Introns play an essential role in splicing-dependent formation of the exon junction complex

Pre-mRNA splicing specifically deposits the exon junction complex (EJC) onto spliced mRNA, which is important for downstream events. Here, we show that EJC components are primarily recruited to the spliceosome by association with the intron via the intron-binding protein, IBP160. This initial association of EJC components occurs in the absence of the final EJC-binding site on the exon. RNA interference (RNAi) knockdown of IBP160 arrested EJC association with cytoplasmic RNAs following nonsense-mediated decay. We propose that the intron has a crucial role in the early steps of EJC formation and is indispensable for the subsequent formation of a functional EJC.     

Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites.     

A ribosomal function is necessary for efficient splicing of the T4 phage thymidylate synthase intron in vivo

Splicing of the group I intron of the T4 thymidylate synthase (td) gene was uncoupled from translation by introducing stop codons in the upstream exon. This resulted in severe splicing deficiency in vivo. Overexpression of a UGA suppressor tRNA partially rescued splicing, suggesting that this in vitro self-splicing intron requires translation for splicing in vivo. Inhibition of translation by the antibiotics chloramphenicol and spectinomycin also resulted in splicing deficiency. Ribosomal protein S12, a protein with RNA chaperone activity, and CYT-18, a protein that stabilizes the three-dimensional structure of group I introns, efficiently rescued the stop codon mutants. We identified a region in the upstream exon that interferes with splicing. Point mutations in this region efficiently alleviate the effect of a nonsense codon. We infer from these results that the ribosome acts as an RNA chaperone to facilitate proper folding of the intron.     

Spectrum of splicing errors caused by CHRNE mutations affecting introns and intron/exon boundaries

Background: Mutations in CHRNE, the gene encoding the muscle nicotinic acetylcholine receptor ε subunit, cause congenital myasthenic syndromes. Only three of the eight intronic splice site mutations of CHRNE reported to date have had their splicing consequences characterised. Methods: We analysed four previously reported and five novel splicing mutations in CHRNE by introducing the entire normal and mutant genomic CHRNEs into COS cells. Results and conclusions: We found that short introns (82–109 nucleotides) favour intron retention, whereas medium to long introns (306–1210 nucleotides) flanking either or both sides of an exon favour exon skipping. Two mutations are of particular interest. Firstly, a G→T substitution at the 3'' end of exon 8 predicts an R286M missense mutation, but instead results in skipping of exon 8. In human genes, a mismatch of the last exonic nucleotide to U1 snRNP is frequently compensated by a matching nucleotide at intron position +6. CHRNE intron 8 has a mismatch at position +6, and accordingly fails to compensate for the exonic mutation at position –1. Secondly, a 16 bp duplication, giving rise to two 3'' splice sites (g.IVS10-9_c.1167dup16), results in silencing of the downstream 3'' splice site. This conforms to the scanning model of recognition of the 3'' splice site, which predicts that the first "ag" occurring after the branch point is selected for splicing.     

Ab initio identification of functionally interacting pairs of cis-regulatory elements

Cooperatively acting pairs of cis-regulatory elements play important roles in many biological processes. Here, we describe a statistical approach, compositionally orthogonalized co-occurrence analysis (coCOA) that detects pairs of oligonucleotides that preferentially co-occur in pairs of sequence regions, controlling for correlations between the compositions of the analyzed regions. coCOA identified three clusters of oligonucleotide pairs that frequently co-occur at 5′ and 3′ ends of human and mouse introns. The largest cluster involved GC-rich sequences at the 5′ ends of introns that co-occur and are co-conserved with specific AU-rich sequences near intron 3′ ends. These motifs are preferentially conserved when they occur together, as measured by a new co-conservation measure, supporting common in vivo function. These motif pairs are also enriched in introns flanking alternative “cassette” exons, suggesting a role in silencing of intervening exons, and we showed that these motifs can cooperatively silence splicing of an intervening exon in a splicing reporter assay. This approach can be easily generalized to problems beyond RNA splicing.     

5' exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

A general consequence of pre-mRNA splicing is the stable deposition of several proteins 20-24 nucleotides (nt) upstream of exon-exon junctions on spliced mRNAs. This exon junction complex (EJC) contains factors involved in mRNA export, cytoplasmic localization, and nonsense-mediated mRNA decay. Here we probed the mechanism and timing of EJC assembly. Over the course of splicing, the 5' exon is subject to numerous dynamic protein-RNA interactions involving at least nine distinct polypeptides. Within the fully assembled spliceosome, these interactions afford protection to the last 25-27 nt of the 5' exon intermediate. Coincident with exon ligation, interactions at the 3' end of the 5' exon disappear, and new species associate with position -24. Mass spectrometry and Western blotting of purified H, C, and mRNP complexes revealed that at least one EJC component, REF/Aly, can interact with pre-mRNA prior to spliceosome assembly, whereas Y14, Magoh, RNPS1, UAP56, and SRm160 are found in intermediate-containing spliceosomes. Upon exon ligation, association of RNPS1, UAP56, and SRm160 is destabilized. In contrast, REF/Aly, Y14, and Magoh remain stably bound to spliced mRNA, indicating that these three proteins are components of the EJC core.     

Mutation of the AAUAAA polyadenylation signal depresses in vitro splicing of proximal but not distal introns.

To investigate the relationship between splicing and polyadenylation during the production of vertebrate mRNAs, we examined the effect of mutation of a poly(A) site on splicing of upstream introns. Mutation of the AAUAAA polyadenylation consensus sequence inhibited in vitro splicing of an upstream intron. The magnitude of the depression depended on the magnesium concentration. Dependence of splicing on polyadenylation signals suggests the existence of interaction between polyadenylation and splicing factors. In multi-intron precursor RNAs containing duplicated splice sites, mutation of the poly(A) site inhibited removal of the last intron, but not the removal of introns farther upstream. Inhibition of removal of only the last intron suggests segmental recognition of multi-exon precursor RNAs and is consistent with previous suggestions that signals at both ends of an exon are required for effective splicing of an upstream intron.     

Crystal structure of the Drosophila Mago nashi-Y14 complex

Pre-mRNA splicing is essential for generating mature mRNA and is also important for subsequent mRNA export and quality control. The splicing history is imprinted on spliced mRNA through the deposition of a splicing-dependent multiprotein complex, the exon junction complex (EJC), at approximately 20 nucleotides upstream of exon-exon junctions. The EJC is a dynamic structure containing proteins functioning in the nuclear export and nonsense-mediated decay of spliced mRNAs. Mago nashi (Mago) and Y14 are core components of the EJC, and they form a stable heterodimer that strongly associates with spliced mRNA. Here we report a 1.85 A-resolution structure of the Drosophila Mago-Y14 complex. Surprisingly, the structure shows that the canonical RNA-binding surface of the Y14 RNA recognition motif (RRM) is involved in extensive protein-protein interactions with Mago. This unexpected finding provides important insights for understanding the molecular mechanisms of EJC assembly and RRM-mediated protein-protein interactions.     

DNA sequence and organization of the mitochondrial ND1 gene from Podospora anserina: analysis of alternate splice sites

Summary Earlier, we reported that the ND1 mitochondrial gene of Podospora anserina is mosaic, containing at least three class I introns. We have now completed the sequence of the ND1 gene and have determined that it contains four class I introns of 1,820, 2,631, 2,256 and 2,597 by with the entire gene complex containing 10,505 bp, only 1,101 of which are exon sequences. Introns 1 and 3 appear to be related in that their open reading frames (ORFS) exhibit extensive amino acid sequence similarity and like the URFN sequence from Neurospora crassa have multiple sequence repetitions. Introns 2 and 4 are similar in that both appear to be mosaic introns. Where intron 2 has many short ORFS, intron 4 has two, 391 and 262 as respectively. The first ORF has some patch work sequence similarity with one of the intron 2 ORFs but the second ORF is strikingly similar to the single intron ORF in the ND1 gene of N. crassa. Just upstream of the sequences necessary to form the central core of the P. anserina intron 4 secondary structure, there is a 17 bp sequence which is an exact replica of the exon sequence abutting the 5 flank of the 1,118 by N. crassa ND1 intron. Secondary structure analysis suggests that the 2,597 by intron 4 can fold as an entity but a similar structure can be constructed just from an 1,130 bp portion by utilizing the 17 bp element as an alternate splice site. Detailed structural analysis suggests that intron 4 (as well as the single ND1 intron from N. crassa) can utilize helical configurations which bring the downstream open reading frame into juxtaposition with the exon sequences.     

Self-splicing activity of the mitochondrial group-I introns from Aspergillus nidulans and related introns from other species

The mitochondrial genome of Aspergillus nidulans contains several group-I introns. Each one has been assayed for its ability to self-splice in vitro in the absence of proteins. The intron from the apocytochrome b gene is unusual among subgroup IB4 introns in being able to self-splice, unlike a similar intron from Saccharomyces cerevisiae. The first intron in the cytochrome oxidase subunit-1 gene self-splices but only correctly completes the first step of splicing; cryptic 3′ splice-sites are recognized instead and these are also used at a low frequency in vivo. The highly homologous intron from Podospora anserina completes both steps in vitro. The remaining introns do not self-splice. The correlation between subgroup category, the likely presence of specific tertiary interactions, and self-splicing activity is discussed. Received: 16 May / 25 August 1997     

Genetic variation at a splicing branch point in intron 9 of the low density lipoprotein (LDL)-receptor gene: a rare mutation that disrupts mRNA splicing in a patient with familial hypercholesterolaemia and a common polymorphism

Mutations in the coding sequence, splice junctions or promoter of the gene for the low density lipoprotein (LDL) receptor are known to be the underlying cause of familial hypercholesterolaemia (FH), but mutations of this type cannot be identified in all patients with a clinical diagnosis of FH. We show here that minor sequence changes elsewhere in introns can be deleterious. A minor rearrangement 30 bp upstream from the junction of intron 9 with exon 10 was detected as a heteroduplex in amplified genomic DNA from one out of 300 heterozygous FH patients. The mutation destroys the only consensus sequence for a splicing branch point in intron 9 and analysis of mRNA from cells from the patient showed that it causes retention of intron 9 or, more rarely, in the use of cryptic splice sites in exon 10. The effect of the mutation on mRNA splicing was confirmed by analysis of mRNA in cells transfected with LDL-receptor mini-gene constructs expressing exons 9 and 10, together with the normal or mutant intron 9. A common C/T polymorphism within this branch point in intron 9 of the LDL-receptor gene does not affect mRNA splicing in vitro and is not associated with significant differences in mean plasma cholesterol concentration in a healthy population.