5′ cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5′ splice region, not by the conserved 5′ GU

doi:10.1016/0092-8674(87)90219-4

Cell

Volume 50, Issue 2, 17 July 1987, Pages 237-246

https://doi.org/10.1016/0092-8674(87)90219-4 Get rights and content

Abstract

We have generated all possible single point mutations of the invariant 5′ GT of the large β-globin intron and determined their effect on splicing in vitro. None of the mutants prevented cleavage in the 5′ splice region, but many reduced or abolished exon joining. The mutations GT→TT and GT→CT resulted in a shift of the 5′ cleavage site one nucleotide upstream; in the case of the mutation GT→TT, this shift was reverted by a second site mutation within the 5′ splice region. Our results suggest that the 5′ cleavage site is determined not by the conserved GU sequence but by the 5′ splice region as a whole, most probably via base-pairing to the 5′ end of the U1 snRNA.

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For the AS of pre-RNA, it is critical to select the appropriate splice sites; the usage of different alternative splice sites leads to different splicing outcomes, thereby affecting the stability of RNA or the function of protein products. The 5′ SS mainly consists of the canonical GT; however, it is determined not by GT dinucleotide but by the whole 5′ splice region, which may be the −3∼+6 region recognized by U1 snRNA using base-pairing at the exon–intron junction (Aebi et al., 1987; Laura et al., 2013). Compared with 5′ SS, the selection and regulation of 3′ SS is more complicated, and 3′ SS is comprised of three consensus motifs (BP, Py, and AG dinucleotide) that are differently conserved, required or spaced among different species (Sohail and Xie, 2015).
Alternative splicing (AS) selects different alternative splice sites and produces a variety of transcripts with different exon/intron combinations, which may result in multiple protein isoforms. The splicing signals include cis-elements and RNA structures; however, the mechanisms of AS regulation in plants have yet to be elucidated. Previous studies have shown that in Platanus acerifolia, the FLOWERING LOCUS T (FT) homolog PaFT has a unique and complex AS pattern, in which most of the splice forms of PaFT involve the first and/or second intron, and the FD homolog PaFDL1 produces two transcripts via AS, whereas the other FT homolog PaFTL is not regulated by AS. In this study, the regulatory mechanism of the AS of PaFT was demonstrated to be conserved in different plant species. To define the distribution of the AS regulatory signals, the intron-swap, site-directed mutagenesis of alternative splice sites, and deletion experiment were performed. For the PaFT gene, all the signals that regulate the AS of the first intron were located within this intron, while the usage of the first alternative splice site in the second intron was determined by the first intron. Meanwhile, the AS of PaFDL1 might be co-regulated by exons and the first intron. Additionally, the first alternative splice site and adjacent region in PaFT intron 1 might contain cis-elements and/or RNA structures that affect the use of the other sites. This study had provided a deeper insight into the distribution of AS signals in plants, namely the AS signals of different splice sites might exist in the intron where the sites were present, and might also be distributed in exons or other introns.
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The cell wall is a distinctive feature of filamentous fungi, providing them with structural integrity and protection from both biotic and abiotic factors. Unlike plant cell walls, fungi rely on structurally strong hydrophobic chitin core for mechanical strength together with alpha- and beta-glucans, galactomannans and glycoproteins. Cell wall stress conditions are known to alter the cell wall through the signaling cascade of the cell wall integrity (CWI) pathway and can result in increased cell wall chitin deposition. A previously isolated set of Aspergillus niger cell wall mutants was screened for increased cell wall chitin deposition. UV-mutant RD15.8#16 was found to contain approximately 60% more cell wall chitin than the wild type. In addition to the chitin phenotype, RD15.8#16 exhibits a compact colony morphology and increased sensitivity towards SDS. RD15.8#16 was subjected to classical genetic approach for identification of the underlying causative mutation, using co-segregation analysis and SNP genotyping. Genome sequencing of RD15.8#16 revealed eight SNPs in open reading frames (ORF) which were individually checked for co-segregation with the associated phenotypes, and showed the potential relevance of two genes located on chromosome IV. In situ re-creation of these ORF-located SNPs in a wild type background, using CRISPR/Cas9 genome editing, showed the importance Rab GTPase dissociation inhibitor A (gdiA) for the phenotypes of RD15.8#16. An alteration in the 5′ donor splice site of gdiA reduced pre-mRNA splicing efficiency, causing aberrant cell wall assembly and increased chitin levels, whereas gene disruption attempts showed that a full gene deletion of gdiA is lethal.
Molecular choreography of pre-mRNA splicing by the spliceosome
2019, Current Opinion in Structural Biology
The spliceosome executes eukaryotic precursor messenger RNA (pre-mRNA) splicing to remove noncoding introns through two sequential transesterification reactions, branching and exon ligation. The fidelity of this process is based on the recognition of the conserved sequences in the intron and dynamic compositional and structural rearrangement of this multi-megadalton machinery. Since atomic visualization of the splicing active site in an endogenous Schizosaccharomyces pombe spliceosome in 2015, high-resolution cryoelectron microscopy (cryo-EM) structures of other spliceosome intermediates began to uncover the molecular mechanism. Recent advances in the structural biology of the spliceosome make it clearer the mechanisms of its assembly, activation, disassembly and exon ligation. Together, these discrete structural images give rise to a molecular choreography of the spliceosome.
Structures of the Catalytically Activated Yeast Spliceosome Reveal the Mechanism of Branching
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Pre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B^∗) is pivotal for understanding the branching reaction. In this study, we assembled the B^∗ complexes on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B^∗ complexes at overall resolutions of 2.9–3.8 Å. The duplex between U2 small nuclear RNA (snRNA) and the branch point sequence (BPS) is discretely away from the 5′-splice site (5′SS) in the three B^∗ complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5′SS, with the BPS nucleophile positioned 4 Å away from the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in branching. These structures on different pre-mRNAs reveal substrate-specific conformations of the spliceosome in a major functional state.
Activation of a cryptic splice site in a potentially lethal coagulation defect accounts for a functional protein variant
2012, Biochimica et Biophysica Acta - Molecular Basis of Disease
Citation Excerpt :
This has been shown by us in coagulation factor VII (FVII) deficiency [2] and by Pacho and co-workers in LAMA3 deficiency [3]. Mutations at the completely conserved guanine at the intronic + 1 position (Fig. 1A, top) of donor splice sites (5'ss), a relatively frequent cause of severe human genetic diseases [4], are also generally considered null mutations as they are predicted to disrupt the splicing process [5]. However, for most of them, the complete loss-of-function feature has not been demonstrated.
Changes at the invariable donor splice site + 1 guanine, relatively frequent in human genetic disease, are predicted to abrogate correct splicing, and thus are classified as null mutations. However, their ability to direct residual expression, which might have pathophysiological implications in several diseases, has been poorly investigated. As a model to address this issue, we studied the IVS6 + 1G > T mutation found in patients with severe deficiency of the protease triggering coagulation, factor VII (FVII), whose absence is considered lethal. In expression studies, the IVS6 + 1G > T induced exon 6 skipping and frame-shift, and prevented synthesis of correct FVII transcripts detectable by radioactive/fluorescent labelling or real-time RT-PCR. Intriguingly, the mutation induced the activation of a cryptic donor splice site in exon 6 and production of an in-frame 30 bp deleted transcript (8 ± 2%). Expression of this cDNA variant, lacking 10 residues in the activation domain, resulted in secretion of trace amounts (0.2 ± 0.04%) of protein with appreciable specific activity (48 ± 16% of wt-FVII). Altogether these data indicate that the IVS6 + 1G > T mutation is compatible with the synthesis of functional FVII molecules (~ 0.01% of normal, 1 pM), which could trigger coagulation. The low but detectable thrombin generation (352 ± 55 nM) measured in plasma from an IVS6 + 1G > T homozygote was consistent with a minimal initiation of the enzymatic cascade. In conclusion, we provide experimental clues for traces of FVII expression, which might have reverted an otherwise perinatally lethal genetic condition.
RNA Processing in C. elegans
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Citation Excerpt :
In all organisms, introns containing these splice-site consensus sequences are called GT–AG introns. The splicing reaction happens in multiple steps: First, the U1 snRNP binds to the 5′ splice site of the intron via base-pairing between the U1 snRNP sequence 3′-UC/CAUUCA-5′ and the 5′ splice-site consensus 5′-AG/GURAGU-3′ (Aebi et al., 1987; Séraphin et al., 1988). The two subunits of the U2 snRNP auxillary factor (U2AF), U2AF65 and U2AF35, bind the polypryimidine tract and the AG nucleotides of the 3′ splice site, respectively (Merendino et al., 1999; Wu et al., 1999; Zorio and Blumenthal, 1999a).
In Caenorhabditis elegans, newly transcribed RNA is processed in several novel ways. Although introns are removed by a canonical spliceosome, they have evolved several specialized features that reflect the differences in the way they are recognized and the way they are spliced. C. elegans introns are unusually short, in part because they have no specific branch-point sequences and contain minimal polypryimidine tracts. Instead, their 3′ splice site is characterized by a highly conserved consensus sequence, which alone may be sufficient to position all spliceosomal elements at the 3′ end of the intron. Many RNA molecules are also trans-spliced: a capped 22 nt RNA leader is donated by one of a family of specialized snRNPs and spliced to an unpaired 3′ splice site, usually just upstream of the start codon. The RNA upstream of this splice site, the outron, is removed during trans-splicing and presumably degraded, making the identification of the transcriptional start site problematic. Transcripts from approximately 70% of all genes are trans-spliced. Trans-splicing has enabled the evolution of operons – multigene clusters in which a single upstream promoter drives the transcription of a polycistronic pre-mRNA. The C. elegans genome contains more than 1000 such operons. The polycistronic pre-mRNA is processed into individual gene-encoding mRNAs by coordinated upstream 3′ end formation and downstream trans-splicing. An intercistronic RNA sequence, the Ur element, plays a key role in specifying downstream trans-splicing.

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^★: Present address: Max Planck Institut für Molekulare Genetik, Ihnestrasse 63-73, D-1000 Berlin 33, Federal Republic of Germany.

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Cell

Article5′ cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5′ splice region, not by the conserved 5′ GU

Abstract

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J. Mol. Biol.

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J. Biol. Chem.

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Ribonucleoprotein complex formation during pre-mRNA splicing in vitro

Mol. Cell. Biol.

A catalogue of splice junction and putative branch point sequences from plant introns

Nucl. Acids Res.

Article
5′ cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5′ splice region, not by the conserved 5′ GU