Introns play an essential role in splicing-dependent formation of the exon junction complex (original) (raw)
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Nucleic Acids Research, 2021
Vertebrate genomes contain major (>99.5%) and minor (<0.5%) introns that are spliced by the major and minor spliceosomes, respectively. Major intron splicing follows the exon-definition model, whereby major spliceosome components first assemble across exons. However, since most genes with minor introns predominately consist of major introns, formation of exon-definition complexes in these genes would require interaction between the major and minor spliceosomes. Here, we report that minor spliceosome protein U11-59K binds to the major spliceosome U2AF complex, thereby supporting a model in which the minor spliceosome interacts with the major spliceosome across an exon to regulate the splicing of minor introns. Inhibition of minor spliceosome snRNAs and U11-59K disrupted exon-bridging interactions, leading to exon skipping by the major spliceosome. The resulting aberrant isoforms contained a premature stop codon, yet were not subjected to nonsense-mediated decay, but rather boun...
F1000 - Post-publication peer review of the biomedical literature, 2011
Background: A very early step in splice site recognition is exon definition, a process that is as yet poorly understood. Communication between the two ends of an exon is thought to be required for this step. We report genome-wide evidence for exons being defined through the combinatorial activity of motifs located in flanking intronic regions. Results: Strongly co-occurring motifs were found to specifically reside in four intronic regions surrounding a large number of human exons. These paired motifs occur around constitutive and alternative exons but not pseudo exons. Most co-occurring motifs are limited to intronic regions within 100 nucleotides of the exon. They are preferentially associated with weaker exons. Their pairing is conserved in evolution and they exhibit a lower frequency of single nucleotide polymorphism when paired. Paired motifs display specificity with respect to distance from the exon borders and in constitutive versus alternative splicing. Many resemble binding sites for heterogeneous nuclear ribonucleoproteins. Specific pairs are associated with tissue-specific genes, the higher expression of which coincides with that of the pertinent RNA binding proteins. Tested pairs acted synergistically to enhance exon inclusion, and this enhancement was found to be exon-specific. Conclusions: The exon-flanking sequence pairs identified here by genomic analysis promote exon inclusion and may play a role in the exon definition step in pre-mRNA splicing. We propose a model in which multiple concerted interactions are required between exonic sequences and flanking intronic sequences to effect exon definition.
RNA, 2010
The majority of mammalian pre-mRNAs contains multiple introns that are excised prior to export and translation. After intron excision, ligated exon intermediates participate in subsequent intron excisions. However, exon ligation generates an exon of increased size, a feature of pre-mRNA splicing that can interfere with downstream splicing events. These considerations raise the question of whether unique mechanisms exist that permit efficient removal of introns neighboring ligated exons. Kinetic analyses of multiple intron-containing pre-mRNAs revealed that splicing is more efficient following an initial intron removal event, suggesting that either the recruitment of the exon junction complex (EJC) to ligated exons increases the efficiency of multiple intron excisions or that the initial definition of splice sites is sufficient to permit efficient splicing of introns neighboring ligated exons. Knockdown experiments show that the deposition of the EJC does not affect subsequent splicing kinetics. Instead, spliceosomal components that are not involved in the initial splicing event remain associated with the pre-mRNA to ensure efficient removal of neighboring introns. Thus, ligated exons do not require redefinition, providing an additional kinetic advantage for exon defined splice sites. .
Nature Structural & Molecular Biology, 2008
The polypyrimidine tract binding protein (PTB) binds pre-mRNAs to alter splice-site choice. We characterized a series of spliceosomal complexes that assemble on a pre-mRNA under conditions of either PTB-mediated splicing repression or its absence. In the absence of repression, exon definition complexes that were assembled downstream of the regulated exon could progress to pre-spliceosomal A complexes and functional spliceosomes. Under PTB-mediated repression, assembly was arrested at an Alike complex that was unable to transition to spliceosomal complexes. Trans-splicing experiments indicated that, even when the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) are properly bound to the upstream and downstream exons, the presence of PTB prevents the interaction of the two exon complexes. Proteomic analyses of these complexes provide a new description of exon definition complexes, and indicate that splicing regulators can act on the transition between the exon definition complex and an intron-defined spliceosome. Alternative splicing is a common means of regulation for eukaryotic gene expression 1-3. Splicing choices are directed by proteins that bind to specific regulatory sequences in the pre-mRNA to alter spliceosome assembly, but the interactions of these proteins with the splicing apparatus are mostly unknown 4-6. Each intron to be excised from a pre-mRNA must be assembled into a spliceosome containing the five small ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6, and multiple auxiliary proteins 7-9. In vitro, the snRNPs sequentially bind the target intron to form discrete intermediate complexes termed H, E, A, B and C. The H complex contains the U1 snRNP bound to the 5¢ splice site of the pre-mRNA and many sequence-specific RNA binding proteins, including members of the heterogeneous nuclear ribonucleoprotein (hnRNP) group of proteins 10. The subsequent E complex contains the U2 auxiliary factor (U2AF) and splicing factor 1 (SF1) bound to the 3¢ splice site and branchpoint, respectively. The U2 snRNP is also present in this complex, but is relatively loosely associated 11. In the E complex, the 5¢ and 3¢ splice sites are brought together via an interaction between the U1-containing complex and the U2AF complex 12-14. The first ATP-dependent step in spliceosome assembly results in the stable association of the U2 snRNP to the branchpoint through RNA base-pairing, and the formation of the A complex. Binding of the U4/U6-U5 tri-snRNP then forms the B complex. Several structural rearrangements in the B complex lead to loss of the U1 and U4 snRNPs, resulting in the C complex 9,15,16. Here
Genes & development, 2014
Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, we propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splic...
Splicing of designer exons reveals unexpected complexity in pre-mRNA splicing
RNA, 2009
Pre-messengerRNA (mRNA) splicing requires the accurate recognition of splice sites by the cellular RNA processing machinery. In addition to sequences that comprise the branchpoint and the 39 and 59 splice sites, the cellular splicing machinery relies on additional information in the form of exonic and intronic splicing enhancer and silencer sequences. The high abundance of these motifs makes it difficult to investigate their effects using standard genetic perturbations, since their disruption often leads to the formation of yet new elements. To lessen this problem, we have designed synthetic exons comprised of multiple copies of a single prototypical exonic enhancer and a single prototypical exonic silencer sequence separated by neutral spacer sequences. The spacer sequences buffer the exon against the formation of new elements as the number and order of the original elements are varied. Over 100 such designer exons were constructed by random ligation of enhancer, silencer, and neutral elements. Each exon was positioned as the central exon in a 3-exon minigene and tested for exon inclusion after transient transfection. The level of inclusion of the test exons was seen to be dependent on the provision of enhancers and could be decreased by the provision of silencers. In general, there was a good quantitative correlation between the proportion of enhancers and splicing. However, widely varying inclusion levels could be produced by different permutations of the enhancer and silencer elements, indicating that even in this simplified system splicing decisions rest on complex interplays of yet to be determined parameters. .
Listening to Silence and Understanding Nonsense: Exonic Mutations That Affect Splicing
Nature Reviews Genetics, 2002
A typical mammalian gene is composed of several relatively short exons that are interrupted by much longer introns. To generate correct, mature mRNAs, the exons must be identified and joined together precisely and efficiently, in a process that requires the coordinated action of five small nuclear (sn)RNAs (U1, U2 and U4-U6) and more than 60 polypeptides 1,2 . The inaccurate recognition of exon-intron boundaries or the failure to remove an intron generates aberrant mRNAs that are either unstable or code for defective or deleterious protein isoforms.
Molecular Cell, 2006
The splicing machinery associates with genes to facilitate efficient cotranscriptional mRNA processing. We have mapped these associations by genome localization analysis to ascertain how splicing is achieved and regulated on a system-wide scale. Our data show that factors important for intron recognition sample nascent mRNAs and are retained specifically at intron-containing genes via RNA-dependent interactions. Spliceosome assembly proceeds cotranscriptionally but completes posttranscriptionally in most cases. Some intron-containing genes were not bound by the spliceosome, including several developmentally regulated genes. On this basis, we predicted and verified regulated splicing and observed a role for nuclear mRNA surveillance in monitoring those events. Finally, we present evidence that cotranscriptional processing events determine the recruitment of specific mRNA export factors. Broadly, our results provide mechanistic insights into the coordinated regulation of transcription, mRNA processing, and nuclear export in executing complex gene expression programs.