Spliceosomal Prp24 Unwinds A Minimal U2/u6 Complex From Yeast

Splicing plays a major role in eukaryotic gene expression by processing pre-mRNA to form mature mRNA. Pre-mRNAs undergo splicing to remove introns, non–protein coding regions, and religate exons, protein coding regions. This process is catalyzed by the spliceosome, which consists of five small nuclear ribonucleoprotein particles (snRNPs: U1, U2, U4, U5 and U6) and numerous protein factors. Proper assembly of spliceosomal components is critical for function, and thus, defects in assembly can be lethal. Several spliceosomal proteins facilitate structural rearrangements important for spliceosomal assembly and function. Prp24 is an essential factor in U6 snRNP assembly, and it has been proposed to assist in U4/U6 formation and unwinding. Here, we address the question whether Prp24 affects the U2/U6 complex dynamics. Using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we have previously shown that a minimal U2/U6 complex from yeast can adopt at least three distinct conformations in dynamic equilibrium. Our new single molecule data show that Prp24 unwinds U2 from U2/U6 complex and stabilizes U6 in a low FRET conformation. We also show that the RNA Recognition Motifs of Prp24 affect the binding affinity of Prp24 for U6 and unwinding activity. We propose that Prp24 plays an important role in U2 and U6 snRNP recycling by dissociating the U2/U6 complex

Conformational features of the human U2-U6 snRNA complex

The splicing of precursor messenger (pre-m) RNA, during which noncoding intervening sequences are excised and flanking coding regions ligated, is an integral reaction of gene expression. In eukaryotes, it is carried out by a dynamic RNA-protein complex called the spliceosome, in which five small nuclear (sn) RNA components are actively involved in recognition and chemical aspects of the process. A complex formed between U2 and U6 snRNAs is implicated in the chemistry of pre-mRNA splicing. The catalytic activity of the U2-U6 snRNA complex is dependent on the presence of Mg2+ ions, and the complex has been shown to have several specifically bound Mg2+ binding sites in vitro. The overall goal of this research is to characterize the conformational changes of the human U2-U6 snRNA complex upon addition of Mg2+. In order to pursue this question, we attempted to characterize the lowest energy structure of the complex in the absence of spliceosomal proteins using a combination of biophysical and biochemical techniques in the solution state. We first used enzymatic structure probing to evaluate the secondary structural fold of protein-free human U2-U6 snRNA complex. Cleavage patterns resulting from probing reactions were consistent with formation of four stem regions surrounding the junction, therefore favoring the four-helix model consistent with previous results of in vivo studies of the human U2-U6 snRNA complex. However, 19F NMR studies from our laboratory also identified a lesser fraction (up to 14%) of a three- helix conformation. Upon addition of up to 100 mM Mg2+, a slight increase in cleavage by enzymes specific for both single-stranded and double-stranded regions was observed at the junction region, suggesting that this region is becoming more accessible, perhaps because of an increase in the fraction of the three-helix conformation. Analytical ultracentrifugation studies revealed that the Stokes radius of the RNA complex decreased slightly from 31.3 Ã? to 27.9 Ã? in the presence of 100 mM Mg2+, suggesting a slight compaction of the tertiary structure in the presence of divalent metal ions. Hydroxyl radical footprinting experiments on this complex showed signs of increased protection in some areas near and more distant from the junction upon addition of Mg2+, suggesting a change in three-dimensional conformation. Therefore, it appears that Mg2+ induces a small three-dimensional conformational change on human U2-U6 snRNA complex. In order to build a three-dimensional model for the four-helix conformation, we designed a mutant that favors the formation of four-helix conformation and performed SAXS experiments on it. The preliminary SAXS studies suggest that the human U2-U6 snRNA complex and the mutant complex may also be amenable to further study by SAXS. These results act as a good starting point to characterize further the overall global conformation of human U2-U6 snRNA complex and effects of spliceosomal proteins on it

Single Molecule Studies Of Spliceosomal Snrnas U2-U6

Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5 and U6) involved in substrate positioning and catalysis. It has been postulated, but never shown experimentally, that the U2-U6 complex adopts at least two conformations that reflect different activation states. We have used single-molecule fluorescence to probe the structural dynamics of a protein-free RNA complex modeling U2-U6 from yeast and mutants of highly conserved regions of U2-U6. Our data show the presence of at least three distinct conformations in equilibrium. The minimal folding pathway consists of a two-step process with an obligatory intermediate. The first step is strongly magnesium dependent, and we provide evidence suggesting that the second step corresponds to the formation of the genetically conserved helix IB. Site-specific mutations in the highly conserved AGC triad and the U80 base in U6 suggest that the observed conformational dynamics correlate with residues that have an important role in splicing. We also report the first direct structural evidence that supports the existence of the base triples in the spliceosomal snRNA U2/U6. These interactions were proposed according to a corresponding set of base triple interactions discovered in the recent published crystal structure of the self-splicing group II intron.19-16, 24b-17, 48 We proposed that these base triples existing in the spliceosomal RNA U2/U6 complex are in the same family of the ones found in crystal structure of the self-splicing group II intron given the extensive similarities between the spliceosome and the group II intron. Our data agree very well with the hypothesis. There are a large number of proteins in the spliceosome that play vital structural and catalytic roles3,36. RNA Chaperones, like Prp24, help the structural rearrangements of spliceosomal snRNAs. We report single molecule FRET data showing that spliceosomal protein Prp24 can induce a conformational change in U2/U6 complex. This RNA:protein interaction is Mg2+ and protein concentration dependent and inhibits the binding of Mg2+ to U2/U6

snRNA Catalysts in the Spliceosome’s Ancient Core

The spliceosome, an assembly of snRNAs and proteins, catalyzes the removal of introns from premessenger RNAs. A new study identifies specific phosphates in the U2-U6 snRNA complex that position two catalytic metals. Remarkably, these correspond precisely to metal-binding phosphates in a homologous structure of Group II self-splicing introns, long proposed to be the ribozyme progenitor of spliceosome

Conformation of the U12-U6atac snRNA Complex of the Minor Spliceosome and Binding by NTC-related Protein RBM22

Splicing of precursor messenger (pre-m)RNA is a critical process in eukaryotes in which the non-coding regions, called introns, are removed and coding regions, or exons, are ligated to form a mature mRNA. This process is catalyzed by the spliceosome, a multi-mega Dalton ribonucleoprotein complex assembled from five small nuclear ribonucleoproteins (snRNP) in the form of small nuclear (sn)RNA-protein complexes (U1, U2, U4, U5 and U6) and \u3e100 proteins. snRNA components catalyze the two transesterification reactions while proteins perform critical roles in assembly and rearrangement. U2 and U6 snRNAs are the only snRNAs directly implicated in catalyzing the splicing of pre-mRNA. However, assembly and rearrangement steps prior to catalysis require numerous proteins. The catalytic core comprises a paired complex of U2 and U6 snRNAs for the major form of the spliceosome and U12 and U6atac snRNAs for the minor variant (~0.3% of all spliceosomes in higher eukaryotes). The minor spliceosome shares key catalytic sequence elements with the major spliceosome and performs identical chemistry. Previous studies have shown that the protein-free U2-U6 snRNA complex adopts two conformations in equilibrium, characterized by four and three helices surrounding a central junction. The four-helix conformer is strongly favored in the in vitro protein-free state, while the three-helix conformer predominates in spliceosomes. The minor spliceosome does not exhibit the conformational heterogeneity in the junction found in the major spliceosome. Here we use solution NMR techniques to show that the U12-U6atac snRNA complex of both human and Arabidopsis maintain base-pairing patterns similar to those in the three-helix model of the U2-U6 snRNA complex that position key elements to form the spliceosome’s active site. However, in place of the stacked base pairs at the base of the U6 snRNA intramolecular stem loop and the central junction of the U2-U6 snRNA complex, we see elongation in the single stranded hinge region opposing termini of the snRNAs to enable interaction between the key elements. Electrophoretic mobility shift assays and fluorescence assays show that the human spliceosomal protein RBM22, implicated in remodeling the human U2-U6 snRNA complex prior to catalysis but not yet definitively identified in minor spliceosomes, binds the U12-U6atac snRNA complexes specifically and with similar affinity as to U2-U6 snRNA (a mean Kd for the two methods = 3.4 μM and 8.0 μM for U2-U6 and U12-U6atac snRNA complexes, respectively), suggesting that RBM22 performs the same role in both spliceosomes; the small but reproducible difference in the measured affinities may be due to differences in binding of the junction and hinge regions of the U2-U6 and U12-U6atac snRNA complexes, respectively. These findings contribute to our overall understanding of the formation of the RNA catalytic core of the major and minor versions of the spliceosome. The absence of the central junction in the human U12-U6atac snRNA may help explain its somewhat lesser affinity for the protein and the slower rate of catalysis exhibited by the minor spliceosome than its major counterpart, suggesting this region also forms a recognition site with the protein

Characterization of splicing mechanisms by single-molecule fluorescence

Group II introns rank amongst the largest self-splicing ribozymes found in bacteria and organellar genomes of various eukaryotes. Despite the diversity in primary sequences, group II introns posses highly conserved secondary structures consisting of six domains (D1-D6). To perform its function, the large multidomain group II intron RNA must adopt the correctly folded structure. As a result, in vitro splicing of these introns requires high ionic strength and elevated temperatures. In vivo, this process is mainly assisted by protein cofactors. However, the exact mechanism of protein-mediated splicing of group II intron RNA is still not known. In order to elucidate the mechanism of protein-mediated splicing of group II introns, we have studied the folding dynamics of the D135 ribozyme, a minimal active form of the yeast ai5γ group II intron, in the presence of its natural cofactor, the DEAD-box protein Mss116, using single-molecule fluorescence. Consistent with folding studies at very high magnesium concentrations, our single-molecule data show that Mss116 can promote the folding of group II introns under near physiological conditions in vitro. Furthermore, smFRET data indicate that the Mss116-mediated group II intron folding pathway is a multi-step process that consists of both ATP-independent and ATP-dependent steps. Structurally and mechanistically group II introns are similar to spliceosome-catalyzed pre-mRNA splicing. Out of five snRNAs, only the highly conserved U2 and U6 snRNAs are required in both steps of RNA splicing. The U2-U6 snRNA complex forms the active site of the spliceosome and has been shown to undergo splicing-related catalysis in the absence of proteins. Single-molecule studies of yeast U2-U6 snRNAs show a Mg2+ induced conformational change, which may be involved in spliceosomal activation in vivo. In contrast to yeast, human U2 and U6 snRNAs contain a large number of post-transcriptional modifications. Recent studies have shown these modifications make human snRNAs more stable than that of yeast indicating a possibility of having different spliceosomal activation states. In order to understand and compare the catalytic mechanisms, we used single-molecule florescence to characterize the conformational changes of human U2-U6 complex in the presence and absence of modifications using Mg2+ as a divalent metal ion. Our FRET data clearly show a Mg2+ induced conformational change with three FRET states. Based on smFRET data, we propose a minimal two-step folding pathway for human snRNAs similar to yeast. Although unmodified snRNAs exhibit similar folding dynamics as yeast, modified bases destabilize the low FRET state of the U2-U6 complex. However, comparison of FRET and UV melting data suggests modified bases may be involved in protein recognition and/or early assembly of the spliceosome rather than direct stabilization of RNA structures in vivo

The conserved central domain of yeast U6 snRNA: importance of U2-U6 helix Ia in spliceosome assembly

In the pre-mRNA processing machinery of eukaryotic cells, U6 snRNA is located at or near the active site for pre-mRNA splicing catalysis, and U6 is involved in catalyzing the first chemical step of splicing. We have further defined the roles of key features of yeast U6 snRNA in the splicing process. By assaying spliceosome assembly and splicing in yeast extracts, we found that mutations of yeast U6 nt 56 and 57 are similar to previously reported deletions of U2 nt 27 or 28, all within yeast U2-U6 helix Ia. These mutations lead to the accumulation of yeast A1 spliceosomes, which form just prior to the Prp2 ATPase step and the first chemical step of splicing. These results strongly suggest that, at a late stage of spliceosome assembly, the presence of U2-U6 helix Ia is important for promoting the first chemical step of splicing, presumably by bringing together the 5' splice site region of pre-mRNA, which is base paired to U6 snRNA, and the branchsite region of the intron, which is base paired to U2 snRNA, for activation of the first chemical step of splicing, as previously proposed by Madhani and Guthrie [Cell, 1992, 71: 803-817]. In the 3' intramolecular stem-loop of U6, mutation G81C causes an allele-specific accumulation of U6 snRNP. Base pairing of the U6 3' stem-loop in yeast spliceosomes does not extend as far as to include the U6 sequence of U2-U6 helix Ib, in contrast to the human U6 3' stem-loop structure

Kondo screening by the surface modes of a strong topological insulator

We consider a magnetic impurity deposited on the surface of a strong topological insulator and interacting with the surface modes by a Kondo exchange interaction. Taking into account the warping of the Fermi line of the surface modes, we derive a mapping to an effective one dimensional model and show that the impurity is fully screened by the surface electrons except when the Fermi level lies exactly at the Dirac point. Using an Abrikosov fermion mean-field theory, we calculate the shape of the electronic density Friedel oscillation resulting from the presence of the Kondo screening cloud. We analyze quantitatively the observability of a six-fold symmetry in the Friedel oscillations for two prototype compounds: Bi$_2$Se$_3$ and Bi$_2$Te$_3$.Comment: 22 pages, 6 figure