366a Tuesday, February 20, 2018 Symposium: RNA Structure and Function have played a crucial role in eukaryotic evolution as the ancestors of nuclear premessenger introns and their splicing machinery (the spliceosome). A hall- 1805-Symp mark of group II introns, which they share with spliceosomal introns, is their Coupling between Transcription & Splicing Tunes Gene Expression excision as branched molecules called ‘lariats’. This particular conformation Karla Neugebauer. results from a specific 2’-5’ phosphodiester bond between a conserved intron Yale University, New Haven, CT, USA. adenosine and the first intron nucleotide, most often a guanosine. Most bacterial RNA is synthesized by RNA polymerases that travel along template DNA. group II introns encode a reverse transcriptase enzyme that associates with the The growing RNA polymers are referred to as nascent. As transcription pro- intron to promote its genomic mobility through ‘retrotransposition’. This ceeds, nascent RNA grows in length and sequence complexity, increasing op- mobility pathway is initiated by ‘reverse splicing’ of the excised intron lariat portunity for folding and interacting with RNA binding proteins. In into a DNA target. eukaryotes, the capping enzymes modify the mRNA 5’ end, the spliceosome Our recent crystal structures of a group II intron lariat reveal for the first assembles and removes introns, and the polyadenylation machinery cleaves 3’ time that the 2’-5’ branch structure organizes the intron active site for effi- ends to initiate polyadenylation of mRNA and terminate transcription. All of cient and accurate ligation of the flanking exons during the last stage of this takes place in a nascent RNP, which lies close to the DNA axis. I will splicing. Moreover, after the release of the ligated exons, the terminal 3’-hy- illustrate how recent single molecule RNA-Seq strategies have revealed the droxyl group of the excised lariat remains docked in the reaction center, in a in vivo kinetics of pre-mRNA splicing relative to the progress of RNA poly- configuration poised for catalysis of the reverse-splicing reaction, the first merase II (Pol II). In budding and fission yeast, the spliceosome can act on step in retrotransposition. Interestingly, these structures also reveal that introns as soon as they emerge from Pol II, showing that splicing and tran- active-site assembly and catalysis are under tight conformational control: scription rates are matched. This predicts that introns present in multi- (1) a rearrangement of the base-pairing pattern within the adenosine- intron transcripts may be removed in the order of their transcription. Instead, branchpoint helix promotes docking of the 2’-5’ branch into the active sequencing of full-length nascent RNA molecules in S. pombe revealed that site, (2) binding of the 5’-exon drives an induced fit that contributes to intron removal is equally likely to occur ‘‘not in order’’, showing regulation. coordination of one of the two catalytic metal ions. Finally, this work ex- Surprisingly, partially spliced transcripts were rare; most nascent RNA de- tends the parallels between group II introns and the spliceosome by suggest- tected was either fully spliced or unspliced, indicating that splicing of any ing new homologies between active-site nucleotides conserved in both given intron may depend on the splicing status of the other introns in the tran- systems. script. Fully unspliced transcripts failed to cleave at the polyA site, underwent transcriptional read-through at gene 3’ends, and were degraded by the exo- 1808-Symp some. These observations suggest crosstalk among spliceosomes and 3’end A Solid View on RNA: Solid-State NMR of RNA and RNP Complexes processing machineries assembling on the same nascent transcript. I will Alexander Marchanka, Mumdooh Ahmed, Teresa Carlomagno. discuss an example of regulated gene expression in which reduced splicing Leibniz University of Hanover, Hanover, Germany. leads to lower reduced mRNA levels. These findings highlight coordination To date, substantial progresses have been made in the structure determination between transcription and RNA processing steps along the pathway of gene of membrane proteins and amyloid fibrils by solid-state NMR, while signifi- expression. cantly fewer studies have addressed the structure of RNA or protein-RNA com- plexes (RNP). Nevertheless, the application of ssNMR to study large RNP 1806-Symp complexes holds excellent promises, due to the independence of the ssNMR Cryo-Em Snapshots of the Spliceosome line widths from the molecular size. Here, I will present the ssNMR-based Kiyoshi Nagai. structure of the complex consisting of the the 26mer box C/D RNA and the pro- MRC Laboratory of Molecular Biology, Cambridge, United Kingdom. tein L7Ae, together with the experimental strategy that we used to obtain it. In the past two years we have determined near atomic resolution structures This includes experiments for the assignment of RNA resonances and collec- of yeast spliceosomes in several key intermediate states. These structures tion of structural restraints, as well as experiments to determine the protein- have begun to reveal the detailed mechanical working of this intricate mo- RNA contacts. In addition I will discuss the application of proton-detection lecular machine. The yeast pre-catalytic B complex structure (1) shows in ssNMR of RNA. that U2 snRNP is held over the tri-snRNP by U2/U6 helix II and the Finally, I will show first ssNMR spectra of a RNA in the context of the 400 kDa branch helix is bound to SF3b. The single stranded region of U4 snRNA box C/D RNP. is already bound to the active site of the Brr2 helicase ready to unwind the U4/U6 snRNA duplex suggesting how U6 snRNA freed from U4 Symposium: Interrogating Membrane snRNA may fold to form the active site. In the C complex structure imme- diately after the first trans-esterification reaction (2) the cleaved 5’exon is Organization and Dynamics held between the Large and N-terminal domains of Prp8 and the 5’-phos- phate of the first intron nucleotide (G1) positioned at the catalytic center is 1809-Symp linked to the branch point adenosine through the 2’-5’ phosphodiester link- Insight Into Plasma Membrane Organization Acquired with Secondary age. The branch helix is docked into the active site by step 1 factors. The Ion Mass Spectrometry (SIMS) Prp16 helicase located near the intron exit site is poised to release the step Mary L. Kraft. 1 factors and the branch helix. The C* complex structure after Prp16 ac- Chem & Biomol Engr, University of Illinois, Urbana, IL, USA. tion (3) shows that the dissociation of step 1 factors from the active site The plasma membrane is compartmentalized into different domains that induce conformational change of the active site to create a space for the have specialized compositions and functions. Identifying the compositions incoming 3’exon. The RNA-based catalytic core of the spliceosome of these domains, the mechanisms that establish them, and how they relate formed during the B to Bact transition remains unchanged throughout to cellular function is currently a major challenge. Significant advances in the catalytic phase of the splicing and catalyzes both branching and imaging and biotechnology have enabled imaging the distributions of pro- exon-ligation and . The DEAH-box RNA helicases and step specific factors teins in cell membranes with unprecedented spatial resolution and speci- regulate docking and undocking of the substrates (branch site and 3’ splice ficity. Although the cellular abundances of cholesterol, sphingolipids, and site) to the single RNA-based active site to catalyze the two trans- other lipid species influence many important biological processes, less is esterification reactions. known about the distributions of cholesterol and distinct lipid species within the plasma membrane. We are addressing this issue with a high-resolution 1807-Symp secondary ion mass spectrometry (SIMS) approach for imaging isotope- Crystal Structures of a Group II Intron Lariat and Implications for the labeled cholesterol and sphingolipids in parallel with immunolabeled pro- Spliceosome teins in the plasma membranes of mammalian cells with %100-nm-lateral Maria Costa1,Hele`ne Walbott1, Dario Monachello1, Eric Westhof2, resolution. With this approach, we have found that sphingolipids are concen- Franc¸ois Michel1. trated in micrometer-scale domains that are dependent on the cytoskeleton, 1Institute for Integrative Biology of the Cell - CNRS, CEA, University Paris- but cholesterol is evenly distributed within the plasma membrane. We Sud, University Paris-Saclay, Gif-sur-Yvette, France, 2Institute of Molecular have also used this approach to image the distribution of cholesterol, sphin- and Cellular Biology of the CNRS, Strasbourg, France. golipids, and influenza viral envelope proteins in the plasma membranes of Group II introns are catalytic RNAs that can excise by themselves from precur- cells infected with the influenza virus. These experiments are a direct test of sor RNA molecules. These large ribozymes of bacterial origin are believed to the long-standing hypotheses that influenza virus assembles and buds from BPJ 8646_8657 Tuesday, February 20, 2018 367a domains enriched with cholesterol and sphingolipids. The results of these ex- Platform: Intrinsically Disordered Proteins (IDP) periments will be presented. and Aggregates I 1810-Symp Computational Modeling of Realistic Cell Membranes 1813-Plat Siewert J. Marrink. Inferring Properties of Disordered Chains From FRET Transfer