PAPER TYPE EAXTR VIEW Nucleus 5:1, 1–4; January/February 2014; © 2014 Landes Bioscience The timing of pre-mRNA splicing visualized in real-time Maria Carmo-Fonseca1,* and Tomas Kirchhausen2 1Instituto de Medicina Molecular; Faculdade de Medicina; Universidade de Lisboa; Lisboa, Portugal; 2Departments of Cell Biology and Pediatrics; Harvard Medical School and Program in Molecular and Cellular Medicine at Boston Children’s Hospital; Boston, MA USA ince it became clear that intervening isolated RNA molecules can hybridize Ssequences or introns are spliced out to double-stranded DNA by displacing from precursor pre-mRNA molecules one of the DNA strands.1 Using this in the nucleus before mature mRNAs approach, the Roberts and Sharp teams distribute. are exported to the cytoplasm, independently realized that adenoviral questions were raised about the timing polysomal RNAs do not hybridize in a of splicing. Does splicing start while continuous manner with the DNA from not RNA polymerase II is still transcribing? which they are transcribed. Rather, the Is splicing a slow or a fast process? Is mRNA sequence was complementary to Do timing important to control the splicing multiple noncontiguous regions of the reaction? Although our understanding DNA.2,3 They concluded that adenoviral on the mechanism and function of genes were split into several pieces, and splicing is largely based on data obtained because adenovirus is a mammalian using biochemical and large-scale virus, it seemed likely that at least some “omic” approaches, microscopy has been mammalian genes could have a similar instrumental to address questions related structure. This was soon found to be the to timing. Experiments done with the case. Although different mechanisms electron microscope paved the way to could result in joining together certain Bioscience. the discovery of splicing and provided segments of genomic sequence into mature unequivocal evidence that splicing mRNA, evidence quickly accumulated can occur co-transcriptionally. More supporting a model for mRNA synthesis recently, live-cell microscopy introduced that involves splicing of a larger precursor a technical breakthrough that allows molecule or pre-mRNA. real-time visualization of splicing Understanding the splicing mechanism Landes dynamics. We discuss here some of the required the development of cell-free microscopy advances that provided the systems where conditions could be Keywords: splicing, transcription, basis for the current conceptual view of controlled and the components purified RNA, lifetime; live-cell imaging, single- the splicing process, and we outline a and reconstituted. In vitro splicing assays molecule visualization most recent development that permits based on extracts obtained by moderate *Correspondence to: Maria Carmo-Fonseca; direct measurement, in living cells, of salt solubilization of lysed cells or crude ©2014 Email: [email protected] the time it takes to synthesize and excise nuclei were able to accurately splice Submitted: 01/05/2014; Revised: an intron from individual pre-mRNA exogenously added pre-mRNA, indicating 01/26/2014; Accepted: 01/30/2014; molecules. that splicing and transcription are Published Online: 01/30/2014 In the summer of 1977, a set of electron independent processes (for a review see ref. http://dx.doi.org/10.4161/nucl.28056 micrographs shown at the annual Cold 4). Yet, visualizing dispersed chromatin by Spring Harbor Symposium revolutionized electron microscopy showed that splicing Extra View to: Martin RM, Rino J, Carvalho C, Kirchhausen T, Carmo-Fonseca M. Live-cell the way molecular biologists perceived was coupled to transcription in vivo. Back visualization of pre-mRNA splicing with single- the structure of genes. Following the in 1969, Miller and Beatty reported that molecule sensitivity. Cell Rep 2013; 4:1144-55; development of methodologies to observe hypotonic lysis of cells released chromatin PMID:24035393; http://dx.doi.org/10.1016/j. isolated RNA and DNA molecules by from the nucleus.5 The released chromatin celrep.2013.08.013 electron microscopy, it was found that was then centrifuged onto a grid for www.landesbioscience.com Nucleus 1 observation in the electron microscope. The pioneer method relied on synthesis waiting for the opportunity to assemble This procedure resulted in a loosened of radioactive RNA complementary to a new spliceosome. Since actively spliced two-dimensional array of chromatin the DNA of interest. Cells were made genes are intimately associated with fibers that maintained the nucleosomal permeable and incubated with the the periphery of nuclear speckles, one structure. Nascent transcripts remained radioactive probe, resulting in formation of possibility is that clustering of spliceosomal attached to the chromatin and appeared RNA-DNA hybrids. The location of these components in dedicated compartments as ribonucleoprotein fibers extending hybrids was then detected at the cytological increases their local concentration in the from the chromatin backbone. The level by autoradiography.11,12 Over the next neighborhood of nascent transcripts, thus length of nascent transcripts, which could 30 years, this technology progressed from enhancing spliceosome assembly on newly be precisely measured, increased with a laborious, time-consuming method synthesized introns.21 This could be a increasing distance from the transcription limited to the detection of highly abundant reason why splicing is so much faster in start site. Using this approach, Beyer and nucleic acid species with low cytological vivo than in vitro. Osheim showed that the spliceosome resolution, to a relatively fast, highly An imaging breakthrough was formed shortly after synthesis of the 3′ precise, and sensitive imaging of single introduced by the development of splice site and that splicing of pre-mRNA genes and RNA molecules in the cell. An genetically encoded fluorescent tags often occurred on the nascent transcript.6 important advance was the optimization that combined with fluorescence-based This visual demonstration that introns of the in situ hybridization procedure microscopic approaches of increasingly could be removed prior to transcript release for fluorescent detection of nuclear RNA higher spatial and temporal resolution, from the template and thus presumably using biotin-labeled DNA probes and making it possible to analyze protein distribute. prior to polyadenylation challenged the fluorophore-conjugated avidin.13 Next, movement in living cells. Time-series prevailing textbook view that splicing the method was further optimized for recordings of cells expressing the green took place after polyadenylation. Beyer the simultaneous detection of DNA, fluorescent protein (GFP) fused to not and Osheim assumed an elongation rate unspliced precursors, and spliced mRNA. an essential splicing protein provided of 1500 nucleotides per minute to then Using these tools, several laboratories direct evidence that nuclear speckles Do deduce that splicing of introns removed showed that spliced mRNA localized in supply spliceosomal components to cotranscriptionally occurred within close proximity to the DNA from which nearby activated sites of transcription.22 3 min after synthesis of the 3′ splice it was transcribed, consistent with the Subsequent developments of methods site. Similar conclusions were obtained view that many introns are excised while such as FRAP (fluorescence recovery after when studying actively transcribed genes the pre-mRNA is still tethered to the gene photobleaching) unravelled the kinetic in Chironomus tentans salivary gland locus via RNA polymerase.14-16 There properties of splicing proteins in the polytene chromosomes, which can be are, however, situations where splicing is nucleus of live cells.23 FRAP revealed that isolated by microdissection for direct uncoupled from transcription: in the case spliceosomal proteins are continuously 7 observation in the electron microscope. In of regulated alternative splicing events, moving in the nucleus, shuttling in and Bioscience. vivo splicing rates in the range of 5 min or intron removal can be delayed until after out of nuclear speckles within seconds. less were also measured biochemically for release of the pre-mRNA from the site Surprisingly, trafficking kinetics was adenovirus and β-globin transcripts.8-10 of transcription.17 In situ hybridization independent of ongoing transcription Such rates differed substantially from the procedures were also optimized to localize and splicing, challenging the view that results obtained in cell-free assays, where a the small nuclear RNAs that form the spliceosomal components are stored in 18 lag period of 15–45 min was observed at building blocks of the spliceosome. nuclear speckles until a signal triggers their Landes the start of the reaction before the onset By combining in situ hybridization and recruitment to nascent introns; rather, it of splicing, following which spliced RNA immuno-labeling for fluorescent and is more likely that the building blocks of accumulated for 2–3 h.4 Thus, splicing electron microscopic visualization, it the spliceosome are constantly roaming in vivo is at least 10-fold more rapid than became evident that spliceosomal small the nuclear space until they collide and in vitro. This discrepancy highlights nuclear ribonucleoproteins and splicing transiently interact with either a nascent the importance of nuclear organization: protein factors accumulated in dedicated pre-mRNA, to form a spliceosome, or with ©2014 most