Single-molecule pull-down for studying protein interactions

This protocol describes a single-molecule pull-down (SiMPull) assay for analyzing physiological protein complexes. The assay combines the conventional pull-down assay with single-molecule total internal reflection fluorescence (TIRF) microscopy and allows the probing of single macromolecular complexes directly from cell or tissue extracts. In this method, antibodies against the protein of interest are immobilized on a passivated microscope slide. When cell extracts are applied, the surface-tethered antibody captures the protein together with its physiological interaction partners. After washing away the unbound components, single-molecule fluorescence microscopy is used to probe the pulled-down proteins. Captured proteins are visualized through genetically encoded fluorescent protein tags or through antibody labeling. Compared with western blot analysis, this ultrasensitive assay requires considerably less time and reagents and provides quantitative data. Furthermore, SiMPull can distinguish between multiple association states of the same protein. SiMPull is generally applicable to proteins from a variety of cellular contexts and to endogenous proteins. Starting with the cell extracts and passivated slides, the assay requires 1.5-2.5 h for data acquisition and analysis.     

In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes

Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies.     

Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.     

Understanding Protein Mobility in Bacteria by Tracking Single Molecules

Protein diffusion is crucial for understanding the formation of protein complexes in vivo and has been the subject of many fluorescence microscopy studies in cells; however, such microscopy efforts are often limited by low sensitivity and resolution. During the past decade, these limitations have been addressed by new super-resolution imaging methods, most of which rely on single-particle tracking and single-molecule detection; these methods are revolutionizing our understanding of molecular diffusion inside bacterial cells by directly visualizing the motion of proteins and the effects of the local and global environment on diffusion. Here we review key methods that made such experiments possible, with particular emphasis on versions of single-molecule tracking based on photo-activated fluorescent proteins. We also discuss studies that provide estimates of the time a diffusing protein takes to locate a target site, as well as studies that examined the stoichiometries of diffusing species, the effect of stable and weak interactions on diffusion, and the constraints of large macromolecular structures on the ability of proteins and their complexes to access the entire cytoplasm.     

Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles.

The spliceosome is a macromolecular assembly that carries out the excision of introns from nuclear pre-mRNAs. It consists of four large RNA-protein complexes, called the U1, U2, U4/U6 and U5 small nuclear ribonucleoproteins (snRNPs), and many protein factors. Crystal structures of seven protein components and fragments of the U1 and U2 small nuclear RNAs have been determined in the form of RNA-protein and protein-protein complexes. Together with electron microscopy studies of the snRNPs, these structures have begun to provide important insights into the architecture of the snRNPs and the mechanisms of RNA-protein and protein-protein recognition.     

KERA: analysis tool for multi-process,multi-state single-molecule data

Molecular machines within cells dynamically assemble, disassemble and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy, in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA’s utility by analyzing conformational dynamics of two DNA binding proteins: replication protein A and xeroderma pigmentosum complementation group D helicase.     

A three-dimensional model of the U1 small nuclear ribonucleoprotein particle

Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich small nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form small nuclear ribonucleoprotein particles (snRNPs). Initial recognition of the 5′ exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature. Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.     

An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.

We have studied the assembly, composition and structure of splicing complexes using biotin-avidin affinity chromatography and RNase protection assays. We find that U1, U2, U4, U5 and U6 snRNPs associate with the pre-mRNA and are in the mature, functional complex. Association of U1 snRNP with the pre-mRNA is rapid and ATP independent; binding of all other snRNPs occurs subsequently and is ATP dependent. Efficient binding of U1 and U2 snRNPs requires a 5' splice site or a 3' splice site/branch point region, respectively. Both sequence elements are required for efficient U4, U5 and U6 snRNP binding. Mutant RNA substrates containing only a 5' splice site or a 3' splice site/branch point region are assembled into 'partial' splicing complexes, which contain a subset of these five snRNPs. RNase protection experiments indicate that in contrast to U1 and U2 snRNPs, U4, U5 and U6 snRNPs do not contact the pre-mRNA. Based upon the time course of snRNP binding and the composition of sucrose gradient fractionated splicing complexes we suggest an assembly pathway proceeding from a 20S (U1 snRNP only) through a 40S (U1 and U2 snRNPs) to the functional 60S splicing complex (U1, U2, U4, U5 and U6 snRNPs).     

Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP

Two different models currently exist for the assembly pathway of the spliceosome, namely, the traditional model, in which spliceosomal snRNPs associate in a stepwise, ordered manner with the pre-mRNA, and the holospliceosome model, in which all spliceosomal snRNPs preassemble into a penta-snRNP complex. Here we have tested whether the spliceosomal A complex, which contains solely U1 and U2 snRNPs bound to pre-mRNA, is a functional, bona fide assembly intermediate. Significantly, A complexes affinity-purified from nuclear extract depleted of U4/U6 snRNPs (and thus unable to form a penta-snRNP) supported pre-mRNA splicing in nuclear extract depleted of U2 snRNPs, whereas naked pre-mRNA did not. Mixing experiments with purified A complexes and naked pre-mRNA additionally confirmed that under these conditions, A complexes do not form de novo. Thus, our studies demonstrate that holospliceosome formation is not a prerequisite for generating catalytically active spliceosomes and that, at least in vitro, the U1 and U2 snRNPs can functionally associate with the pre-mRNA, prior to and independent of the tri-snRNP. The ability to isolate functional spliceosomal A complexes paves the way to study in detail subsequent spliceosome assembly steps using purified components.     

Arrested yeast splicing complexes indicate stepwise snRNP recruitment during in vivo spliceosome assembly

Pre-mRNA splicing is catalyzed by the spliceosome, a macromolecular machine dedicated to intron removal and exon ligation. Despite an abundance of in vitro information and a small number of in vivo studies, the pathway of yeast (Saccharomyces cerevisiae) in vivo spliceosome assembly remains uncertain. To address this situation, we combined in vivo depletions of U1, U2, or U5 snRNAs with chromatin immunoprecipitation (ChIP) analysis of other splicing snRNPs along an intron-containing gene. The data indicate that snRNP recruitment to nascent pre-mRNA predominantly proceeds via the canonical three-step assembly pathway: first U1, then U2, and finally the U4/U6*U5 tri-snRNP. Tandem affinity purification (TAP) using a U2 snRNP-tagged protein allowed the characterization of in vivo assembled higher-order splicing complexes. Consistent with an independent snRNP assembly pathway, we observed high levels of U1-U2 prespliceosomes under U5-depletion conditions, and we observed significant levels of a U2/U5/U6/Prp19-complex mature splicing complex under wild-type conditions. These complexes have implications for the steady-state distribution of snRNPs within nuclei and also reinforce the stepwise recruitment of U1, U2, and the tri-snRNP during in vivo spliceosome assembly.     

U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6.

To understand how the U5 small nuclear ribonucleoprotein (snRNP) interacts with other spliceosome components, its structure and binding to the U4/U6 snRNP were analyzed. The interaction of the U5 snRNP with the U4/U6 snRNP was studied by separating the snRNPs in HeLa cell nuclear extracts on glycerol gradients. A complex running at 25S and containing U4, U5, and U6 but not U1 or U2 snRNAs was identified. In contrast to results with native gel electrophoresis to separate snRNPs, this U4/U5/U6 snRNP complex requires ATP to assemble from the individual snRNPs. The structure of the U5 RNA within the U5 snRNP and the U4/5/6 snRNP complexes was then compared. Oligonucleotide-targeted RNase H digestion identified one RNA sequence in the U5 snRNP capable of base pairing to other nucleic acid sequences. Chemical modification experiments identified this sequence as well as two other U5 RNA sequences as accessible to modification within the U5 RNP. One of these regions is a large loop in the U5 RNA secondary structure whose sequence is conserved from Saccharomyces cerevisiae to humans. Interestingly, no differences in modification of free U5 snRNP as compared to U5 in the U4/U5/U6 snRNP complex were observed, suggesting that recognition of specific RNA sequences in the U5 snRNP is not required for U4/U5/U6 snRNP assembly.     

Intranuclear binding kinetics and mobility of single native U1 snRNP particles in living cells

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single-molecule sensitivity. The single-particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites, many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay behavior on time scales ranging from milliseconds to seconds, reflecting the involvement of U1 snRNPs in numerous distinct interactions. The average dwell times for U1 snRNPs bound at sites within the nucleoplasm did not differ significantly from those in speckles, indicating that similar processes occur in both compartments. Mobile U1 snRNPs moved with diffusion constants in the range from 0.5 to 8 μm²/s. These values were consistent with uncomplexed U1 snRNPs diffusing at a viscosity of 5 cPoise and U1 snRNPs moving in a largely restricted manner, and U1 snRNPs contained in large supramolecular assemblies such as spliceosomes or supraspliceosomes.     

Composition and functional characterization of the yeast spliceosomal penta-snRNP.

Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.     

The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins.

The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K and A proteins are known to bind directly to stem-loops of the U1 snRNA, whereas the U1-C protein does not bind to naked U1 snRNA, but depends on other U1 snRNP protein components for its association. Focusing on the U1-70K and U1-C proteins, protein-protein interactions contributing to the association of these particle-specific proteins with the U1 snRNP were studied. Immunoprecipitation of complexes formed after incubation of naked U1 snRNA or purified U1 snRNPs lacking their specific proteins (core U1 snRNP) with in vitro translated U1-C protein, revealed that both common snRNP proteins and the U1-70K protein are required for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrated that the U1-70K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lacked the U1-70K RNP-80 motif and did not bind naked U1 RNA, associated stably with core U1 snRNPs. This suggests that a new U1-70K binding site is generated upon association of common U1 snRNP proteins with U1 RNA. The interaction between the N-terminal domain of U1-70K and the core RNP domain was specific for the U1 snRNP; stable binding was not observed with core U2 or U5 snRNPs, suggesting essential structural differences among snRNP core domains. Evidence for direct protein-protein interactions between U1-specific proteins and common snRNP proteins was supported by chemical crosslinking experiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, were detected. A model for the assembly of U1 snRNP is presented in which the complex of common proteins on the RNA backbone functions as a platform for the association of the U1-specific proteins.     

Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are required for pre-mRNA splicing throughout the nucleoplasm, yet snRNPs also concentrate in Cajal bodies (CBs). To address a proposed role of CBs in snRNP assembly, we have used fluorescence resonance energy transfer (FRET) microscopy to investigate the subnuclear distribution of specific snRNP intermediates. Two distinct complexes containing the protein SART3 (p110), required for U4/U6 snRNP assembly, were localized: SART3.U6 snRNP and SART3.U4/U6 snRNP. These complexes segregated to different nuclear compartments, with SART3.U6 snRNPs exclusively in the nucleoplasm and SART3.U4/U6 snRNPs preferentially in CBs. Mutant cells lacking the CB-specific protein coilin and consequently lacking CBs exhibited increased nucleoplasmic levels of SART3.U4/U6 snRNP complexes. Reconstitution of CBs in these cells by expression of exogenous coilin restored accumulation of SART3.U4/U6 snRNP in CBs. Thus, while some U4/U6 snRNP assembly can occur in the nucleoplasm, these data provide evidence that SART3.U6 snRNPs form in the nucleoplasm and translocate to CBs where U4/U6 snRNP assembly occurs.     

New insights into trypanosomatid U5 small nuclear ribonucleoproteins

Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.