egl. Figure 7F,G shows that only 14% of complexes have RNAs with different dyes, meaning that 28% of complexes have two RNAs. Given all the data shows elsewhere in the paper, we can assume that those 28% of complexes come from the 80% containing two BICD2s and Egl. Therefore, 35% (28/80% ) of motile complexes have two RNAs. This does not seem to me to be "substantially lower" than the 65% of complexes that would have one RNA and two BICD2s and Egls, as the authors state. The composition of the activated transport complex is a major point of this work and thus I think this stoichiometry needs to be worked out more convincingly.ILS controls for the SE-AUC and SEC-MALS, or at least the SEC-MALS if the SE-AUC is more demanding experimentally. Right now there is nothing in these experiments to rule out non-specific binding.• It would be useful to include scrambled • The distribution of velocities in Figure 3E, Figure 3—figure supplement 1 and Figure 5—figure supplement 1 all show increases in velocity in the presence of RNA. However, these distributions appear bimodal, with low and high velocity populations. It seems it would be very useful, from a mechanistic point of view, to establish whether the RNA-induced increase in velocity is due to a shift of transport complexes from the slower to the faster population, without changing the velocities of each population. Since the general observation that dynein is faster in the presence of the RNA would not change, this is not a weakness of the manuscript, but if additional analysis of the data could answer the question it would add to it. Could this be an indication of a second dynein being recruited to the transport complex? Essential revisions:Reviewer #3 raised a concern related to the stoichiometry of the RNA in the complex. After an open discussion of the manuscript and of the comments, Reviewers #1 and #2 agreed that this point is very important in terms of understanding the nature of the complex and that it requires further attention.• Figure 6B shows that BICD2 and Egl are fully complexed under the conditions used for SEC-MALS (since the Egl/BICD2 peak does not show a BICD2-like shoulder). This suggests that the RNA (ILS) is not required to recruit (at least some) Egl to BICD2. However, the SDS-PAGE below shows that more Egl is present in the larger complexes when ILS is added. This raises a few questions:1) How much more Egl is recruited to BICD2 in the presence of ILS? Given that the authors have all the components purified, they should be able to quantitate a gel like that shown in this figure and use standards to determine the stoichiometry. Is there a single Egl in the Egl/BICD2 complex with a second one recruited by the ILS?We agree with the interpretation that RNA is not essential for (at least some) Egl to bind BICD2 in the SEC-MALS regime in Figure 6B. This conclusion is supported by the mean masses of the complexes measured in the absence of RNA compared to the BICD2 only sample, as well as the gel images. It is unfortunate that we did not make it clear in our first submission that these data are not consistent with a fixed stoichiometry of a BICD2 dimer and a single Egl in the absence of RNA. Rather, the relatively small increase in mean molar mass of the peak fraction compared to the BICD2 only sample, together with the gel images, indicates a mixture of complexed Egl/BICD2 (of indeterminate stoichiometry) and a large excess of free BICD2 dimer. The homogeneous values for mean molar mass across this peak and the lack of a BICD2-like shoulder suggest rapid association and dissociation of the minority Egl population with BICD2 dimers.eLife 2014 (PMID: 24596152)).Similarly, although there is a clear overall increase in the occupancy of BICD2 with Egl in Figure 6B when RNA is present, the data do not support a uniform recruitment of two Egl proteins per BICD2 dimer. The broad range of mean masses across the Egl/BICD2 peak indicates that our experimental conditions captured an equilibrating mixture of different constituent species, which is likely to include both single and double occupancy of Egl with BICD2. Since these species were not sufficiently resolved by SEC , the MALS analysis provided the abundance-weighted mean mass of all of the species throughout the peak rather than masses of discrete states of complexation. Similar situations have been observed in SEC-MALS analysis of self-associating systems  and the Figure 6 legend to clarify the key points discussed above.The major conclusion we draw from the SEC-MALS experiments is that RNA pushes existing equilibria in favour of the occupancy of the BICD2 dimer with Egl . An important prediction of this model is that the equilibria can be shifted by modulating the concentration of Egl/BICD2. We now include additional data from a SEC-MALS experiment with a series of Egl/BICD2 concentrations , which show that this is indeed the case.ILS, providing an estimate of the relative increase in Egl signal when the RNA is present. This analysis is included in the legend to Figure 6. To further demonstrate the RNA-mediated increase in Egl/BICD2 association, we also include gel images from a new SEC-MALS experiment with lower ionic strength buffer . The quantitative comparison of Egl to BICD2 signals in the gels is included in the legend.As the reviewer indicates, a quantitative representation of this shift can be derived from gel images of peak SEC-MALS fractions. Making these measurements highly quantitative would require standards of Egl alone, which we were unable to purify . We are, however, able to measure the intensity of Egl bands relative to BICD2 bands from the peak fractions in the presence and absence of the ILS in the SECMALS data are compatible with a fraction of complexes containing two Egl molecules and a BICD2 dimer, the analysis of single transport complexes in Figure 7 is much better suited to analyse protein copy number . We believe that the analysis of protein copy number in active transport complexes in Figure 7 has been strengthened significantly as a result of the feedback from the reviewers (please see below). We now also present evidence from SEC-MALS that free Egl is indeed monomeric , which helps clarify possible mechanisms of RNA-mediated assembly of active adaptor protein complexes.Although the upper limits of the mean molar masses of mixtures of Egl, BICD2 and 2) Since the ILS is present in a 10-fold molar excess in these experiments, why do the authors think it does not compete in what appears to be an ILS-mediated recruitment of additional Egl to the Egl/BICD2 complex?ILS-bound Egl. However, RNA-free and RNA-bound Egl complexes cannot be discriminated in our experiments.As our data suggest dynamic association/dissociation of Egl and BICD2 it seems likely that the RNA-free Egl that is bound to BICD2 will be replaced with • The data in Figure 7 suggest that a significant fraction of the transport complexes contain two RNAs. Figure 7B-E show that about 40% of complexes contain BICD2s and Egls with different dyes. Since one would expect 50% of the dimers to have two colors, this means that 80% of the complexes have two copies of BICD2, or Egl. Figure 7F,G shows that only 14% of complexes have RNAs with different dyes, meaning that 28% of complexes have two RNAs. Given all the data shows elsewhere in the paper, we can assume that those 28% of complexes come from the 80% containing two BICD2s and Egl. Therefore, 35% (28/80% ) of motile complexes have two RNAs. This does not seem to me to be "substantially lower" than the 65% of complexes that would have one RNA and two BICD2s and Egls, as the authors state. The composition of the activated transport complex is a major point of this work and thus I think this stoichiometry needs to be worked out more convincingly.We regret that we did not adequately explain the implications of a small fraction of unlabelled protein for the interpretation of this experiment. As stated only in the Materials and methods, we achieved 90% of SNAP polypeptides labelled with a dye with our optimised procedures. Thus, the expected fraction of complexes that are dual-labelled assuming an obligate dimer is 0.405 ((0.9x0.9)/2). Our data therefore fit very well with there being two Egl polypeptides and two BICD2 polypeptides in almost all active transport complexes. We now explain this point in the Results , and include tables illustrating the calculations (Supplementary file 1 and 2).hairy RNA samples. Correcting for these values, our data indicate that 30% of motile complexes have two RNAs and 70% have one RNA . We think that this is a substantial difference but do not make this point explicitly in light of the reviewer’s comment. The key conclusion is that transport of a single mRNA molecule by a complex containing two Egl molecules is not only possible but occurs frequently. This conclusion is consistent with our previous work showing that a single RNA can be transported by the native complex in an extract-based motility assay  (cited in the aforementioned paragraph).To more accurately assess the fraction of transported complexes that contain two RNAs in the experiments documented in Figure 7F,G, we have now calculated the labelling efficiency of the labelled hairy and Cy5-hairy in the absence of the proteins. We find a very low incidence of co-localisation of these RNAs. This result indicates that in our motility assay, in which the RNA is present in a large molar excess, the presence of a second RNA in a subset of complexes is due to association with a protein(s) in the complex. This interaction was presumably blocked in our previous studies with extracts, as we always observed one RNA per transport complex. This observation calls into the question the physiological relevance of the binding of an additional RNA molecule. We have further analysed the data presented in Figure 7F and G to assess what affect, if any, the presence of multiple RNAs has on motility. Although there is a small effect on run length, the velocity distribution of complexes containing both Cy3-hairy and Cy5-hairy is very similar to that of complexes with signal from a single fluorophore. Thus, the presence of an additional RNA does not have substantial functional consequences for transport. We have included these analyses as Figure 7—figure supplement 2 and 3 and have revised the manuscript accordingly (see the aforementioned paragraph).We have also clarified the basis of the association of two RNA molecules with a single transport complex by performing dual colour analysis of a mixture of Cy3-We believe that the new analyses strengthen significantly the section of the manuscript on the composition of active transport complexes.• It would be useful to include scrambled ILS controls for the SE-AUC and SEC-MALS, or at least the SEC-MALS if the SE-AUC is more demanding experimentally. Right now there is nothing in these experiments to rule out non-specific binding.ILS. Our data indicate there is some interaction of the mutant RNA, which is in a 10-fold molar excess, with Egl/BICD2. This does not surprise us based on previous evidence that Egl is not a highly selective RNA-binding protein  .The SE-AUC experiments require large amounts of protein, instrument time and analysis, which would indeed make this experiment very demanding. We have therefore taken up the option of performing this specificity control in the SEC-MALS format. We show in a new panel  that the scrambled RNA elicits a relatively small shift in molar mass of Egl/BICD2 compared to the • The distribution of velocities in Figure 3E, Figure 3—figure supplement 1 and Figure 5—figure supplement 1 all show increases in velocity in the presence of RNA. However, these distributions appear bimodal, with low and high velocity populations. It seems it would be very useful, from a mechanistic point of view, to establish whether the RNA-induced increase in velocity is due to a shift of transport complexes from the slower to the faster population, without changing the velocities of each population. Since the general observation that dynein is faster in the presence of the RNA would not change, this is not a weakness of the manuscript, but if additional analysis of the data could answer the question it would add to it. Could this be an indication of a second dynein being recruited to the transport complex?As suggested, we have performed additional analysis of the velocity distributions. Although it is possible to fit two Gaussian distributions to the data, we do not find the fits compelling. We therefore think it would not be appropriate to include them in the manuscript. We intend to investigate the potential role of dynein copy number in RNA transport in a separate study. We draw attention to the possible influence of dynein copy number in the Discussion (sixth paragraph).


To facilitate proper mitotic cell partitioning, the Golgi disassembles by suppressing vesicle fusion. However, the underlying mechanism has not been characterized previously. Here, we report a Ran pathway-independent attenuation mechanism that allows Importin-α (a nuclear transport factor) to suppress the vesicle fusion mediated by p115 (a vesicular tethering factor) and is required for mitotic Golgi disassembly. We demonstrate that Importin-α directly competes with p115 for interaction with the Golgi protein GM130. This interaction, promoted by a phosphate moiety on GM130, is independent of Importin-β and Ran. A GM130 K34A mutant, in which the Importin-α-GM130 interaction is specifically disrupted, exhibited abundant Golgi puncta during metaphase. Importantly, a mutant showing enhanced p115-GM130 interaction presented proliferative defects and G2/M arrest, demonstrating that Importin-α-GM130 binding modulates the Golgi disassembly that governs mitotic progression. Our findings illuminate that the Ran and kinase-phosphatase pathways regulate multiple aspects of mitosis coordinated by Importin-α . Golgi disassembly is required for mitosis and occurs by vesicle fusion suppression, although the mechanism is unclear. Here, Chang et al. show, with quantitative analyses and crystallography, that Importin-α regulates this process by blocking GM130-p115 interactions in a Ran pathway-independent way. The vesicle tethering factor p115 and the Golgi peripheral membrane protein GM130 facilitate targeting of transport vesicles to the Golgi4. However, during mitosis, p115-mediated vesicle fusion to Golgi membranes is reduced, thereby down-regulating the secretory pathway7 and triggering the Golgi vesiculation necessary for Golgi partitioning during cell division8. Mitotic phosphorylation of GM130 on serine 25 (Ser-25) has been proposed to account for the reduced binding of p115 to Golgi membranes10, but whether serine phosphorylation alone is solely responsible for inhibiting p115 binding has not been proven empirically.During interphase, the Golgi apparatus of the cytoplasmic endomembrane system carries out critical functions in the protein secretory pathway, such as receiving transport vesicles from the endoplasmic reticulum (ER) and directing them to their destinations13. Conventionally, Importin-α-dependent classical NLS (cNLS) includes: (1) a monopartite element that consists of 4–8 positively charged residues and primarily binds to the major binding site of Importin-α (e.g. SV40 large T antigen14); and (2) a bipartite element that contains two clusters of basic amino acids separated by a linker region and that binds to the major and minor binding sites of Importin-α (e.g. nucleoplasmin16). In the nuclear import pathway, cNLS-containing proteins assemble into a ternary complex, with Importin-α and Importin-β in equal stoichiometries, which is transported across the nuclear envelope (NE) through nuclear pore complexes (NPCs) via interaction with nucleoporins that contain phenylalanine–glycine (FG) repeats17. Subsequently, in the nucleus, GTP-bound Ran binds to Importin-β and disassembles the ternary complex, releasing the imported cargo proteins17. Guanine nucleotide exchange factors (GEFs) (e.g. chromatin-associated protein RCC1) are required to switch Ran from GDP-bound to GTP-bound states and to confine localization of RanGTP in the nucleus17. Although the NLS-like motif of GM130 does interact with Importin-α13, sub-stoichiometries of Importin-α and Importin-β have been associated with Xenopus egg membrane fractions and can be pulled down by GM130 from mitotic cell lysates18. These observations suggest that binding of Importin-α to GM130 without Importin-β may be carried out by a non-canonical NLS.Recently, an evolutionarily conserved and nuclear localization signal (NLS)-like motif was identified in the N-terminal region of GM130 that binds p11519. Structural and biochemical analyses have further suggested that the NLS of the INM proteins Heh1/Heh2 (S. cerevisiae) and Pom121 (metazoans) adopts a non-canonical conformation and bears unique biochemical properties, allowing displacement of the N-terminal Importin-β binding domain (IBB) of Importin-α that competes for binding with the NLS-proteins and thereby bypassing auto-inhibition21. Thus, these INM proteins bind Importin-α in the absence of Importin-β21. As both Golgi and NE are part of the cellular endomembrane system, it is plausible that the Golgi membrane-associated GM130 protein carries an NLS-like motif that has evolutionarily conserved structural and biochemical properties shared with the NLS of INM proteins.Numerous membrane proteins situated in the inner membrane (INM) of the NE are actively transported to the nucleus by the Importin-α/-β-mediated pathway, facilitated by NLS13. Since both Importin-α and p115 bind to the GM130 N-terminus and Importin-α pulldown by GM130 is enriched during mitosis, we hypothesize that binding of Importin-α to GM130 during mitosis may suppress the p115 and GM130 interaction, contributing to Golgi disassembly. Importantly, this non-canonical interaction may be sustainably resistant to mitotic RanGTP-induced liberation, ensuring Golgi vesiculation.The NLS-like motif of GM130 was proposed to sequester Importin-α from TPX2, thereby stimulating microtubule nucleation during mitosisTo test our hypothesis, we first biochemically demonstrate that binding of Importin-α and p115 to GM130 is mutually exclusive. GM130 directly interacts with full-length Importin-α in the absence of Importin-β, which is enhanced by phosphorylation of the Ser-25 residue, and this interaction is resistant to Ran pathway liberation. Next, our crystal structure of the Importin-α and GM130-NLS complex reveals a non-conventional binding mode that accounts for a higher affinity for full-length Importin-α compared to the IBB domain. Moreover, crystal structures of Importin-α with recombinant GM130 either carrying a phosphomimetic residue or a phosphate moiety and molecular dynamic (MD) simulations show that the phosphate moiety indirectly enhances the affinity of Importin-α for GM130 by extending the intermolecular interaction network.Next, we introduce a single point mutation (K34A) in GM130 to biochemically disrupt its interaction with Importin-α. Interestingly, the heterozygous mutant shows proliferative defects and punctate Golgi during metaphase. Furthermore, in vitro immuno-precipitation and in situ proximity ligation assays (PLA) reveal elevated levels of p115 and GM130 during mitosis in the mutant cell line. Our data describe a molecular mechanism whereby Importin-α modulates mitotic Golgi morphology via a Ran pathway-independent interaction with GM130.10. The N-terminal region of GM130 and the p115 C-terminus interact12, so we purified recombinant proteins containing these two regions and then assessed their binding affinity by isothermal titration calorimetry (ITC). The wild type GM130 N-terminal region  directly bound p115   mutant in which Ser-25 was substituted by Asp (hereafter GM130-S25D) )  Fig. . Further5D) Fig. . Thus, uB)) Fig. . AdditioB)) Fig. . Therefo12121 orthorhombic space group that diffracted to 2.8 Å  complex to understand their binding mode. The co-purified GM130 peptide (a.a. 1–48)•Importin-α (a.a. 70–498) complex yielded crystals of the P2 Å Table .Table 1DXenopus to human and provide salt bridges and hydrogen bonds that interact with residues Glu-354, Glu-396, and Lys-353 on Importin-α  comprised two stretches of basic residues representing the major and minor NLS-binding sites for Importin-α Fig. . Interes12121 space group that diffracted to 1.75 and 2.4 Å respectively (Table GM130-S25D peptide (a.a. 1–48) or GM130-NLS phospho-S25 was co-purified with Importin-α (70–498) and the protein•peptide complexes were subjected to crystallographic analysis. Both complexes yielded crystals in the P2ly Table . Interesly Table : Asp-25 ly Table , whereasly Table .Tm) of protein complexes. The temperature scans (wavelength = 222 nm) for Importin-α in complex with GM130-WT, S25D, or phospho-S25 revealed that the phosphorylated protein complexes exhibited increased melting temperatures  of the GM130-NLS S25D complex is substantially lower than that of the wild-type complex  was performed to determine the melting temperatures . Interestingly, similar to what we observed for Importin-α (ΔIBB) Fig. , GM130-NTo examine whether Importin-α FL also competes with p115-CTD for GM130 binding, we carried out competition binding assays in the presence of Importin-α FL, p115-CTD, and GM130. We found that whereas Importin-α pulldown by GST-GM130-WT was elevated, the percentage of p115 remaining on GM130 was reduced when we added increasing amounts of Importin-α FL Fig. . Hence, We then assessed whether the GM130-S25D phosphomimetic mutant could enhance Importin-α pulldown from interphase mammalian HEK293T cell lysates. Interestingly, Importin-α pulldown from these cell lysates by GST-GM130-S25D was increased ~1.3-fold compared with that of wild-type  revealed structural similarities  the GM130-NLS displayed a higher binding affinity for Importin-α (ΔIBB) compared to the IBB domain alone Fig. ; and (3)SEC Fig. , suggestSEC Fig. . Hence, Xenopus egg extracts, has been pulled down by GM130 from HeLa cell lysates, and was recapitulated by our in vitro pulldown analysis  phosphorylation on GM130 reducing its binding to Importin-β or (2) Ran-GTP in the extracts/lysates releasing GM130 from Importin-β but not from Importin-α. To test these hypotheses, we first carried out ITC to determine the Kd value between GM130-NLS phospho-S25 peptide and Importin-β. Interestingly, binding of the GM130 phospho-peptide with Importin-β not only displayed an endothermic curve but also a binding constant that is at least three orders of magnitude lower than that of the wild-type GM130 peptide  binding of GM130 to Importin-α FL is resistant to IBB displacement and is enhanced by GM130 phosphorylation; or (2) both the Ran pathway and GM130 phosphorylation suppress the interaction of GM130 and Importin-β.Next, we performed a GST pulldown experiment to examine whether GST-GM130 can interact with Importin-α or Importin-β in the presence of RanQ69L (a constitutively active mutant). We found that RanQ69L mediates release of Importin-β from Importin-β•GM130 complex and this is enhanced by the S25D mutation  or minor (K14A) binding sites of GM130-S25D to establish whether they could disrupt binding of Importin-α FL. Importin-α FL was virtually undetectable when we used either of these GST-GM130 mutants to pull down recombinant protein or from HEK293T cell lysates Fig. . FurtherTo determine the cellular relevance of our biochemical findings, we used CRISPR/Cas9 genome editing to introduce the K34A point mutation of GM130 into a HEK293T cell line. Although no colonies representing homozygous mutations were found, we independently identified three cell lines with heterozygous mutations among ~500 screened colonies  analysis. Interestingly, the mutant cell lines presented a scattered, punctate pattern of greater fluorescence intensity during metaphase compared to the more evenly dispersed GM130 signal in wild-type metaphase cells  can also increase the number of cells containing monopolar spindles during mitosis. Overexpression and knockdown efficiencies of GM130 were assessed by Western blot analyses  resulting from GM130 overexpression and depletion.Both GM130 mutant overexpression and endogenous GM130 depletion also resulted in elevation of mitotic cell percentages relative to controls, as found for the K34A mutant Fig. . Next, wFinally, K34A mutant cells also presented more compact Golgi compared to the rather dispersed Golgi of wild-type cells in interphase upon staining with GM130 antibody , the heterozygous K34A mutant colonies we isolated allowed us to study the role of this interaction beyond microtubule nucleation13. The heterozygous GM130 mutant may retain the ability to partially compete for Importin-α due to high local protein concentration, thereby minimizing defects in the microtubule nucleation function of GM13013. However, GM130 complex containing either wild-type or mutant peptides can interact with p115, still enabling p115-mediated vesicle fusion during mitosis and leading to impairment of Golgi disassembly and a higher percentage of monopolar spindles.Conventionally, GTP-bound Ran binds to Importin-β to either directly dissociate cargos or by competing with the IBB domain (which self-binds to the major NLS-binding site of Importin-α) to indirectly trigger the release of cargos. Although GM130 carries an IBB-like NLS that interacts with both full-length Importin-α and Importin-β, GM130 and Importin-β binding is perturbed by Ran and GM130 phosphorylation. Notably, the interaction between GM130 and Importin-α, which is enhanced by phosphorylation, is independently regulated by the Ran-Importin-β pathway, explaining why Importin-α but not Importin-β associates with GM130 in membrane fractions from either Our crystal structure of the Importin-α•GM130-NLS complex showed that three regions of the GM130-NLS are involved in binding Importin-α Fig. . The pos29. However, the GM130 NLS contains an α-helix that may interfere with the GM130 and Importin-α interaction. Hence, phosphorylation of apo-GM130 shortens the α-helix, promoting GM130 and Importin-α binding  by destabilizing the conformation of free GM130-NLS and (2) by stabilizing the conformation of GM130-NLS bound to Importin-α.MD simulations further revealed destabilization of the free GM130 peptide in solution upon phosphorylation, in addition to water-mediated intramolecular interactions of the bound phosphorylated peptide. Most cNLS (e.g. IBB) sequences lack secondary structural elements and are likely to be unstructured within proteinsing Fig. . Notablyn-β Fig. . Previou32. Notably, Cdk1 also phosphorylates the Ser-25 residue within the NLS-like motif of GM130 during early mitosis, and this GM130 phosphorylation event has been correlated with mitotic Golgi disassembly9. Here, we have demonstrated that phosphorylation of Ser-25 enhances the GM130 and Importin-α interaction, thereby sterically blocking the p115-binding site of GM130. During early mitosis, RanGTP releases Importin-α/-β from spindle assembly factors (SAFs) (e.g. TPX227 and NuMA26), activating the mitotic functions of these SAFs and in turn promoting mitotic spindle assembly. Thus, the Importin-α liberated by RanGTP can instead inhibit the p115-binding function of Cdk1-phosphorylated GM130, suppressing p115-mediated vesicle–Golgi fusion and allowing Golgi disassembly. Taken together, our findings provide a model whereby the Ran and kinase-phosphatase pathways coordinately regulate major events during mitosis  to ensure mitotic progression, facilitated by Cdk1 and the nuclear transport factor Importin-α  at the onset of open mitosis, requiring phosphorylation of numerous proteins residing in the NE (e.g. nuclear lamins) by Cdk1n-α Fig. .Homo sapiens GM130 (a.a. 1–48), GM130 (a.a. 1–85), and Importin-β were amplified by PCR and ligated into pGEX-6P-1 . H. sapiens full-length Importin-α and Mus musculus Importin-α were amplified by PCR and ligated into modified pGEX-6P-1  that produces proteins with 6-His at the C–terminus. Mouse Importin-α shares ~95% identity with human Importin-α over the full protein sequence, so we used the former for biochemical and structural studies. GM130-S25D (a.a. 1–48), GM130-S25D (a.a. 1–85), GM130-S25D/K14A (a.a. 1–85), and GM130-S25D/K34A (a.a. 1–85) mutants were created by PCR-based mutagenesis and were verified by sequencing. A list of primer sequences used in this study is shown in Supplementary Table Escherichia coli Rosetta strain (Novagen). Protein production was induced by 0.5 mM IPTG at 18 °C overnight. The cell pellet was re-suspended, disrupted by French press and clarified by centrifugation at 15,000 × g at 4 °C for 20 min. Recombinant proteins were then purified by chromatography.Bacterial expression constructs were transformed into Cells containing GST-tagged GM130 (a.a. 1–48)•His-Importin-α (a.a. 70–498) were harvested and re-suspended in a buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, and 3 mM DTT. Clarified lysate was loaded onto a GST column  and protein samples were eluted by a buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 50 mM reduced glutathione, and 3 mM DTT. The GST-GM130•Importin-α(ΔIBB) complex was incubated with PreScission protease at 4 °C overnight to remove the GST tag. PreScission-digested samples were incubated with nickel resin (Sigma), followed by a pre-wash of 25 mM imidazole. Next, protein was eluted by a buffer containing 250 mM imidazole and then subjected to SEC (Superdex 200 16/600) analysis. The peak fractions were analyzed by SDS–PAGE. Purified protein fractions were collected and concentrated to 25 mg/ml using Amicon Ultra 15 (Millipore) for crystallization. Purification of the GM130-S25D (a.a. 1–48)•Importin-α (a.a. 70–498) complex was carried out using the same method. To produce the GM130-NLS phospho-S25•His-Importin-α(ΔIBB) (a.a. 70–498) complex for crystallization, GST-tagged His-Importin-α (a.a. 70–498) was purified by GST-, nickel- and SEC chromatography using the same protocol for the purification of other GM130-containing complexes. The Importin-α protein was then mixed with a synthetic GM130-NLS phospho-S25 peptide (synthesized by the peptide synthesis core facility of Academic Sinica) in a molar ratio of 1:3. The excess peptide was removed by loading the sample into SEC (Superdex 200 10/300). Purified protein fractions were collected and concentrated to 25 mg/ml using Amicon Ultra 15 (Millipore) for crystallization. The same expression and purification protocol was applied to purify GST-tagged GM130-WT, GM130-S25D, GM130-S25D/K14A, and GM130-S25D/K34A, as well as non-tagged Importin-β, GM130-WT, and GM130-S25D. GST tags were removed by PreScission protease cleavage.To purify p115 (a.a. 780–930), cells containing p115 (a.a. 780–940) were resuspended in a buffer containing 50 mM K-phosphate (pH 7.4), 150 mM NaCl, and 2 mM β-mercaptoethanol. The cells were lysed with a cell disrupter. After clarification, lysates were applied to nickel resin (Sigma), followed by a pre-wash of 25 mM imidazole. Proteins were eluted with a buffer containing 250 mM imidazole. Fractions containing p115 protein were pooled and loaded into SEC (Superdex 75 16/600) and analyzed by SDS–PAGE. Purified protein fractions were collected and concentrated using Amicon Ultra 15 (Millipore). RanQ69L and GFP-Importin-α-NLS were obtained using an identical expression and purification protocol.12121 and contained a heterodimer in the asymmetric unit. For cryoprotection, crystals were stabilized in 0.1 M sodium malonate, 12% w/v polyethylene glycol 3350 (pH 7.0), and 20% (v/v) glycerol.The purified GM130 (a.a. 1–48)•Importin-α (a.a. 70–498) complex was screened for crystallization. Crystals were grown at a concentration of 25 mg/ml at 20 °C using the hanging drop method. Crystals grew to their maximum size after one week in 0.1 M sodium malonate and 12% w/v polyethylene glycol 3350 (pH 7.0). Crystals grew in the orthorhombic space group P2Crystals of the GM130-S25D (a.a. 1–48)•Importin-α (a.a. 70–498) and GM130-NLS phospho-S25 (a.a. 13–38)•Importin-α (a.a. 70–498) complexes were also grown at concentrations of 25 mg/ml at 20 °C using the hanging drop method. Crystals of these two complexes grew in 0.2 M potassium thiocyante and 20% w/v polyethylene glycol 3350 (for GM130-S25D (a.a. 1–48)•Importin-α (a.a. 70–498)) or 0.1 M sodium citrate tribasic dihydrate and 16% w/v polyethylene glycol 8000 (pH 5.5) (for GM130-NLS phospho-S25 (a.a. 13–38)•Importin-α (a.a. 70–498)), respectively.33, and molecular replacement was performed using Phaser in the Phenix software, with Importin-α (PDB code: 1IQ1 [https://www.rcsb.org/structure/1iq1]) as a search model34. For all of these structures, electron densities for GM130 could only be observed and assigned for residues 13–38 or 39. No clear electron densities outside this region could be identified. All the residues were in favored or allowed regions, and thereby no residues in the disallowed region of the Ramachandran plot. Coordinates and structure factors have been deposited in the Protein Data Bank.X-ray data were collected at beamline TPS 05A and BL13B1 at NSRRC  using a wavelength of 0.99984 Å. X-ray intensities were processed using HKL2000A 1:1 molar ratio of GST-GM130-WT or mutant GM130 and Importin-α FL was incubated with glutathione-coupled sepharose beads . After pre-washing with 150 mM NaCl, proteins were eluted by a buffer containing 50 mM reduced glutathione and analyzed by SDS–PAGE. To examine the interactions between GST-GM130-S25D and Importin-α FL or Importin-β in the presence of RanQ69L, we incubated GST-GM130-S25D, RanQ69L and Importin-α FL or Importin-β in a molar ratio of 1:4:1.GST-GM130-WT and GST-GM130-S25D were, respectively, incubated with Importin-β or Importin-α FL in a molar ratio of 1:1. Samples were injected into an SEC (Superdex 200 16/60) column  pre-equilibrated with a buffer containing 20 mM HEPES (pH 7.4), 50 mM NaCl, and 3 mM DTT. Peak fractions were analyzed by SDS–PAGE.Binding affinities of different GM130 proteins to Importin-α and Importin-β were measured by ITC . All proteins were dialyzed against ITC buffer  with either 50 or 200 mM NaCl. Protein concentrations were determined spectrophotometrically with respective extinction coefficients. ITC was performed at 25 °C. The respective heat changes were fitted using a one-sites binding model.The CD spectra of the GM130-WT(1–48)•Importin-α (a.a. 70–498), GM130-S25D(1–48)•Importin-α (a.a. 70–498), and GM130-NLS phospho-S25 (a.a. 13–38)•Importin-α (a.a. 70–498) complexes were recorded using an AVIV CD400 spectrometer. Thermal melting temperatures were determined by measuring the ellipticity at a wavelength of 222 nm as a function of temperature from 20 to 75 °C. The protein concentration used in the CD measurements was 0.2 mg/ml in a buffer of 20 mM HEPES (pH 7.4), 50 mM NaCl, and 3 mM DTT. Measurements were taken three times and averaged.35. Each system was solvated in a box of TIP3P36 water molecules at an ionic strength of 0.15 M using Na+ and Cl− ions. All bonds to hydrogen atoms were constrained by the SHAKE37 algorithm. Long-range electrostatic forces were treated using the particle mesh Ewald method38 with a grid spacing of 1 Å and a cutoff of 12 Å. The non-bonded interactions were updated every 1 fs. The simulations employed the CHARMM36 force-field39 using the NAMD 2.1240 program with an integration time step of 2 fs. For each complex simulation, the first 10 ns was excluded and hydrogen bonds were analyzed using the CHARMM37 program41 and an acceptor to hydrogen distance of ≤2.4 Å and a donor–hydrogen–acceptor angle of ≥130°. Each 200-ns apo simulation was divided into 1 ns sub-windows from which 200 conformations were extracted and the secondary structure of each residue was assigned using DSSP42.Starting from the crystallographic structure of the native GM130-NLS•Importin-α complex, eight 200-ns simulations at 300 K and 1 atm were performed for the apo peptides (native and phosphorylated GM130-NLS), whereas ten 50-ns simulations were performed for the GM130-NLS•Importin-α and GM130-NLS phospho-S25•Importin-α complexes. All histidines were found to be neutral according to Propka 3.1 and were thus deprotonated®CRL11268) were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum and 1% penicillin and streptomycin. Cells were seeded in a 24-well plate with a concentration of 5000 cells per well. Cell numbers were determined by counting trypan blue-negative cells using a hemocytometer under microscopy. Cells were counted every 24 h after seeding, and growth curves were generated based on three independent experiments.HEK293T cells  according to the manufacturer’s instructions. In brief, cells were dissociated and washed once with 1X PBS and fixed with 70% ethanol overnight. Cells were then centrifuged and washed once with 1X PBS. Cells were then re-suspended in 200 μl of Muse cell cycle assay kit and analyzed using Muse Cell Analyzer software .g for 10 min at 4 °C. Cell lysates were incubated with 5 µM of GST-GM130-WT, GST-GM130-S25D, or GST-GM130-S25D/K34A, and glutathione-coupled sepharose beads . After three pre-washes with buffer containing 150 mM NaCl, proteins were eluted using a buffer containing 50 mM of reduced glutathione. Eluted proteins were analyzed by SDS–PAGE, followed by Western blotting using antibodies against GM130 (Abcam ab52649), Importin-α (R&D Systems MAB6207), and Importin-β (Abcam ab2811). Uncropped scans of Western blots are provided in Supplementary Fig. HEK293T cells were lysed in RIPA buffer containing 50 mM Tris, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethyl sulphonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml leupeptin. Supernatants were collected after centrifugation at 21,000×TM 6 Fast Flow . Beads were then washed three times with buffer . Protein samples were boiled in SDS sample buffer and analyzed by SDS–PAGE, followed by Western blotting analysis.To pull down endogenous GM130, HEK293T cells were synchronized via nocodazole treatment and lysed in a buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton, 0.15% SDS, protease inhibitor cocktail (Roche), and 1 mM PMSF. Clarified lysates were pre-incubated with GM130 antibody (Abcam ab52649) for 12 h at 4 °C, followed by one hour incubation with IgG sepharoseTo prepare Cdk1 inhibitor treated lysates for GM130 pull-down assays, WT HEK293T cells were synchronized by 30 µM of nocodazole for 13 h, followed by treatment of Cdk1 inhibitor RO-3306 (10 µM/ml)  for 2 h.Golga2 was designed using the Optimized CRISPR Design website (http://crispr.mit.edu) followed by ligation into PX330 vector (Addgene 42230). HEK293T cells were dissociated as single cells using 0.05% Trypsin-EDTA. We co-transfected 1 × 106 million cells with 4 µg Cas9-sgRNA plasmid, 400 nM single-stranded DNA and a puromycin vector (pLVX-mCherry-C1) using Nucleofector Kit 2 (Lonza VPH-5022). Cells were then selected with puromycin. Approximately 10 days later, we selected single colonies for expansion and subsequent genotype sequencing. Sequences of the oligonucleotides used in the CRISPR/Cas9 genome editing experiment are shown in Supplementary Table SgRNA targeting human We used GM130 , GRASP65 , Giantin , α-Tubulin , and Importin-α  antibodies, followed by Alexa Fluor 488-conjugated and Alexa Fluor 568-conjugated donkey secondary antibodies against mouse or rabbit . Nuclei were counterstained with DAPI. Fluorescence images were collected using an Olympus fluorescence microscope (IX83), 3D images were collected using a Zeiss confocal microscope (LSM780), and all images were analyzed using Imaris software.Cells were synchronized with nocodazole and fixed with 4% PFA. Fixed cells were then stained with Duolink . Our PLA protocol was performed according to the manufacturer’s instructions using GM130  and p115  antibodies. Nuclei were counterstained with DAPI. Images were taken using an Olympus fluorescence microscope (IX83). Data were quantified by counting fluorescent spots.GM130 SMART pool and Non-Targeting siRNAs were purchased from Dharmacon. HEK293T cells were transfected with siRNA oligonucleotides using Lipofectamine RNAiMax  according to the manufacturer’s protocol. After 24-h transfection, transfected cells were replated on coverslides for further IF staining after 24-h seeding.6 per well into six-well plates. Next day cells were co-transfected with Lenti-viral GM130 K34A and lentiviral packaging plasmid psPAX2 (Addgene# 12260) and pMD2.G (Addgene #12259) using a standard CaCl2 transfection method described previously43. After 48 h, virus supernatants were collected and passed through 0.45 μm pore size filters . GM130 K34A mutation virus and 4 μg/ml of polybrene (Sigma) were added to the HEK293T cells. Infection efficiency was enhanced by spinning the plates at 1100×g for 30 min. Infected cells surviving puromycin selection (2 µg/ml) were then expanded and harvested for further analysis.Full length of GM130 K34A mutation was cloned into Lenti-virus vector pCDH-CMV-MCS-EF1-GFP-T2A-Puro . For virus production, HEK293T cells were plated at 1.5 × 10GM130 WT and K34A mutant cells were replated on six-well plates. Next day cells were treated with 100 µg/ml of cycloheximide  for 0, 4, and 8 h.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Furthermore, clinical outcomes correlated with DC migration to vaccine-site draining lymph nodes measured by Indium-111 labeling of RNA-loaded DCs and SPECT/CT imaging. Although these studies demonstrated that tracking DC migration may be an important clinical biomarker for response to DC vaccination, the complexity and regulatory requirements associated with nuclear labelling to track DC migration limits widespread application of this technique. We have therefore developed RNA-loaded magnetic nanoparticles (RNA-NPs) to enhance DC migration to LNs and track that migration with a widely available imaging modality . METHODS/STUDY POPULATION: Cationic liposomes were loaded with iron oxide nanoparticles with or without cholesterol. The resulting nanoparticles were complexed with RNA and used to transfect DCs ex vivo. RNA-NP-loaded DsRed+ DCs were then injected intradermally into mice and tracked noninvasively with T2-weighted 11T MRI before excision and quantification with flow cytometry. RESULTS/ANTICIPATED RESULTS: In vitro experiments demonstrate that iron oxide loading does not reduce RNA-NP-mediated transfection of DCs. Additionally, replacement of cationic lipids with cholesterol increased RNA-NP transfection of the DC2.4 cell line and enhanced the T cell stimulatory capacity of treated bone marrow-derived dendritic cells (BMDCs). Compared to electroporation, RNA-NPs enhanced DC migration to lymph nodes and reduced T2 MRI intensity in DC-bearing lymph nodes. DISCUSSION/SIGNIFICANCE OF IMPACT: This data suggests that iron oxide-loaded RNA-NPs enable noninvasive cell tracking with MRI and enhance DC migration to lymph nodes. We have further shown that inclusion of cholesterol in RNA-NPs augments the stimulatory capacity of transfected DCs. Future work will consider effects of RNA-NPs on antitumor immune responses and the utility of MRI-detected DC migration as a biomarker of vaccine efficacy.OBJECTIVES/SPECIFIC AIMS: Despite aggressive chemotherapy, surgical resection, and radiation therapy, glioblastoma remains almost universally fatal. In a pilot, randomized, and blinded clinical trial, we recently demonstrated that administration of RNA-loaded DC vaccines was associated with significantly improved progression-free and overall survival in patients with glioblastoma (Mitchell