Nan-Sheng Li

RNA catalyses nuclear pre-mRNA splicing

In nuclear pre-messenger RNA splicing, introns are excised by the spliceosome, a dynamic machine composed of both proteins and small nuclear RNAs (snRNAs). Over thirty years ago, after the discovery of self-splicing group II intron RNAs, the snRNAs were proposed to catalyse splicing. However, no definitive evidence for a role of either RNA or protein in catalysis by the spliceosome has been reported so far. By using metal rescue strategies in spliceosomes from budding yeast, here we show that the U6 snRNA catalyses both of the two splicing reactions by positioning divalent metals that stabilize the leaving groups during each reaction. Notably, all of the U6 catalytic metal ligands we identified correspond to the ligands observed to position catalytic, divalent metals in crystal structures of a group II intron RNA. These findings indicate that group II introns and the spliceosome share common catalytic mechanisms and probably common evolutionary origins. Our results demonstrate that RNA mediates catalysis within the spliceosome.

A G-quadruplex–containing RNA activates fluorescence in a GFP-like fluorophore

Spinach is an in vitro selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence.Spinach is thus an RNA analog of GFP, and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2 and 2.4 Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially pre-formed binding site for the fluorophore.The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.

Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pKa = 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5′ oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolate-modified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2′-O nucleophile by G638 and protonation of the 5′-O leaving group by A756.

Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design

Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a “monobody,” a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with Kd values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

Metal-ion rescue revisited: Biochemical detection of site-bound metal ions important for RNA folding

Within the three-dimensional architectures of RNA molecules, divalent metal ions populate specific locations, shedding their water molecules to form chelates. These interactions help the RNA adopt and maintain specific conformations and frequently make essential contributions to function. Defining the locations of these site-bound metal ions remains challenging despite the growing database of RNA structures. Metal-ion rescue experiments have provided a powerful approach to identify and distinguish catalytic metal ions within RNA active sites, but the ability of such experiments to identify metal ions that contribute to tertiary structure acquisition and structural stability is less developed and has been challenged. Herein, we use the well-defined P4-P6 RNA domain of the Tetrahymena group I intron to reevaluate prior evidence against the discriminatory power of metal-ion rescue experiments and to advance thermodynamic descriptions necessary for interpreting these experiments. The approach successfully identifies ligands within the RNA that occupy the inner coordination sphere of divalent metal ions and distinguishes them from ligands that occupy the outer coordination sphere. Our results underscore the importance of obtaining complete folding isotherms and establishing and evaluating thermodynamic models in order to draw conclusions from metal-ion rescue experiments. These results establish metal-ion rescue as a rigorous tool for identifying and dissecting energetically important metal-ion interactions in RNAs that are noncatalytic but critical for RNA tertiary structure.

Synthesis of pyridine, pyrimidine and pyridinone C-nucleoside phosphoramidites for probing cytosine function in RNA.

In the structures of the HDV ribozyme a cytosine nucleobase resides at the active site poised to participate directly in catalysis. Defining the functional role of the nucleobase requires nucleoside analogues that perturb the functional groups in a strategic manner. Herein, we have developed efficient methods for the synthesis of five C-nucleoside phosphoramidite derivatives that, when used in combination, provide strategies for probing the potential functional role of cytosines keto group and imino nitrogen. Phosphoramidites 15a and 15b were synthesized in 11 steps starting from 2-amino-5-bromopyrimidine (1a) and 2-amino-5-bromopyridine (1b), respectively, with overall yields of 10.8% and 6.6%, respectively. Phosphoramidite 21 was prepared from intermediate 11b in seven steps with an overall yield of 33.7%. Phosphoramidites 23 and 25 were prepared from 2,4-diamino-5-(beta-d-ribofuranosyl)-1,3-pyrimidine (22) and pseudoisocytidine (24), respectively, with an overall yield of 15.9% (six steps) and 37.9% (four steps), respectively. These phosphoramidites were incorporated into oligonucleotides by solid-phase synthesis.

Molecular Analysis of Lipid-Reactive Vδ1 γδ T Cells Identified by CD1c Tetramers

CD1c is abundantly expressed on human dendritic cells (DC) and B cells, where it binds and displays lipid Ags to T cells. In this study, we report that CD1c tetramers carrying Mycobacterium tuberculosis phosphomycoketide bind γδ TCRs. An unbiased method of ligand-based TCR selection detects interactions only with Vδ1+ TCRs, and mutational analyses demonstrate a role of the Vδ1 domain during recognition. These results strengthen evidence for a role of CD1c in the γδ T cell response, providing biophysical evidence for CD1c–γδ TCR interactions and a named foreign Ag. Surprisingly, TCRs also bind CD1c complexes formed with diverse lipids such as lysophosphatidylcholine, sulfatide, or mannosyl-phosophomycoketide, but not lipopeptide ligands. Dissection of TCR interactions with CD1c carrying foreign Ags, permissive ligands, and nonpermissive lipid ligands clarifies the molecular basis of the frequently observed but poorly understood phenomenon of mixed self- and foreign Ag reactivity in the CD1 system.

Highly Stereocontrolled Total Synthesis of β-d-Mannosyl Phosphomycoketide: A Natural Product from Mycobacterium tuberculosis

β-D-mannosyl phosphomycoketide (C32-MPM), a naturally occurring glycolipid found in the cell walls of Mycobacterium tuberculosis, acts as a potent antigen to activate T-cells upon presentation by CD1c protein. The lipid portion of C32-MPM contains a C32-mycoketide, consisting of a saturated oligoisoprenoid chain with five chiral methyl branches. Here we develop several stereocontrolled approaches to assemble the oligoisoprenoid chain with high stereopurity (>96%) using Julia-Kocienski olefinations followed by diimide reduction. By careful choice of olefination sites, we could derive all chirality from a single commercial compound, methyl (2S)-3-hydroxy-2-methylpropionate (>99% ee). Our approach is the first highly stereocontrolled method to prepare C32-MPM molecule with >96% stereopurity from a single >99% ee starting material. We anticipate that our methods will facilitate the highly stereocontrolled synthesis of a variety of other natural products containing chiral oligoisoprenoid-like chains, including vitamins, phytol, insect pheromones, and archaeal lipids.

Arginine as a General Acid Catalyst in Serine Recombinase-mediated DNA Cleavage

Background: Sin resolvase is a site-specific DNA recombinase that catalyzes phosphotransfer without the use of divalent cations. Results: Mutation of Arg-69 in the active site is partially rescued by a 3′ phosphorothiolate substrate. Conclusion: Arg-69 is linked to protonation of the leaving group and most likely acts as a general acid catalyst. Significance: Serine recombinases employ a different catalytic strategy than most other phosphoryl transfer enzymes. Members of the serine family of site-specific DNA recombinases use an unusual constellation of amino acids to catalyze the formation and resolution of a covalent protein-DNA intermediate. A recent high resolution structure of the catalytic domain of Sin, a particularly well characterized family member, provided a detailed view of the catalytic site. To determine how the enzyme might protonate and stabilize the 3′O leaving group in the strand cleavage reaction, we examined how replacing this oxygen with a sulfur affected the cleavage rate by WT and mutant enzymes. To facilitate direct comparison of the cleavage rates, key experiments used suicide substrates that prevented religation after cleavage. The catalytic defect associated with mutation of one of six highly conserved arginine residues, Arg-69 in Sin, was partially rescued by a 3′ phosphorothiolate substrate. We conclude that Arg-69 has an important role in stabilizing the 3′O leaving group and is the prime candidate for the general acid that protonates the 3′O, in good agreement with the position it occupies in the high resolution structure of the active site of Sin.

A general and efficient approach for the construction of RNA oligonucleotides containing a 5′-phosphorothiolate linkage

Oligoribonucleotides containing a 5′-phosphorothiolate linkage have provided effective tools to study the mechanisms of RNA catalysis, allowing resolution of kinetic ambiguity associated with mechanistic dissection and providing a strategy to establish linkage between catalysis and specific functional groups. However, challenges associated with their synthesis have limited wider application of these modified nucleic acids. Here, we describe a general semisynthetic strategy to obtain these oligoribonucleotides reliably and relatively efficiently. The approach begins with the chemical synthesis of an RNA dinucleotide containing the 5′-phosphorothiolate linkage, with the adjacent 2′-hydroxyl group protected as the photolabile 2′-O-o-nitrobenzyl or 2′-O-α-methyl-o-nitrobenzyl derivative. Enzymatic ligation of the 2′-protected dinucleotide to transcribed or chemically synthesized 5′ and 3′ flanking RNAs yields the full-length oligoribonucleotide. The photolabile protecting group increases the chemical stability of these highly activated oligoribonucleotides during synthesis and long-term storage but is easily removed with UV irradiation under neutral conditions, allowing immediate use of the modified RNA in biochemical experiments.