Colocalization Single-molecule Spectroscopy Research Articles

Identification of transient intermediates during spliceosome activation by single molecule fluorescence microscopy

Spliceosome activation is the process of creating the catalytic site for RNA splicing and occurs de novo on each intron following spliceosome assembly. Dozens of factors bind to or are released from the activating spliceosome including the Lsm2-8 heteroheptameric ring that binds the U6 small nuclear RNA 3'-end. Lsm2-8 must be released to permit active site stabilization by the Prp19-containing complex (NineTeen Complex, NTC); however, little is known about the temporal order of events and dynamic interactions that lead up to and follow Lsm2-8 release. We have used colocalization single molecule spectroscopy (CoSMoS) to visualize Lsm2-8 dynamics during activation of Saccharomyces cerevisiae spliceosomes in vitro. Lsm2-8 is recruited as a component of the tri-snRNP and is released after integration of the Prp19-containing complex (NTC). Despite Lsm2-8 and the NTC being mutually exclusive in existing cryo-EM structures of yeast B complex spliceosomes, we identify a transient intermediate containing both ([Formula: see text]) and provide a kinetic framework for its formation and transformation during activation. Prior to [Formula: see text] assembly, the NTC rapidly and reversibly samples the spliceosome suggesting a mechanism for preventing NTC sequestration by defective spliceosomes that fail to properly activate. In complementary ensemble assays, we show that a base-pairing-dependent ternary complex can form between Lsm2-8 and U2 and U6 helix II RNAs. We propose that this interaction may play a role in formation of transient spliceosome intermediates formed during activation.

Multi-step recognition of potential 5' splice sites by the Saccharomyces cerevisiae U1 snRNP.

In eukaryotes, splice sites define the introns of pre-mRNAs and must be recognized and excised with nucleotide precision by the spliceosome to make the correct mRNA product. In one of the earliest steps of spliceosome assembly, the U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5' splice site (5' SS) through a combination of base pairing, protein-RNA contacts, and interactions with other splicing factors. Previous studies investigating the mechanisms of 5' SS recognition have largely been done in vivo or in cellular extracts where the U1/5' SS interaction is difficult to deconvolute from the effects of trans-acting factors or RNA structure. In this work we used colocalization single-molecule spectroscopy (CoSMoS) to elucidate the pathway of 5' SS selection by purified yeast U1 snRNP. We determined that U1 reversibly selects 5' SS in a sequence-dependent, two-step mechanism. A kinetic selection scheme enforces pairing at particular positions rather than overall duplex stability to achieve long-lived U1 binding. Our results provide a kinetic basis for how U1 may rapidly surveil nascent transcripts for 5' SS and preferentially accumulate at these sequences rather than on close cognates.

A helicase-tethered ORC flip enables bidirectional helicase loading.

Replication origins are licensed by loading two Mcm2-7 helicases around DNA in a head-to-head conformation poised to initiate bidirectional replication. This process requires origin-recognition complex (ORC), Cdc6, and Cdt1. Although different Cdc6 and Cdt1 molecules load each helicase, whether two ORC proteins are required is unclear. Using colocalization single-molecule spectroscopy combined with single-molecule Förster resonance energy transfer (FRET), we investigated interactions between ORC and Mcm2-7 during helicase loading. In the large majority of events, we observed a single ORC molecule recruiting both Mcm2-7/Cdt1 complexes via similar interactions that end upon Cdt1 release. Between first- and second-helicase recruitment, a rapid change in interactions between ORC and the first Mcm2-7 occurs. Within seconds, ORC breaks the interactions mediating first Mcm2-7 recruitment, releases from its initial DNA-binding site, and forms a new interaction with the opposite face of the first Mcm2-7. This rearrangement requires release of the first Cdt1 and tethers ORC as it flips over the first Mcm2-7 to form an inverted Mcm2-7-ORC-DNA complex required for second-helicase recruitment. To ensure correct licensing, this complex is maintained until head-to-head interactions between the two helicases are formed. Our findings reconcile previous observations and reveal a highly coordinated series of events through which a single ORC molecule can load two oppositely oriented helicases.

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.

Single-Molecule Dynamics of the Human Mitochondrial RNA Polymerase Preinitiation Complex

Mitochondrial mRNA transcription requires assembly of the preinitiation complex (PIC), which contains promoter DNA, mitochondrial RNA polymerase (mtRNAP), and the two General Transcription Factors (GTFs), TFAM and TF2BM. After TFAM-initiated DNA bending and the assembly of the PIC, mtRNAP, with the help of TFB2M, unwinds the promoter DNA, begins RNA synthesis, escapes the promoter, and begins elongation of the nascent RNA. The existence of structural models for mtRNAP, the GTFs, and even PIC intermdiates do not fully explain the order of PIC assembly, the rate-limiting steps that must be overcome to initiate transcription and the fates of residual GTFs after mtRNAP escape. In order to investigate these questions, we visualised the initiation of mtRNAP in vitro using Co-localization Single Molecule Spectroscopy (CoSMoS) in real time, at a one second time resolution. Further to this, we dynamically captured the entry of TFAM and mtRNAP into single PICs, mtRNAP promoter escape, mtRNAP elongation, and mtRNAP re-initiation. By super-resolving the dynamic positions of mtRNAP and TFAM on the DNA during initiation, we have indicated that it is feasible to use single-molecule, real-time, structural analysis of the formation of the PIC by mitochondrial transcription components.

Preparation of SNAPf-Beads for Colocalization Single-Molecule Spectroscopy (CoSMoS) of RNA-Protein Complexes.

The SNAPf-tag is a chemical tag that allows rapid and highly specific covalent labeling of proteins even in the full complexity of the cellular environment. The SNAPf-tag has been instrumental to study native RNA-protein complexes at single-molecule resolution in their cellular environment as efficient labeling of the RNAs and proteins of interest is essential for this colocalization single-molecule spectroscopy (CoSMoS) technique. However, removal of excessive benzylguanine dye after the labeling reaction has remained challenging. Here, we describe a strategy to remove excessive benzylguanine dye using SNAPf-tag coated beads as sponges.

Single-Molecule Analysis of Spliceosome Activation Kinetics Reveals Multiple Intermediate States

During pre-mRNA splicing, the spliceosome needs to be activated in an ATP-dependent manner in order to carry out the catalytic steps of the splicing reaction. During activation, the protein composition of the spliceosome changes dramatically and includes loss of the B complex proteins (Spp381, Snu23 and Prp38) and Snu66. Using colocalization single molecule spectroscopy (CoSMoS), we show that Spp381, Snu23, and Snu66 release is dependent on ATP by carrying out reactions at both high and low ATP concentrations. By comparing the characteristic dwell times for these proteins at high ATP, we found that Spp381 and Snu23 behave similar to each other and with kinetics distinct from Snu66. Specifically, at both ATP concentrations, Snu66 shows much longer-lived populations than Spp381 and Snu23. This suggests that Snu66 is bound more stably to the spliceosome than the other two. These results are consistent with previous biochemical and structural studies suggesting that Spp381, Snu23, and Prp38 form a protein subcomplex of the spliceosome. Together, our results suggest that this subcomplex and Snu66 likely have two different roles in the activation step and give rise to multiple activation intermediates as they are sequentially released from the spliceosome.

Function and Dynamics of the LSm2-8 Protein Ring during Spliceosome Activation

LSm2-8, a heteroheptameric protein ring, decorates the 3′ end of the U6 snRNA and is a stable component of U6 snRNP, U4/U6 di-snRNP, U4/U6.U5 tri-snRNP, and the spliceosome B complex. However, it is released during spliceosome activation, during which the active site for pre-mRNA splicing is created. While Lsm2-8 is known to stabilize the U6 snRNA in the cell, any potential function during spliceosome assembly or activation is unclear. Here, we characterize Lsm2-8 ejection during spliceosome activation using Colocalization Single Molecule Spectroscopy (CoSMoS). Our CoSMoS data reveals that LSm ring leaves after both release of U4 snRNP and arrival of Nine Teen Complex (NTC) on pre-mRNA. This indicates that a conformational change must occur within the NTC after spliceosome binding since available cryo-EM structures indicate that Lsm2-8 and NTC binding are mutually exclusive with one another. Thus, our data provides evidence for an intermediate spliceosome complex between B and Bact that has not been yet observed by cryo-EM. This unexpected late release of the Lsm2-8 ring also suggests an extended role during spliceosome activation. Based on sequence homology to bacterial Hfq proteins, it is possible that Lsm2-8 may be promoting formation of a RNA duplex between the U2 and U6 snRNAs. Using in vitro annealing assay, we observed that LSm ring forms a ternary complex in the presence U6 and U2 RNAs, which is dependent on base pairing. These results indicate that LSm ring may help in stabilizing U2/U6 helix II during activation and is retained on spliceosome until U4 unwinding is complete. After U4 is unwound from U6, LSm is replaced by NTC components that takeover stabilization of helix II during later stages of activation and splicing.

Mechanism and timing of Mcm2-7 ring closure during DNA replication origin licensing.

Opening and closing of two ring-shaped Mcm2–7 DNA helicases is necessary to license eukaryotic origins of replication although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process, establishing a topological link between each Mcm2–7 and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule FRET (Förster resonance energy transfer), we monitored S. cerevisiae Mcm2–7 ring opening and closing during origin licensing. The two Mcm2–7 rings are open during initial DNA association and close sequentially, concomitant with release of their associated Cdt1. ATP hydrolysis by Mcm2–7 is coupled to ring closure and Cdt1 release, and failure to load the first Mcm2–7 prevents recruitment of the second Mcm2–7. Our findings identify key mechanisms controlling the Mcm2–7 DNA-entry gate during origin licensing and reveal that the two Mcm2–7 complexes are loaded by a coordinated series of events with implications for bidirectional replication initiation and quality control.

Single-Molecule Analysis of Pre-mRNA Splicing with Colocalization Single-Molecule Spectroscopy (CoSMoS).

Recent development of single-molecule techniques to study pre-mRNA splicing has provided insights into the dynamic nature of the spliceosome. Colocalization single-molecule spectroscopy (CoSMoS) allows following spliceosome assembly in real time at single-molecule resolution in the full complexity of cellular extracts. A detailed protocol of CoSMoS has been published previously (Anderson and Hoskins, Methods Mol Biol 1126:217-241, 2014). Here, we provide an update on the technical advances since the first CoSMoS studies including slide surface treatment, data processing, and representation. We describe various labeling strategies to generate RNA reporters with multiple dyes (or other moieties) at specific locations.

Single molecule analysis reveals reversible and irreversible steps during spliceosome activation.

The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation.

Single Molecule Evidence for Long Range Interactions During Commitment Complex Formation

The spliceosome is a multimegadalton ribonucleoprotein (RNP) consisting of five small nuclear RNPs (snRNPS) that assemble on precursor mRNA (pre-mRNA) in an ordered, dynamic pathway to remove introns. Commitment complex (CC) is the earliest stage of spliceosome assembly and is composed of pre-mRNA, the U1 snRNP, the branchpoint bridging protein (BBP)/mud2 heterodimer, and the cap binding complex (CBC). During CC formation U1 recognizes a short conserved sequence that identifies the 5’ end of the intron known the 5’ splice site (5′SS) through snRNA-RNA and RNA-protein interactions. The U1/5′SS interaction is thought to be additionally stabilized by interactions with the CBC and BBP/mud2; however, the quantitative contribution of each participant is not well understood. Here we use Colocalization Single Molecule Spectroscopy (CoSMoS) to monitor the interaction between fluorescently labeled U1 and pre-mRNA. By selectively disrupting individual CC interactions and monitoring the stability of the U1/5′SS complex we have been able to quantify the contributions from individual CC components. Our results provide the first description of how cross-intron interactions formed between the U1 snRNP and other CC components impact the kinetics of the U1/5′SS interaction. Furthermore, our single molecule results provide kinetic evidence for the synergy between the CBC, BBP/Mud2, and the U1 snRNP: long-lived U1/5′SS association requires the presence of either CBC or BBP/Mud2 even though each individually only makes minor contributions. This is in agreement with a “buffering” model obtained using yeast genetics by Schwer et al. for CC interactions. Finally, we have used this system to dissect the roles of snRNP proteins and the snRNA in U1/5′SS association. Our data is consistent with a multi-step pathway for U1 recruitment to the pre-mRNA that may rely on CC components to efficiently trigger stable U1 binding.

Design and construction of a multiwavelength, micromirror total internal reflectance fluorescence microscope.

Colocalization single-molecule spectroscopy (CoSMoS) has proven to be a useful method for studying the composition, kinetics and mechanisms of complex cellular machines. Key to the technique is the ability to simultaneously monitor multiple proteins and/or nucleic acids as they interact with one another. Here we describe a protocol for constructing a CoSMoS micromirror total internal reflection fluorescence microscope (mmTIRFM). Design and construction of a scientific microscope often requires a number of custom components and a substantial time commitment. In our protocol, we have streamlined this process by implementation of a commercially available microscopy platform designed to accommodate the optical components necessary for an mmTIRFM. The mmTIRF system eliminates the need for machining custom parts by the end user and facilitates optical alignment. Depending on the experience level of the microscope builder, these time savings and the following protocol can enable mmTIRF construction to be completed within 2 months.

Reconstitution and Single Molecule Characterization of Yeast Spliceosomal E Complex

The formation of yeast commitment complex (CC2 or “E complex”) is one of the earliest stages in spliceosome assembly on pre-mRNA, and is the only assembly step that forms independent of the activity of an ATPase. Previous studies have shown that E complex components, the nuclear cap binding complex (CBC) and the branchpoint binding Msl5•Mud2 heterodimer, are required to stabilize U1 small nuclear ribonucleoprotein (snRNP) complex to the 5′SS of pre-mRNA. However the kinetics and nature of the interactions between these components have not been established, in part because E complex has never been reconstituted separate from other spliceosomal proteins. Here we present work towards the characterization of E complex interactions by Colocalization Single Molecule Spectroscopy (CoSMoS) experiments with reconstituted fluorescently-labeled CBC, Msl5•Mud2, and U1 snRNP. Intact and functional U1 snRNP was isolated from all other spliceosomal proteins by a novel purification method and fluorescently labeled with a tri-functional SNAP-ligand derivative for single molecule study. The stoichiometry of the seventeen U1 snRNP proteins and the efficiency of the fluorescent labeling reaction is being determined by AQUA mass spectrometry. A combination of splicing assays, primer extension, and CoSMoS experiments with fluorescently labeled pre-mRNA constructs are used to show the functionality of purified U1 snRNP. Our results show that the multi-protein components of E complex can be reconstituted and fluorescently tagged for CoSMoS and other single molecule experiments which elucidate the kinetics of early spliceosome assembly.

Visualizing cellular machines with colocalization single molecule microscopy.

Many of the cell's macromolecular machines contain multiple components that transiently associate with one another. This compositional and dynamic complexity presents a challenge for understanding how these machines are constructed and function. Colocalization single molecule spectroscopy enables simultaneous observation of individual components of these machines in real-time and grants a unique window into processes that are typically obscured in ensemble assays. Colocalization experiments can yield valuable information about assembly pathways, compositional heterogeneity, and kinetics that together contribute to the development of richly detailed reaction mechanisms. This review focuses on recent advances in colocalization single molecule spectroscopy and how this technique has been applied to enhance our understanding of transcription, RNA splicing, and translation.

Alternative Spliceosome Assembly Pathways Revealed by Single-Molecule Fluorescence Microscopy

Removal of introns from nascent transcripts (pre-mRNAs) by the spliceosome is an essential step in eukaryotic gene expression. Previous studies have suggested that the earliest steps in spliceosome assembly in yeast are highly ordered and the stable recruitment of U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site necessarily precedes recruitment of U2 snRNP to the branch site to form the "prespliceosome." Here, using colocalization single-molecule spectroscopy to follow initial spliceosome assembly on eight different S. cerevisiae pre-mRNAs, we demonstrate that active yeast spliceosomes can form by both U1-first and U2-first pathways. Both assembly pathways yield prespliceosomes functionally equivalent for subsequent U5·U4/U6 tri-snRNP recruitment and for intron excision. Although fractional flux through the two pathways varies on different introns, both are operational on all introns studied. Thus, multiple pathways exist for assembling functional spliceosomes. These observations provide insight into the mechanisms of cross-intron coordination of initial spliceosome assembly.

RNA polymerase approaches its promoter without long-range sliding along DNA

Sequence-specific DNA binding proteins must quickly bind target sequences, despite the enormously larger amount of nontarget DNA present in cells. RNA polymerases (or associated general transcription factors) are hypothesized to reach promoter sequences by facilitated diffusion (FD). In FD, a protein first binds to nontarget DNA and then reaches the target by a 1D sliding search. We tested whether Escherichia coli σ(54)RNA polymerase reaches a promoter by FD using the colocalization single-molecule spectroscopy (CoSMoS) multiwavelength fluorescence microscopy technique. Experiments directly compared the rates of initial polymerase binding to and dissociation from promoter and nonpromoter DNAs measured in the same sample under identical conditions. Binding to a nonpromoter DNA was much slower than binding to a promoter-containing DNA of the same length, indicating that the detected nonspecific binding events are not on the pathway to promoter binding. Truncating one of the DNA segments flanking the promoter to a length as short as 7 bp or lengthening it to ~3,000 bp did not alter the promoter-specific binding rate. These results exclude FD over distances corresponding to the length of the promoter or longer from playing any significant role in accelerating promoter search. Instead, the data support a direct binding mechanism, in which σ(54)RNA polymerase reaches the local vicinity of promoters by 3D diffusion through solution, and suggest that binding may be accelerated by atypical structural or dynamic features of promoter DNA. Direct binding explains how polymerase can quickly reach a promoter, despite occupancy of promoter-flanking DNA by bound proteins that would impede FD.

Single-molecule colocalization FRET evidence that spliceosome activation precedes stable approach of 5′ splice site and branch site

Removal of introns from the precursors to messenger RNA (pre-mRNAs) requires close apposition of intron ends by the spliceosome, but when and how apposition occurs is unclear. We investigated the process by which intron ends are brought together using single-molecule fluorescence resonance energy transfer together with colocalization single-molecule spectroscopy, a combination of methods that can directly reveal how conformational transitions in macromolecular machines are coupled to specific assembly and disassembly events. The FRET measurements suggest that the 5' splice site and branch site remain physically separated throughout spliceosome assembly, and only approach one another after the spliceosome is activated for catalysis, at which time the pre-mRNA becomes highly dynamic. Separation of the sites of chemistry until very late in the splicing pathway may be crucial for preventing splicing at incorrect sites.

CoSMoS Unravels Mysteries of Transcription Initiation

Using a fluorescence method called colocalization single-molecule spectroscopy (CoSMoS), Friedman and Gelles dissect the kinetics of transcription initiation at a bacterial promoter. Ultimately, CoSMoS could greatly aid the study of the effects of DNA sequence and transcription factors on both prokaryotic and eukaryotic promoters.

Into the CoSMoS: Single Molecule Analysis of Spliceosome Assembly and Activation

Excision of introns from pre-mRNAs is mediated by the spliceosome, a large, dynamic complex consisting of five small ribonucleoprotein particles (snRNPs) and scores of associated proteins. Current understanding of spliceosome assembly is based largely on the procession of stable complexes that can be resolved from in vitro splicing reactions. Such ensemble experiments have suggested a highly ordered, linear assembly pathway in which initial binding of U1 snRNP to the 5′ splice site is followed by stable U2 association with the branch site and subsequent U4/U5/U6 tri-snRNP and Nineteen Complex (NTC) addition to form the fully assembled spliceosome. Previously unknown, however, were the detailed forward and reverse kinetics of each assembly step, the extent to which branched and/or dead-end assembly pathways exist, and whether or not different introns utilize the same or alternate assembly pathway(s). We are now addressing these questions by combining yeast genetic engineering, chemical biology, and multi-wavelengthfluoresence microscopy to follow assembly of single spliceosomes in real time. Because no protein purification or reconstitution is required for such Colocalization Single Molecule Spectroscopy (CoSMoS), this experimental strategy should prove widely useful for mechanistic analysis of many other macromolecular machines in environments approaching the complexity of living cells.