Home
This Title All WIREs
WIREs RSS Feed
How to cite this WIREs title:
WIREs RNA
Impact Factor: 4.928

Lights, camera, action! Capturing the spliceosome and pre‐mRNA splicing with single‐molecule fluorescence microscopy

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

The process of removing intronic sequences from a precursor to messenger RNA (pre‐mRNA) to yield a mature mRNA transcript via splicing is an integral step in eukaryotic gene expression. Splicing is carried out by a cellular nanomachine called the spliceosome that is composed of RNA components and dozens of proteins. Despite decades of study, many fundamentals of spliceosome function have remained elusive. Recent developments in single‐molecule fluorescence microscopy have afforded new tools to better probe the spliceosome and the complex, dynamic process of splicing by direct observation of single molecules. These cutting‐edge technologies enable investigators to monitor the dynamics of specific splicing components, whole spliceosomes, and even cotranscriptional splicing within living cells. WIREs RNA 2016, 7:683–701. doi: 10.1002/wrna.1358 This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Methods > RNA Analyses in Cells
Structural insights into U6 snRNA conformational changes during splicing. (a, top) Structure of a portion of the yeast U6 snRNA (red) bound to yeast Prp24 (gray). (a, bottom) Proposed basepairing structure of the yeast U6 snRNA. Darker regions are those observed in the U6/Prp24 crystal structure, lighter regions were either not observed or crystallized. (b, top) Cryo‐EM structure of the yeast tri‐snRNP highlighting the U6 (red) and U4 (orange) snRNAs. (b, bottom) Proposed basepairing structure of the yeast U4/U6 di‐snRNA. Darker regions are those modeled in the cryo‐EM structure at top, lighter regions were either not observed or modeled. (c, top) Cryo‐EM structure of the S. pombe U2/U6.U5 spliceosomal complex highlighting the U6 (red) and U2 (green) snRNAs. (c, bottom) Proposed basepairing structure of the S. cerevisiae U2/U6 snRNA. The boxed region represents helix III, which was not observed in the S. pombe spliceosome structure. Structures in (a)–(c) are all shown to scale. Figures for (a) and (c) were prepared from 4NOT.pdb and 3JB9.pdb, respectively. Coordinates for the tri‐snRNP model in (b) were generously provided by K. Nagai (MRC, Cambridge, UK).
[ Normal View | Magnified View ]
Studies of splicing in fixed and live cells. (a) Schematic of the smFISH‐based strategy developed by Vargas et al. to study cotranscriptional splicing in fixed cells. Reporter RNAs were labeled by insertion of repetitive sequences that can be targeted by multiple fluorescent, complementary oligos. (b) Microscopic images (left) and colocalization data (right) obtained by Vargas et al. using the approach described in (a). Bright spots correspond to single transcripts bound to the fluorescent probes. Red circles in the colocalization data denote mRNA locations, green circles represent processed introns, and yellow circles represent unspliced pre‐mRNAs or spliceosome‐bound mRNA/intron complexes. The nuclear boundary is shown with a blue, dashed line. (c) Schematic of the RNA binding protein‐based strategy developed by Coulon et al. to study cotranscriptional splicing in live cells. Reporter RNAs were labeled by insertion of MS2 or PP7 stemloops that can be targeted by PP7‐mCherry or MS2‐GFP fusion proteins. (d) Microscopic images (top) and fluorescence time trajectories (bottom) obtained by Coulon and coworkers using the approach described in (c). In the microscopic images (top), a pre‐mRNA or spliceosomal complex containing both an intron and 3′ UTR signal is identified by colocalization of the green and red spots of fluorescence (arrow head). In the fluorescence trajectories (bottom), multiple rounds of transcription and splicing can be observed (peaks). In some cases (peaks B and C), the red, intronic signal disappeared before the green 3′ UTR signal, consistent with cotranscriptional splicing. In other cases (peak A), both signals disappeared simultaneously consistent with the completion of splicing after the end of transcription. Data in (b) and (d) were obtained from Refs and , respectively, and are used with permission.
[ Normal View | Magnified View ]
Insights into spliceosomes from single‐molecule fluorescence. (a) Using a combination of labeled snRNPs, pre‐mRNA substrates, and CoSMoS, Shcherbakova et al. were able to show that functional yeast spliceosomes can assemble via either U1‐first or U2‐first pathways. (b) smFRET experiments from Krishnan and coworkers revealed pre‐mRNA dynamics that persist after activation of the spliceosome by Prp2. Cwc25 is hypothesized to bias the conformational dynamics of the spliceosome toward a structure in which the 5′ SS and BS are proximal, thus promoting the first chemical step in splicing. (c) By making use of purified spliceosomes and dcFCCS, Ohrt et al. were able to study the binding properties of a number of splicing factors as the spliceosome progressed through activation and the chemical steps of splicing. In some cases, Ohrt et al. were able to use dcFCCS to study the dynamics of GFP labeled proteins (e.g., Cwc24) while in other cases the proteins were labeled with organic fluorophores (e.g., Cwc25). Prp2 activity destabilizes a number of splicing factors while promoting Cwc25 association, which in turn facilitates 5′ SS cleavage. [Adapted from Refs , and ].
[ Normal View | Magnified View ]
Insights into RNA dynamics from smFRET of model systems. (a) Using a model for the U2 snRNA stem II region (green), Rodgers et al. were able to show that the RNA toggles spontaneously between IIc and IIa conformations. Addition of Mg2+ or Cus2 protein strongly promoted accumulation of IIa conformers. (b) By employing a model for the U2 (green)/U6(red) di‐snRNA, Guo et al. revealed Mg2+ dynamics that may correspond to switching of the RNA model between 3‐helix and 4‐helix structures. (c) Hardin et al. used a model for the U4 (orange)/U6(red) di‐snRNA to show that, despite few intrinsic dynamics, addition of U4/U6 di‐snRNP proteins (Prp3, 4, and 31) were able to change the relative orientation of the U4 5′ stem loop (SL) and U4/U6 stem II. (d) Lamichhane et al. developed a smFRET‐based RNA looping assay to study the interaction of a fragment of PTB with labeled RNAs.
[ Normal View | Magnified View ]
Schematic overview of the major stages in splicing. Splicing occurs through distinct stages of assembly, activation, catalysis, product release, and recycling of the splicing machinery. During assembly, the snRNPs (U1, U2, and the U4/U6.U5 tri‐snRNP) assemble on a pre‐mRNA substrate to form the spliceosomal B complex. During activation, U1 and U4 are expelled, and the Prp19‐associated complex (NTC) joins the spliceosome. After action of the ATPase Prp2, the first chemical step of splicing occurs, and the spliceosomal C complex is formed. After exon ligation, mRNA and lariat intron products are released from the spliceosome and the splicing machinery is recycled to begin a new round of splicing on another intron.
[ Normal View | Magnified View ]
Overview of single‐molecule fluorescence techniques. (a) Illustration of simple, tethered molecule smFRET (top) and colocalization (bottom) single‐molecule fluorescence experiments. In smFRET, donor (green trace and star) and acceptor (red trace and star) fluorescence intensities are anti‐correlated and can be used to calculate EFRET. In colocalization experiments, peaks of fluorescence intensity (green) represent binding events occurring between a freely diffusing molecule and a tethered substrate. (b) Illustration of a single‐molecule experiment carried out with a freely diffusing fluorescent molecule and using confocal microscopy. As the molecule passes through the confocal volume (shown), fluorescence can be detected. Many individual events can be studied using correlation analysis to produce a plot of correlation [G(τ)] versus a given time constant. The shape of the correlation curve is indicative of the diffusive properties of the molecule as well as molecular concentration. (c) In cells, single RNA transcripts can be imaged by targeting many fluorophores (n = dozens or hundreds of fluorescent molecules) to the RNA. In live cells, this can be accomplished by encoding a binding site for an RNA binding protein‐FP fusion (top). In fixed cells, transcripts can be detected by hybridization of complementary, fluorescent oligos.
[ Normal View | Magnified View ]

Related Articles

Techniques for following the movement of single RNAs in living cells
RNA biology in a test tube—an overview of in vitro systems/assays
Pre‐mRNA splicing during transcription in the mammalian system

Browse by Topic

RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry
RNA Methods > RNA Analyses in Cells

Access to this WIREs title is by subscription only.

Recommend to Your
Librarian Now!

The latest WIREs articles in your inbox

Sign Up for Article Alerts