Co-purification of a small RNA species with multicatalytic proteinase (proteasome) from rat liver

AbstractPrevious studies have come to different conclusions about the presence of RNA in particles known variously as prosomes, proteasomes or multicatalytic proteinase (MCP). To determine the reason for this, MCP was isolated from rat liver by 4 different purification protocols. One major band of RNA, about 80 nucleotides in length, co-purified in all preparations. The amount of RNA detected was less than one molecule per MCP particle suggesting that there may be more than one population of MCP in rat liver cells

A genome-wide survey demonstrates widespread non-linear mRNA in expressed sequences from multiple species

We describe here the results of the first genome-wide survey of candidate exon repetition events in expressed sequences from human, mouse, rat, chicken, zebrafish and fly. Exon repetition is a rare event, reported in <10 genes, in which one or more exons is tandemly duplicated in mRNA but not in the gene. To identify candidates, we analysed database sequences for mRNA transcripts in which the order of the spliced exons does not follow the linear genomic order of the individual gene [events we term rearrangements or repetition in exon order (RREO)]. Using a computational approach, we have identified 245 genes in mammals that produce RREO events. RREO in mRNA occurs predominantly in the coding regions of genes. However, exon 1 is never involved. Analysis of the open reading frames suggests that this process may increase protein diversity and regulate protein expression via nonsense-mediated RNA decay. The sizes of the exons and introns involved around these events suggest a gene model structure that may facilitate non-linear splicing. These findings imply that RREO affects a significant subset of genes within a genome and suggests that non-linear information encoded within the genomes of complex organisms could contribute to phenotypic variation

Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways

The selection of 3' splice sites (3?ss) is an essential early step in mammalian RNA splicing reactions, but the processes involved are unknown. We have used single molecule methods to test whether the major components implicated in selection, the proteins U2AF35 and U2AF65 and the U2 snRNP, are able to recognize alternative candidate sites or are restricted to one pre-specified site. In the presence of adenosine triphosphate (ATP), all three components bind in a 1:1 stoichiometry with a 3?ss. Pre-mRNA molecules with two alternative 3?ss can be bound concurrently by two molecules of U2AF or two U2 snRNPs, so none of the components are restricted. However, concurrent occupancy inhibits splicing. Stoichiometric binding requires conditions consistent with coalescence of the 5? and 3? sites in a complex (I, initial), but if this cannot form the components show unrestricted and stochastic association. In the absence of ATP, when complex E forms, U2 snRNP association is unrestricted. However, if protein dephosphorylation is prevented, an I-like complex forms with stoichiometric association of U2 snRNPs and the U2 snRNA is base-paired to the pre-mRNA. Complex I differs from complex A in that the formation of complex A is associated with the loss of U2AF65 and 3

Human ribosomal protein S13 regulates expression of its own gene at the splicing step by a feedback mechanism

The expression of ribosomal protein (rp) genes is regulated at multiple levels. In yeast, two genes are autoregulated by feedback effects of the protein on pre-mRNA splicing. Here, we have investigated whether similar mechanisms occur in eukaryotes with more complicated and highly regulated splicing patterns. Comparisons of the sequences of ribosomal protein S13 gene (RPS13) among mammals and birds revealed that intron 1 is more conserved than the other introns. Transfection of HEK 293 cells with a minigene-expressing ribosomal protein S13 showed that the presence of intron 1 reduced expression by a factor of four. Ribosomal protein S13 was found to inhibit excision of intron 1 from rpS13 pre-mRNA fragment in vitro. This protein was shown to be able to specifically bind the fragment and to confer protection against ribonuclease cleavage at sequences near the 5′ and 3′ splice sites. The results suggest that overproduction of rpS13 in mammalian cells interferes with splicing of its own pre-mRNA by a feedback mechanism

Design principles for bifunctional targeted oligonucleotide enhancers of splicing

Controlling the patterns of splicing of specific genes is an important goal in the development of new therapies. We have shown that the splicing of a refractory exon, SMN2 exon 7, could be increased in fibroblasts derived from patients with spinal muscular atrophy by using bifunctional targeted oligonucleotide enhancers of splicing (TOES) oligonucleotides that anneal to the exon and contain a ‘tail’ of enhancer sequences that recruit activating proteins. We show here that there are striking agreements between the effects of oligonucleotides on splicing in vitro and on both splicing and SMN2 protein expression in patient-derived fibroblasts, indicating that the effects on splicing are the major determinant of success. Increased exon inclusion depends on the number, sequence and chemistry of the motifs that bind the activator protein SRSF1, but it is not improved by increasing the strength of annealing to the target site. The optimal oligonucleotide increases protein levels in transfected fibroblasts by a mean value of 2.6-fold (maximum 4.6-fold), and after two rounds of transfection the effect lasted for a month. Oligonucleotides targeted to the upstream exon (exon 6 in SMN) are also effective. We conclude that TOES oligonucleotides are highly effective reagents for restoring the splicing of refractory exons and can act across long introns

Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses

Splicing is regulated by complex interactions of numerous RNA-binding proteins. The molecular mechanisms involved remain elusive, in large part because of ignorance regarding the numbers of proteins in regulatory complexes. Polypyrimidine tract-binding protein (PTB), which regulates tissue-specific splicing, represses exon 3 of α-tropomyosin through distant pyrimidine-rich tracts in the flanking introns. Current models for repression involve either PTB-mediated looping or the propagation of complexes between tracts. To test these models, we used single-molecule approaches to count the number of bound PTB molecules both by counting the number of bleaching steps of GFP molecules linked to PTB within complexes and by analysing their total emissions. Both approaches showed that five or six PTB molecules assemble. Given the domain structures, this suggests that the molecules occupy primarily multiple overlapping potential sites in the polypyrimidine tracts, excluding propagation models. As an alternative to direct looping, we propose that repression involves a multistep process in which PTB binding forms small local loops, creating a platform for recruitment of other proteins that bring these loops into close proximity

Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68

Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome

2′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions

Protein-RNA interactions are central to numerous cellular processes. In this work, we present an easy and straightforward NMR-based approach to determine the RNA binding site of RNA binding proteins and to evaluate the binding of pairs of proteins to a single-stranded RNA (ssRNA) under physiological conditions, in this case in nuclear extracts. By incorporation of a 19F atom on the ribose of different nucleotides along the ssRNA sequence, we show that, upon addition of an RNA binding protein, the intensity of the 19F NMR signal changes when the 19F atom is located near the protein binding site. Furthermore, we show that the addition of pairs of proteins to a ssRNA containing two 19F atoms at two different locations informs on their concurrent binding or competition. We demonstrate that such studies can be done in a nuclear extract that mimics the physiological environment in which these protein-ssRNA interactions occur. Finally, we demonstrate that a trifluoromethoxy group (-OCF3) incorporated in the 2′ribose position of ssRNA sequences increases the sensitivity of the NMR signal, leading to decreased measurement times, and reduces the issue of RNA degradation in cellular extracts

Biophysical characterisation of the Bcl-x pre-mRNA and binding specificity of the ellipticine derivative GQC-05 : implication for alternative splicing regulation

The BCL2L1 gene expresses two isoforms of Bcl-x protein via the use of either of two alternative 5′ splice sites (5′ss) in exon 2. These proteins have antagonistic actions, Bcl-XL being anti-apoptotic and Bcl-XS pro-apoptotic. In a number of cancers the Bcl-XL isoform is over-expressed, resulting in cancer cell survival and growth, so switching splicing to the Xs isoform could have therapeutic benefits. We have previously proposed that a putative G-quadruplex (G4) exists downstream of the XS 5′ss and shown that the ellipticine derivative GQC-05, a previously identified DNA G4-specific ligand, induces an increase in the XS/XL ratio both in vitro and in cells. Here, we demonstrate that this G4 forms in vitro and that the structure is stabilised in the presence of GQC-05. We also show that GQC-05 binds RNA non-specifically in buffer conditions, but selectively to the Bcl-x G4 in the presence of nuclear extract, highlighting the limitations of biophysical measurements taken outside of a functional environment. We also demonstrate that GQC-05 is able to shift the equilibrium between competing G4 and duplex structures towards the G4 conformation, leading to an increase in accessibility of the XS 5′ss, supporting our previous model on the mechanism of action of GQC-05

Surface passivation with a perfluoroalkane brush improves the precision of single-molecule measurements

Single-molecule imaging is invaluable for investigating the heterogeneous behavior and interactions of biological molecules. However, an impediment to precise sampling of single molecules is the irreversible adsorption of components onto the surfaces of cover glasses. This causes continuous changes in the concentrations of different molecules dissolved or suspended in the aqueous phase from the moment a sample is dispensed, which will shift, over time, the position of chemical equilibria between monomeric and multimeric components. Interferometric scattering microscopy (iSCAT) is a technique in the single-molecule toolkit that has the capability to detect unlabeled proteins and protein complexes both as they adsorb onto and desorb from a glass surface. Here, we examine the reversible and irreversible interactions between a number of different proteins and glass via analysis of the adsorption and desorption of protein at the single-molecule level. Furthermore, we present a method for surface passivation that virtually eliminates irreversible adsorption while still ensuring the residence time of molecules on surfaces is sufficient for detection of adsorption by iSCAT. By grafting high-density perfluoroalkane brushes on cover-glass surfaces, we observe approximately equal numbers of adsorption and desorption events for proteins at the measurement surface (±1%). The fluorous–aqueous interface also prevents the kinetic trapping of protein complexes and assists in establishing a thermodynamic equilibrium between monomeric and multimeric components. This surface passivation approach is valuable for in vitro single-molecule experiments using iSCAT microscopy because it allows for continuous monitoring of adsorption and desorption of protein without either a decline in detection events or a change in sample composition due to the irreversible binding of protein to surfaces