Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes.

Premessenger RNA splicing is catalyzed by the spliceosome, a multimegadalton RNA-protein complex that assembles in a highly regulated process on each intronic substrate. Most studies of splicing and spliceosomes have been carried out in human or model systems. There exists, however, a large diversity of spliceosomes, particularly in organisms with reduced genomes, that suggests a means of analyzing the essential elements of spliceosome assembly and regulation.

Structural basis for recognition of transcriptional terminator structures by ProQ/FinO domain RNA chaperones.

The ProQ/FinO family of RNA binding proteins mediate sRNA-directed gene regulation throughout gram-negative bacteria. Here, we investigate the structural basis for RNA recognition by ProQ/FinO proteins, through the crystal structure of the ProQ/FinO domain of the Legionella pneumophila DNA uptake regulator, RocC, bound to the transcriptional terminator of its primary partner, the sRNA RocR. The structure reveals specific recognition of the 3' nucleotide of the terminator by a conserved pocket involving a β-turn-α-helix motif, while the hairpin portion of the terminator is recognized by a conserved α-helical N-cap motif.

Radical probing of spliceosome assembly.

Here we describe the synthesis and use of a directed hydroxyl radical probe, tethered to a pre-mRNA substrate, to map the structure of this substrate during the spliceosome assembly process. These studies indicate an early organization and proximation of conserved pre-mRNA sequences during spliceosome assembly. This methodology may be adapted to the synthesis of a wide variety of modified RNAs for use as probes of RNA structure and RNA-protein interaction.

Silencing of natural transformation by an RNA chaperone and a multitarget small RNA.

A highly conserved DNA uptake system allows many bacteria to actively import and integrate exogenous DNA. This process, called natural transformation, represents a major mechanism of horizontal gene transfer (HGT) involved in the acquisition of virulence and antibiotic resistance determinants. Despite evidence of HGT and the high level of conservation of the genes coding the DNA uptake system, most bacterial species appear non-transformable under laboratory conditions.

Analysis of CRISPR Pre-crRNA Cleavage.

We have examined the processing of precursor-clustered regularly interspaced short palindromic repeat (CRISPR) RNAs (pre-crRNAs) of the Type I CRISPR-Cas system by incubation of radiolabeled model RNAs with recombinant CRISPR-associated (Cas) endoribonucleases, followed by denaturing polyacrylamide gel electrophoresis (PAGE) of the products. Determination of cleavage position is based on comparison with RNase T1 digestion and base hydrolysis products. The mechanism of cleavage is investigated by chemical and enzymatic characterization of the reaction products as well as by the demonstration that a specific 2'-deoxy substitution 5' to the scissile phosphate blocks endonucleolytic cleavage.

A conformational switch in PRP8 mediates metal ion coordination that promotes pre-mRNA exon ligation.

Splicing of pre-mRNAs in eukaryotes is catalyzed by the spliceosome, a large RNA-protein metalloenzyme. The catalytic center of the spliceosome involves a structure comprising the U2 and U6 snRNAs and includes a metal bound by U6 snRNA. The precise architecture of the splicesome active site, however, and the question of whether it includes protein components, remains unresolved.

Cas5d processes pre-crRNA and is a member of a larger family of CRISPR RNA endonucleases.

Small RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in bacteria and archaea are involved in an adaptable and heritable gene-silencing pathway. Resistance to invasive genetic material is conferred by the incorporation of short DNA sequences derived from this material into the genome as CRISPR spacer elements separated by short repeat sequences. Processing of long primary transcripts (pre-crRNAs) containing these repeats by a CRISPR-associated (Cas) RNA endonuclease generates the mature effector RNAs that target foreign nucleic acid for degradation.

Role of pri-miRNA tertiary structure in miR-17~92 miRNA biogenesis.

MicroRNAs (miRNAs) regulate gene expression in a variety of biological pathways such as development and tumourigenesis. miRNAs are initially expressed as long primary transcripts (pri-miRNAs) that undergo sequential processing by Drosha and then Dicer to yield mature miRNAs. miR-17~92 is a miRNA cluster that encodes 6 miRNAs and while it is essential for development it also has reported oncogenic activity.

Recognition and maturation of effector RNAs in a CRISPR interference pathway.

In bacteria and archaea, small RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci are involved in an adaptable and heritable gene-silencing pathway. Resistance to phage infection is conferred by the incorporation of short invading DNA sequences into the genome as CRISPR spacer elements separated by short repeat sequences. Processing of long primary transcripts (pre-crRNAs) containing these repeats by an RNA endonuclease generates the mature effector RNAs that interfere with phage gene expression.

Structural model of the p14/SF3b155 · branch duplex complex.

Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex.

Context-dependent remodeling of structure in two large protein fragments.

Protein folding involves the formation of secondary structural elements from the primary sequence and their association with tertiary assemblies. The relation of this primary sequence to a specific folded protein structure remains a central question in structural biology. An increasing body of evidence suggests that variations in homologous sequence ranging from point mutations to substantial insertions or deletions can yield stable proteins with markedly different folds.

Spliceosome structure: piece by piece.

Processing of pre-mRNAs by RNA splicing is an essential step in the maturation of protein coding RNAs in eukaryotes. Structural studies of the cellular splicing machinery, the spliceosome, are a major challenge in structural biology due to the size and complexity of the splicing ensemble. Specifically, the structural details of splice site recognition and the architecture of the spliceosome active site are poorly understood.

Structural elucidation of a PRP8 core domain from the heart of the spliceosome.

The spliceosome is a complex ribonucleoprotein (RNP) particle containing five RNAs and more than 100 associated proteins. One of these proteins, PRP8, has been shown to interact directly with the splice sites and branch region of precursor-mRNAs (pre-mRNAs) and spliceosomal RNAs associated with catalysis of the two steps of splicing. The 1.

Pre-mRNA splicing: a complex picture in higher definition.

Intron excision from pre-mRNAs of higher eukaryotes requires a transition from splice-site recognition across short exons to organization of the spliceosome across long introns. Recently, insight into this transition has been provided and, in addition, it has been shown that an alternative splicing factor, the polypyrimidine-tract-binding protein, can exert its control on splice-site choice by blocking this key step in the assembly of the splicing machinery.

Synthesis of oligo-RNAs with photocaged adenosine 2'-hydroxyls.

This protocol describes a general method for the preparation of RNAs in which the reactivity or hydrogen-bonding properties of the molecule are modified in a photoreversible fashion by use of a caging strategy. A single caged adenosine, modified at the 2' position as a nitro-benzyl ether, can be incorporated into short RNAs by chemical synthesis or into long RNAs by a combination of chemical and enzymatic synthesis. The modified RNAs can be uncaged by photolysis under a variety of conditions including the use of a laser or xenon lamp, and the course of this uncaging reaction may be readily followed by HPLC or thin-layer chromatography.

FRET analysis of in vivo dimerization by RNA-editing enzymes.

Members of the ADAR (adenosine deaminase that acts on RNA) enzyme family catalyze the hydrolytic deamination of adenosine to inosine within double-stranded RNAs, a poorly understood process that is critical to mammalian development. We have performed fluorescence resonance energy transfer experiments in mammalian cells transfected with fluorophore-bearing ADAR1 and ADAR2 fusion proteins to investigate the relationship between these proteins. These studies conclusively demonstrate the homodimerization of ADAR1 and ADAR2 and also show that ADAR1 and ADAR2 form heterodimers in human cells.

Crystal structure of a core spliceosomal protein interface.

The precise excision of introns from precursor mRNAs (pre-mRNAs) in eukaryotes is accomplished by the spliceosome, a complex assembly containing five small nuclear ribonucleoprotein (snRNP) particles. Human p14, a component of the spliceosomal U2 and U11/U12 snRNPs, has been shown to associate directly with the pre-mRNA branch adenosine early in spliceosome assembly and within the fully assembled spliceosome. Here we report the 2.

Characterization of a U2AF-independent commitment complex (E') in the mammalian spliceosome assembly pathway.

Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5' splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3' splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E' which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway.

RNAi: running interference for the cell.

RNA interference or RNAi is a recently characterized mechanism of eukaryotic gene regulation in which a short sequence of double-stranded RNA (dsRNA) specifically down-regulates expression of the associated gene. Preliminary characterization of this phenomenon has revealed a set of inter-related cellular pathways which appear to represent both a response to foreign RNA and a mechanism of endogenous gene regulation. Introduction of dsRNA into cells by a variety of means, including transfection of synthetic RNA duplexes, triggers the RNAi response resulting in specific suppression of target gene expression.

Structuring of the 3' splice site by U2AF65.

Recognition of the 3' splice site in mammalian introns is accomplished by association of the splicing factor U2AF with the precursor mRNA (pre-mRNA) in a multiprotein splicing commitment complex. It is well established that this interaction involves binding of the large U2AF65 subunit to sequences upstream of the 3' splice site, but the orientation of the four domains of this protein with respect to the RNA and hence their role in structuring the commitment complex remain unclear and the basis of contradictory models. We have examined the interaction of U2AF65 with an RNA representing the 3' splice site using a series of U2AF deletion mutants modified at the N terminus with the directed hydroxyl radical probe iron-EDTA.

Adenosine to inosine editing by ADAR2 requires formation of a ternary complex on the GluR-B R/G site.

RNA editing by members of the ADAR (adenosine deaminase that acts on RNA) enzyme family involves hydrolytic deamination of adenosine to inosine within the context of a double-stranded pre-mRNA substrate. Editing of the human GluR-B transcript is catalyzed by the enzyme ADAR2 at the Q/R and R/G sites. We have established a minimal RNA substrate for editing based on the R/G site and have characterized the interaction of ADAR2 with this RNA by gel shift, kinetic, and cross-linking analyses.

Early organization of pre-mRNA during spliceosome assembly.

Intron excision from precursor mRNAs (pre-mRNAs) in eukaryotes requires juxtaposition of reactive functionalities within the substrate at the heart of the spliceosome where the two chemical steps of splicing occur. Although a series of interactions between pre-mRNAs, pre-spliceosomal and spliceosomal factors is well established, the molecular mechanisms of splicing machinery assembly, as well as the temporal basis for organization of the substrate for splicing, remain poorly understood. Here we have used a directed hydroxyl radical probe tethered to pre-mRNA substrates to map the structure of the pre-mRNA substrate during the spliceosome assembly process.