Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes

The product of the yeast PRP22 gene acts late in the splicing of yeast pre-messenger RNA, mediating the release of the spliced mRNA from the spliceosome. The predicted PRP22 protein sequence shares extensive homology with that of PRP2 and PRP16 proteins, which are also involved in nuclear pre-mRNA splicing. The homologous region contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases. A putative RNA-binding motif originally identified in bacterial ribosomal protein S1 and Escherichia coli polynucleotide phosphorylase has also been found in PRP22.     

Independent evolution of structural and coding regions in a Neurospora mitochondrial intron

The discovery of intervening sequences (introns) in eukaryotic genes has raised questions about the origin and evolution of these sequences. Hypotheses concerning these topics usually consider the intron as a unit that could be lost or gained over time, or as a region within which recombination can occur to facilitate the production of new proteins by exon shuffling. Additional complexities are observed in introns of mitochondrial and chloroplast genes which contain secondary structures required for messenger RNA splicing and open-reading frames encoding proteins. Here we describe differences in the organization of protein-coding sequences in the intron of the mitochondrial ND1 gene in two closely related species of Neurospora. These differences show that intron sequences involved in secondary structure formation and in protein coding can evolve as physically distinct elements. Indeed, the secondary structure elements of the ND1 intron can contain two different coding sequences located at two different positions within the intron.     

Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA

The 'RNA world' hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter's domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts ('RNPzymes'). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure. One such protein, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (TyrRS; CYT-18), is bifunctional and both aminoacylates mitochondrial tRNA(Tyr) and promotes the splicing of mitochondrial group I introns. Here we determine a 4.5-A co-crystal structure of the Twort orf142-I2 group I intron ribozyme bound to splicing-active, carboxy-terminally truncated CYT-18. The structure shows that the group I intron binds across the two subunits of the homodimeric protein with a newly evolved RNA-binding surface distinct from that which binds tRNA(Tyr). This RNA binding surface provides an extended scaffold for the phosphodiester backbone of the conserved catalytic core of the intron RNA, allowing the protein to promote the splicing of a wide variety of group I introns. The group I intron-binding surface includes three small insertions and additional structural adaptations relative to non-splicing bacterial TyrRSs, indicating a multistep adaptation for splicing function. The co-crystal structure provides insight into how CYT-18 promotes group I intron splicing, how it evolved to have this function, and how proteins could have incrementally replaced RNA structures during the transition from an RNA world to an RNP world.     

New components of the spliced leader RNP required for nematode trans-splicing

Pre-messenger-RNA maturation in nematodes and in several other lower eukaryotic phyla involves spliced leader (SL) addition trans-splicing. In this unusual RNA processing reaction, a short common 5' exon, the SL, is affixed to the 5'-most exon of multiple pre-mRNAs. The nematode SL is derived from a trans-splicing-specific approximately 100-nucleotide RNA (SL RNA) that bears striking similarities to the cis-spliceosomal U small nuclear RNAs U1, U2, U4 and U5 (refs 3, 4); for example, the SL RNA functions only if it is assembled into an Sm small nuclear ribonucleoprotein (snRNP). Here we have purified and characterized the SL RNP and show that it contains two proteins (relative molecular masses 175,000 and 30,000 (M(r) 175K and 30K)) in addition to core Sm proteins. Immunodepletion and reconstitution with recombinant proteins demonstrates that both proteins are essential for SL trans-splicing; however, neither protein is required either for conventional cis-splicing or for bimolecular (trans-) splicing of fragmented cis constructs. The M(r) 175K and 30K SL RNP proteins are the first factors identified that are involved uniquely in SL trans-splicing. Several lines of evidence indicate that the SL RNP proteins function by participating in a trans-splicing specific network of protein protein interactions analogous to the U1 snRNP SF1/BBP U2AF complex that comprises the cross-intron bridge in cis-splicing.     

Mechanism of recognition of the 5' splice site in self-splicing group I introns

Group I introns include many mitochondrial ribosomal RNA and messenger RNA introns and the nuclear rRNA introns of Tetrahymena and Physarum. The splicing of precursor RNAs containing these introns is a two-step reaction. Cleavage at the 5' splice site precedes cleavage at the 3' splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5' exon must somehow be held in place for ligation. We have now tested the reactivity of two self-splicing group I RNAs, the Tetrahymena pre-rRNA and the intron 1 portion of the Neurospora mitochondrial cytochrome b (cob) pre-mRNA, in the intermolecular exon ligation reaction (splicing in trans) described by Inoue et al. The different sequence specificity of the reactions supports the idea that the nucleotides immediately upstream from the 5' splice site are base-paired to an internal, 5' exon-binding site, in agreement with RNA structure models proposed by Davies and co-workers and others. The internal binding site is proposed to be involved in the formation of a structure that specifies the 5' splice site and, following the first step of splicing, to hold the 5' exon in place for exon ligation.     

A suppressor of a yeast splicing mutation (prp8-1) encodes a putative ATP-dependent RNA helicase

Five small nuclear RNAs (snRNAs) are required for nuclear pre-messenger RNA splicing: U1, U2, U4, U5 and U6. The yeast U1 and U2 snRNAs base-pair to the 5' splice site and branch-point sequences of introns respectively. The role of the U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs) in splicing is not clear, though a catalytic role for the U6 snRNA has been proposed. Less is known about yeast splicing factors, but the availability of genetic techniques in Saccharomyces cerevisiae has led to the identification of mutants deficient in nuclear pre-mRNA splicing (prp2-prp27). Several PRP genes have now been cloned and their protein products characterized. The PRP8 protein is a component of the U5 snRNP and associates with the U4/U6 snRNAs/snRNP to form a multi-snRNP particle believed to be important for spliceosome assembly. We have isolated extragenic suppressors of the prp8-1 mutation of S. cerevisiae and present here the preliminary characterization of one of these suppressors, spp81. The predicted amino-acid sequence of the SPP81 protein shows extensive similarity to a recently identified family of proteins thought to possess ATP-dependent RNA helicase activity. The possible role of this putative helicase in nuclear pre-mRNA splicing is discussed.     

An eIF-4A-like protein is a suppressor of an Escherichia coli mutant defective in 50S ribosomal subunit assembly

The assembly of ribosomes in bacterial cells is a complex process that remains poorly characterized. The in vitro assembly of active ribosomal subunits from purified RNA and protein components indicates that all of the information for proper assembly resides in the primary sequences of these macromolecules. On the other hand, the in vitro requirement of unphysiological heating steps suggests that this pathway may not accurately reflect the in vivo pathway, and that other proteins may be required. One approach to identify any additional proteins is to isolate second-site revertants of mutants defective in ribosome assembly. Ribosomal protein L24 is essential in the assembly of 50S subunits. We have identified an Escherichia coli gene, srmB, that, when expressed at high copy number, can suppress the effect of a temperature-sensitive lethal mutation in L24. The SrmB amino-acid sequence has sequence identity with mouse translation initiation factor eIF-4A and with the human nuclear protein, p68. The purified SrmB protein is a nucleic acid-dependent ATPase, like eIF-4A, but can also bind RNA in the absence of ATP and other auxiliary protein factors. The RNA dependent ATPase activity of SrmB suggests that like, eIF-4A, it could be involved in specific alterations of RNA secondary structure.     

Comprehensive proteomic analysis of the human spliceosome

The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome 'core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify approximately 145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.     

Single-molecule analysis of Mss116-mediated group II intron folding

DEAD-box helicases are conserved enzymes involved in nearly all aspects of RNA metabolism, but their mechanisms of action remain unclear. Here, we investigated the mechanism of the DEAD-box protein Mss116 on its natural substrate, the group II intron ai5γ. Group II introns are structurally complex catalytic RNAs considered evolutionarily related to the eukaryotic spliceosome, and an interesting paradigm for large RNA folding. We used single-molecule fluorescence to monitor the effect of Mss116 on folding dynamics of a minimal active construct, ai5γ-D135. The data show that Mss116 stimulates dynamic sampling between states along the folding pathway, an effect previously observed only with high Mg(2+) concentrations. Furthermore, the data indicate that Mss116 promotes folding through discrete ATP-independent and ATP-dependent steps. We propose that Mss116 stimulates group II intron folding through a multi-step process that involves electrostatic stabilization of early intermediates and ATP hydrolysis during the final stages of native state assembly.