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 S. cerevisiae 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. In this review, we characterize changes in spliceosome composition across phyla, describing those that are most frequently observed and highlighting an analysis of the reduced spliceosome of the red alga Cyanidioschyzon merolae We used homology modeling to predict what effect splicing protein loss would have on the spliceosome, based on currently available cryo-EM structures. We observe strongly correlated loss of proteins that function in the same process, for example, in interacting with the U1 snRNP (which is absent in C. merolae), regulation of Brr2, or coupling transcription and splicing. Based on our observations, we predict splicing in C. merolae to be inefficient, inaccurate, and post-transcriptional, consistent with the apparent trend toward its elimination in this lineage. This work highlights the striking flexibility of the splicing pathway and the spliceosome when viewed in the context of eukaryotic diversity.

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. In naturally transformable species, the DNA uptake system is only expressed when bacteria enter a physiological state called competence, which develops under specific conditions. Here, we investigated the mechanism that controls expression of the DNA uptake system in the human pathogen Legionella pneumophila We found that a repressor of this system displays a conserved ProQ/FinO domain and interacts with a newly characterized trans-acting sRNA, RocR. Together, they target mRNAs of the genes coding the DNA uptake system to control natural transformation. This RNA-based silencing represents a previously unknown regulatory means to control this major mechanism of HGT. Importantly, these findings also show that chromosome-encoded ProQ/FinO domain-containing proteins can assist trans-acting sRNAs and that this class of RNA chaperones could play key roles in post-transcriptional gene regulation throughout bacterial species.

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.

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. Here we describe functional studies of a Cas5d ortholog, and high-resolution structural studies of a second Cas5d family member, demonstrating that Cas5d is a sequence-specific RNA endonuclease that cleaves CRISPR repeats and is thus responsible for processing of pre-crRNA. Analysis of the structural homology of Cas5d with the previously characterized Cse3 protein allows us to model the interaction of Cas5d with its RNA substrate and conclude that it is a member of a larger family of CRISPR RNA endonucleases.

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. Here we report a structural model of the p14 · bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein-RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14 · duplex interaction must be disrupted before the first step of splicing.

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.

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 ß-turn-α-helix motif, while the hairpin portion of the terminator is recognized by a conserved α-helical N-cap motif. Structure-guided mutagenesis reveals key RNA contact residues that are critical for RocC/RocR to repress the uptake of environmental DNA in L. pneumophila. Structural analysis and RNA binding studies reveal that other ProQ/FinO domains also recognize related transcriptional terminators with different specificities for the length of the 3' ssRNA tail.

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. To date, the role of RNA structure in miRNA biogenesis has only been considered in terms of the secondary structural elements required for processing of pri-miRNAs by Drosha. Here we report that the miR-17~92 cluster has a compact globular tertiary structure where miRNAs internalized within the core of the folded structure are processed less efficiently than miRNAs on the surface of the structure. Increased miR-92 expression resulting from disruption of the compact miR-17~92 structure results in increased repression of integrin α5 mRNA, a known target of miR-92a. In summary, we describe the first example of pri-miRNA structure modulating differential expression of constituent miRNAs.

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. X-ray and NMR techniques have been successfully used to address these questions defining the structure of individual domains, isolated splicing proteins, spliceosomal RNA fragments and recently the U1 snRNP multiprotein.RNA complex. These results combined with extant biochemical and genetic data have yielded important insights as well as posing fresh questions with respect to the regulation and mechanism of this critical gene regulatory process.

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. The E' complex is characterized by a U1 snRNA-5' splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5' splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.

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.

Separation of Spliceosome Assembly from Catalysis with Caged pre-mRNA Substrates.

Getting spliced? Pre-mRNA splicing is catalyzed by the spliceosome, a complex assembly of proteins and RNA, which forms in an ordered fashion on the substrate. If one of the residues of the substrate is caged with a photolabile o-nitrobenzyl group, the splicing reaction can be transiently blocked, until subsequent initiation by photolysis of the complexes. This RNA-caging approach effectively separates the spliceosome assembly from the catalytic reaction and allows the two processes to be studied independently.

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. A wealth of evidence places the protein PRP8 at the heart of the spliceosome through assembly and catalysis. Here we provide evidence that the RNase H domain of PRP8 undergoes a conformational switch between the two steps of splicing, rationalizing yeast prp8 alleles that promote either the first or second step. We also show that this switch unmasks a metal-binding site involved in the second step. Together, these data establish that PRP8 is a metalloprotein that promotes exon ligation within the spliceosome.

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. Here we describe structural and functional analyses of the Thermus thermophilus CRISPR Cse3 endonuclease. High-resolution X-ray structures of Cse3 bound to repeat RNAs model both the pre- and post-cleavage complexes associated with processing the pre-crRNA. These structures establish the molecular basis of a specific CRISPR RNA recognition and suggest the mechanism for generation of effector RNAs responsible for gene silencing.

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. Here we report the structural characterization of domain IV (D4) and ΔD4 (polypeptides with 222 and 160 amino acids, respectively) that differ by virtue of an N-terminal deletion of 62 amino acids (28% of the overall D4 sequence). The high-resolution crystal structures of the monomeric D4 and the dimeric ΔD4 reveal substantially different folds despite an overall conservation of secondary structure. These structures show that the formation of tertiary structures, even in extended polypeptide sequences, can be highly context dependent, and they serve as a model for structural plasticity in protein isoforms.

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 1.85-A X-ray structure of the core of PRP8 domain IV, implicated in key spliceosomal interactions, reveals a bipartite structure that includes the presence of an RNase H fold linked to a five-helix assembly. Analysis of mutant yeast alleles and cross-linking results in the context of this structure, coupled with RNA binding studies, suggests that domain IV forms a surface that interacts directly with the RNA structures at the catalytic core of the spliceosome.