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Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how human U1 snRNP recognizes particular 5′ splice sites (5′SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5′SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5′SS modulation. We have determined a sequential binding mechanism for modulation of −1A bulged 5′SS by branaplam using a combination of ensemble kinetic measurements and colocalization single molecule spectroscopy (CoSMoS). Our mechanism establishes that U1-C protein binds reversibly to U1 snRNP, and branaplam binds to the U1 snRNP/U1-C complex only after it has engaged with a −1A bulged 5′SS. Obligate orders of binding and unbinding explain how reversible branaplam interactions cause formation of long-lived U1 snRNP/5′SS complexes. Branaplam targets a ribonucleoprotein, not only an RNA duplex, and its action depends on fundamental properties of 5′SS recognition. Splice sites must be correctly identified for introns to be removed from pre-mRNAs and for expression of the correct mRNA isoforms. Here, the authors elucidate how the human U1 snRNP splicing factor recognizes 5′ splice sites and how a splicing modulating drug can enhance splice site recognition.

The spliceosome must identify the correct splice sites (SS) and branchsite (BS) used during splicing. E complex is the earliest spliceosome precursor in which the 5' SS and BS are defined. Definition occurs by U1 small nuclear ribonucleoprotein (snRNP) binding the 5' SS and recognition of the BS by the E complex protein (ECP) branchpoint bridging protein (BBP). We have used single molecule fluorescence to study Saccharomyces cerevisiae U1 and BBP interactions with RNAs. E complex is dynamic and permits frequent redefinition of the 5' SS and BS. BBP influences U1 binding at the 5' SS by promoting long-lived complex formation. ECPs facilitate U1 association with RNAs with weak 5' SS and prevent U1 accumulation on RNAs containing hyperstabilized 5' SS. The data reveal a mechanism for how U1 binds the 5' SS and suggest that E complex harnesses this mechanism to stimulate recruitment and retention of U1 on introns. Our genes contain coded instructions for making the molecules in our bodies, but this information must be extensively processed before it can be used. The instructions from each gene are first copied into a molecule called a pre-mRNA, before a process known as splicing removes certain sections to form a mature mRNA molecule. Splicing can remove different sections of the pre-mRNA to make different mRNA molecules from the same gene depending on the current needs of the cell. Splicing is controlled by a combination of proteins and other molecules, collectively called the spliceosome. A part of the spliceosome called U1 recognizes the start of pre-mRNA sections that need to be removed, which is referred to as the five-prime splice site (or “5’ SS” for short). The attachment of U1 to such a site allows other molecules to also attach to the pre-mRNA, which eventually assemble a spliceosome. The very first steps in this process involve U1 and a set of other proteins that create what is called the “Early” or “E” complex. Although there are many molecules involved in the E complex, it was not known how they interact with each other and how this affects which splice sites are used for splicing in different cells. Using advanced microscopy, Larson and Hoskins examined individual U1 molecules from yeast cells while the molecules formed E complexes and identified two different ways U1 can bind to five-prime splice sites. One process involved U1 attaching to pre-mRNA for a short time, whilst the other involved a longer association between U1 and pre-mRNA. Sometimes U1 could also transition between the first process and the second. The results showed that other parts of the E complex affected which process was used at different sites by affecting the type or duration of U1’s attachment. All U1 particles use the same components to attach to splice sites in all pre-mRNAs, but the most used splice sites are not always those that are predicted to have the strongest attachments to U1. This work helps to reveal how other proteins involved in splicing influence this effect, altering U1’s ability to attach to pre-mRNAs to suit each new situation. This also allows cells to change gene splicing to fit different situations. Many genes in our bodies rely on splicing and understanding this process in detail could be the key to diagnosing and treating a range of different illnesses.

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.

Mutations in the splicing machinery have been implicated in a number of human diseases. Most notably, the U2 small nuclear ribonucleoprotein (snRNP) component SF3b1 has been found to be frequently mutated in blood cancers such as myelodysplastic syndromes (MDS). SF3b1 is a highly conserved HEAT repeat (HR)-containing protein and most of these blood cancer mutations cluster in a hot spot located in HR4-8. Recently, a second mutational hotspot has been identified in SF3b1 located in HR9-12 and is associated with acute myeloid leukemias, bladder urothelial carcinomas, and uterine corpus endometrial carcinomas. The consequences of these mutations on SF3b1 functions during splicing have not yet been tested. We incorporated the corresponding mutations into the yeast homolog of SF3b1 and tested their impact on splicing. We find that all of these HR9-12 mutations can support splicing in yeast, and this suggests that none of them are loss of function alleles in humans. The Hsh155V502F mutation alters splicing of several pre-mRNA reporters containing weak branch sites as well as a genetic interaction with Prp2 and physical interactions with Prp5 and Prp3. The ability of a single allele of Hsh155 to perturb interactions with multiple factors functioning at different stages of the splicing reaction suggests that some SF3b1-mutant disease phenotypes may have a complex origin on the spliceosome.

Pre-mRNA splicing is catalyzed in two steps: 5ʹ splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (first and second step factors). We recently identified Fyv6 (FAM192A in humans) as a second step factor in Saccharomyces cerevisiae ; however, we did not determine how widespread Fyv6’s impact is on the transcriptome. To answer this question, we have used RNA sequencing (RNA-seq) to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3ʹ SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only second step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the first step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3ʹ SS.

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.

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.

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. The genes in an organism’s DNA may be expressed to form a protein via an intermediate molecule called RNA. In many organisms including humans, gene expression often begins by making a precursor molecule called a pre-mRNA. The pre-mRNA contains regions called exons that code for the protein product and regions called introns that do not. A machine in the cell called the spliceosome has the job of removing the introns in the pre-mRNA and stitching the exons together by a process known as splicing. The spliceosome is made up of dozens of components that assemble on the pre-mRNAs. Before a newly assembled spliceosome can carry out splicing, it must be activated. The activation process involves several steps that are powered by the cell's universal power source (a molecule called ATP), including the release of many components from the spliceosome. Many of the details of the activation process are unclear. Spliceosomes in the yeast species Saccharomyces cerevisiae are similar to spliceosomes from humans, and so are often studied experimentally. Hoskins et al. have now used a technique called colocalization single molecule fluorescence spectroscopy to follow, in real time, a single yeast spliceosome molecule as it activates. This technique uses a specialized microscope and a number of colored lasers to detect different spliceosome proteins at the same time. Hoskins et al. found that one of the steps during activation is irreversible – once that step occurs, the spliceosome must either perform the next activation steps or start the processes of assembly and activation over again. Hoskins et al. also discovered that ATP causes some spliceosomes to be discarded during activation and not used for splicing. This indicates that before spliceosomes are allowed to activate, they may undergo 'quality control', which may be important for making sure that gene expression occurs efficiently and correctly. Future studies will investigate how this quality control process works in further detail.

Colocalization single-molecule spectroscopy (CoSMoS) enables multiple molecules to be simultaneously monitored. This protocol describes how to construct a CoSMoS micromirror total internal reflection 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.

U6 small nuclear ribonucleoprotein (snRNP) biogenesis is essential for spliceosome assembly, but not well understood. Here, we report structures of the U6 RNA processing enzyme Usb1 from yeast and a substrate analog bound complex from humans. Unlike the human ortholog, we show that yeast Usb1 has cyclic phosphodiesterase activity that leaves a terminal 3′ phosphate which prevents overprocessing. Usb1 processing of U6 RNA dramatically alters its affinity for cognate RNA-binding proteins. We reconstitute the post-transcriptional assembly of yeast U6 snRNP in vitro, which occurs through a complex series of handoffs involving 10 proteins (Lhp1, Prp24, Usb1 and Lsm2–8) and anti-cooperative interactions between Prp24 and Lhp1. We propose a model for U6 snRNP assembly that explains how evolutionarily divergent and seemingly antagonistic proteins cooperate to protect and chaperone the nascent snRNA during its journey to the spliceosome. The mechanism of U6 small nuclear ribonucleoprotein (snRNP) biogenesis is not well understood. Here the authors characterize the enzymatic activities and structures of yeast and human U6 RNA processing enzyme Usb1, reconstitute post-transcriptional assembly of yeast U6 snRNP in vitro, and propose a model for U6 snRNP assembly.