HITS-CLIP yields genome-wide insights into brain alternative RNA processing

Protein-RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3' untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.     

Splicing-related catalysis by protein-free snRNAs

Removal of intervening sequences from eukaryotic messenger RNA precursors is carried out by the spliceosome, a complex assembly of five small nuclear RNAs (snRNAs) and a large number of proteins. Although it has been suggested that the spliceosome might be an RNA enzyme, direct evidence for this has been lacking, and the identity of the catalytic domain of the spliceosome is unknown. Here we show that a protein-free complex of two snRNAs, U2 and U6, can bind and position a small RNA containing the sequence of the intron branch site, and activate the branch adenosine to attack a catalytically critical domain of U6 in a reaction that is related to the first step of splicing. Our data provide direct evidence for the catalytic potential of spliceosomal snRNAs.     

PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome.

The assembly of the spliceosome is an ATP-dependent process. The splicing factor PRP16 contains variations of several motifs that define the eIF-4A-like ATP-dependent RNA helicase family. The protein has now been purified and shown to exhibit RNA-dependent ATPase activity. PRP16 is required specifically for the second catalytic step of the splicing reaction in vitro. This function requires ATP binding and/or hydrolysis, which appears to be concomitant with release of the protein from the spliceosome. PRP16 may be the prototype for a set of splicing factors which use ATP to drive a cycle of conformational changes.     

Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA.

Splicing of pre-messenger RNA in eukaryotic cells occurs in a multicomponent complex termed the spliceosome, which contains small nuclear ribonucleoprotein particles (snRNPs), protein factors and substrate pre-mRNA. Assembly of the spliceosome involves the stepwise binding of snRNPs and protein factors to the pre-mRNA through a poorly understood mechanism which probably involves specific RNA-RNA, RNA-protein and protein-protein interactions. Of particular interest are the interactions between snRNPs, which are likely to be important not only for assembly of the spliceosome but also for catalysis. U1 snRNP interacts with the 5' splice site and U2 snRNP with the branch site of the pre-mRNA; both of these interactions involve Watson-Crick base pairing. But very little is known about how other factors such as the U4/U6 and U5 snRNPs reach the spliceosome and function in splicing. Here we report evidence that U6 snRNA interacts directly with U2 snRNA by a mechanism involving base-pairing, and that this interaction can be necessary for splicing of a mammalian pre-mRNA in vivo.     

Isolation of an active step I spliceosome and composition of its RNP core

Formation of catalytically active RNA structures within the spliceosome requires the assistance of proteins. However, little is known about the number and nature of proteins needed to establish and maintain the spliceosome's active site. Here we affinity-purified human spliceosomal C complexes and show that they catalyse exon ligation in the absence of added factors. Comparisons of the composition of the precatalytic versus the catalytic spliceosome revealed a marked exchange of proteins during the transition from the B to the C complex, with apparent stabilization of Prp19-CDC5 complex proteins and destabilization of SF3a/b proteins. Disruption of purified C complexes led to the isolation of a salt-stable ribonucleoprotein (RNP) core that contained both splicing intermediates and U2, U5 and U6 small nuclear RNA plus predominantly U5 and human Prp19-CDC5 proteins and Prp19-related factors. Our data provide insights into the spliceosome's catalytic RNP domain and indicate a central role for the aforementioned proteins in sustaining its catalytically active 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.     

Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF

Many cellular functions involve multi-domain proteins, which are composed of structurally independent modules connected by flexible linkers. Although it is often well understood how a given domain recognizes a cognate oligonucleotide or peptide motif, the dynamic interaction of multiple domains in the recognition of these ligands remains to be characterized. Here we have studied the molecular mechanisms of the recognition of the 3'-splice-site-associated polypyrimidine tract RNA by the large subunit of the human U2 snRNP auxiliary factor (U2AF65) as a key early step in pre-mRNA splicing. We show that the tandem RNA recognition motif domains of U2AF65 adopt two remarkably distinct domain arrangements in the absence or presence of a strong (that is, high affinity) polypyrimidine tract. Recognition of sequence variations in the polypyrimidine tract RNA involves a population shift between these closed and open conformations. The equilibrium between the two conformations functions as a molecular rheostat that quantitatively correlates the natural variations in polypyrimidine tract nucleotide composition, length and functional strength to the efficiency to recruit U2 snRNP to the intron during spliceosome assembly. Mutations that shift the conformational equilibrium without directly affecting RNA binding modulate splicing activity accordingly. Similar mechanisms of cooperative multi-domain conformational selection may operate more generally in the recognition of degenerate nucleotide or amino acid motifs by multi-domain proteins.     

The RNA splicing factor hSlu7 is required for correct 3' splice-site choice

The production of correctly spliced messenger RNA requires two catalytic splicing steps. During step II, exon 1 attacks an adenine-guanine (AG) dinucleotide at the 3' splice site. This AG is usually located between 18 and 40 nucleotides downstream from the branch site, and closer AGs are skipped in favour of AGs located more optimally downstream. At present, little is understood about how the correct AG is distinguished from other AGs. Here we describe a metazoan splicing factor (hSlu7) that is required for selection of the correct AG. In the absence of hSlu7, use of the correct AG is suppressed and incorrect AGs are activated. We investigated this loss of fidelity by analysing spliceosomes assembled in the absence of hSlu7. These studies reveal that exon 1 is loosely associated with these spliceosomes. Thus, the improperly held exon cannot access the correct AG, but can attack other AGs indiscriminately. We conclude that hSlu7 is required to hold exon 1 tightly within the spliceosome for attack on a prespecified AG.     

Scanning from an independently specified branch point defines the 3' splice site of mammalian introns

During pre-messenger RNA splicing the lariat branch point in mammalian introns is specified independently of the 3' splice site by the sequence surrounding the branch point and by an adjacent downstream polypyrimidine tract. The 3' splice site is dispensable for spliceosome assembly and cleavage at the 5' splice site, and is itself determined by a scanning process that recognizes the first AG located 3' of the branch point/polypyrimidine tract, irrespective of distance or sequence environment.     

Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome

Introns are removed from nuclear messenger RNA precursors through two sequential phospho-transesterification reactions in a dynamic RNA-protein complex called the spliceosome. But whether splicing is catalysed by small nuclear RNAs in the spliceosome is unresolved. As the spliceosome is a metalloenzyme, it is important to determine whether snRNAs coordinate catalytic metals. Here we show that yeast U6 snRNA coordinates a metal ion that is required for the catalytic activity of the spliceosome. With Mg2+, U6 snRNA with a sulphur substitution for the pro-Rp or pro-Sp non-bridging phosphoryl oxygen of nucleotide U80 reconstitutes a fully assembled yet catalytically inactive spliceosome. Adding a thiophilic ion such as Mn2+ allows the first transesterification reaction to occur in the U6/sU80(Sp)- but not the U6/sU80(Rp)-reconstituted spliceosome. Mg2+ competitively inhibits the Mn2+-rescued reaction, indicating that the metal-binding site at U6/U80 exists in the wild-type spliceosome and that the site changes its metal requirement for activity in the Sp spliceosome. Thus, U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome.     

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.     

The U1 snRNP protein U1C recognizes the 5' splice site in the absence of base pairing

Splicing of precursor messenger RNA takes place in the spliceosome, a large RNA/protein macromolecular machine. Spliceosome assembly occurs in an ordered pathway in vitro and is conserved between yeast and mammalian systems. The earliest step is commitment complex formation in yeast or E complex formation in mammals, which engages the pre-mRNA in the splicing pathway and involves interactions between U1 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA 5' splice site. Complex formation depends on highly conserved base pairing between the 5' splice site and the 5' end of U1 snRNA, both in vivo and in vitro. U1 snRNP proteins also contribute to U1 snRNP activity. Here we show that U1 snRNP lacking the 5' end of its snRNA retains 5'-splice-site sequence specificity. We also show that recombinant yeast U1C protein, a U1 snRNP protein, selects a 5'-splice-site-like sequence in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast 5'-splice-site consensus sequence. We propose that a U1C 5'-splice-site interaction precedes pre-mRNA/U1 snRNA base pairing and is the earliest step in the splicing pathway.     

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1-HIND interaction, cannot use certain non-canonical 5' splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.     

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.     

Human U2 snRNA can function in pre-mRNA splicing in yeast

The removal of introns from messenger RNA precursors requires five small nuclear RNAs (snRNAs), contained within ribonucleoprotein particles (snRNPs), which complex with the pre-mRNA and other associated factors to form the spliceosome. In both yeast and mammals, the U2 snRNA base pairs with sequences surrounding the site of lariat formation. Binding of U2 snRNP to the highly degenerate branchpoint sequence in mammalian introns is absolutely dependent on an auxiliary protein, U2AF, which recognizes a polypyrimidine stretch adjacent to the 3' splice site. The absence of this sequence motif in yeast introns has strengthened arguments that the two systems are fundamentally different. Deletion analyses of the yeast U2 gene have confirmed that the highly conserved 5' domain is essential, although the adjacent approximately 950 nucleotides can be deleted without any phenotypic consequence. A 3'-terminal domain of approximately 100 nucleotides is also required for wild-type growth rates; the highly conserved terminal loop within this domain (loop IV) may provide specific binding contacts for two U2-specific snRNP proteins. We have replaced the single copy yeast U2 (yU2) gene with human U2 (hU2), expecting that weak or no complementation would provide an assay for cloning additional splicing factors, such as U2AF. We report here that hU2 can complement the yeast deletion with surprising efficiency. The interactions governing spliceosome assembly and intron recognition are thus more conserved than previously suspected. Paradoxically, the conserved loop IV sequence is dispensable in yeast.