G Run-mediated Recognition of Proteolipid Protein and DM20 5′ Splice Sites by U1 Small Nuclear RNA Is Regulated by Context and Proximity to the Splice Site

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.

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U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2666-2676.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2666-2676

Author(s):

J B Cohen ◽

S D Broz ◽

A D Levinson

Keyword(s):

Splice Site ◽

Mammalian Cells ◽

Mrna Processing ◽

Mrna Splicing ◽

Small Nuclear Rna ◽

Splice Sites ◽

Gene Complementation ◽

Small Nuclear Rnas ◽

Nuclear Rna ◽

Mutant Genes

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.

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The Pre-mRNA 5′ Cap Determines Whether U6 Small Nuclear RNA Succeeds U1 Small Nuclear Ribonucleoprotein Particle at 5′ Splice Sites

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7510 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7510-7520 ◽

Cited By ~ 21

Author(s):

Laura O’Mullane ◽

Ian C. Eperon

Keyword(s):

Splice Site ◽

Major Effect ◽

Small Nuclear Rna ◽

Splice Sites ◽

U6 Snrna ◽

Small Nuclear Ribonucleoprotein ◽

U1 Snrna ◽

U1 Snrnp ◽

Nuclear Rna ◽

Nuclear Ribonucleoprotein

ABSTRACT Efficient splicing of the 5′-most intron of pre-mRNA requires a 5′ m7G(5′)ppp(5′)N cap, which has been implicated in U1 snRNP binding to 5′ splice sites. We demonstrate that the cap alters the kinetic profile of U1 snRNP binding, but its major effect is on U6 snRNA binding. With two alternative wild-type splice sites in an adenovirus pre-mRNA, the cap selectively alters U1 snRNA binding at the site to which cap-independent U1 snRNP binding is stronger and that is used predominantly in splicing; with two consensus sites, the cap acts on both, even though one is substantially preferred for splicing. However, the most striking quantitative effect of the 5′ cap is neither on U1 snRNP binding nor on the assembly of large complexes but on the replacement of U1 snRNP by U6 snRNA at the 5′ splice site. Inhibition of splicing by a cap analogue is correlated with the loss of U6 interactions at the 5′ splice site and not with any loss of U1 snRNP binding.

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Splicing of the Meiosis-Specific HOP2Transcript Utilizes a Unique 5′ Splice Site

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.7933 ◽

1999 ◽

Vol 19 (12) ◽

pp. 7933-7943 ◽

Cited By ~ 9

Author(s):

Jun-Yi Leu ◽

G. Shirleen Roeder

Keyword(s):

Saccharomyces Cerevisiae ◽

Splice Site ◽

Meiotic Prophase ◽

Nucleotide Substitution ◽

Small Nuclear Rna ◽

Single Nucleotide Substitution ◽

Coding Region ◽

Single Nucleotide ◽

Nuclear Rna ◽

Splicing Efficiency

ABSTRACT The Saccharomyces cerevisiae HOP2 gene is required to prevent formation of synaptonemal complex between nonhomologous chromosomes during meiosis. The HOP2 gene is expressed specifically in meiotic cells, with the transcript reaching maximum abundance early in meiotic prophase. The HOP2 coding region is interrupted by an intron located near the 5′ end of the gene. This intron contains a nonconsensus 5′ splice site (GUUAAGU) that differs from the consensus 5′ splice signal (GUAPyGU) by the insertion of a nucleotide and by a single nucleotide substitution. Bases flanking the HOP2 5′ splice site have the potential to pair with sequences in U1 small nuclear RNA, and mutations disrupting this pairing reduce splicing efficiency. HOP2pre-mRNA is spliced efficiently in the absence of the Mer1 and Nam8 proteins, which are required for splicing the transcripts of two other meiosis-specific genes.

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The U1 small nuclear RNA-protein complex selectively binds a 5′ splice site in vitro

Cell ◽

10.1016/0092-8674(83)90432-4 ◽

1983 ◽

Vol 33 (2) ◽

pp. 509-518 ◽

Cited By ~ 269

Author(s):

Stephen M. Mount ◽

Ingvar Pettersson ◽

Monique Hinterberger ◽

Aavo Karmas ◽

Joan A. Steitz

Keyword(s):

Splice Site ◽

Protein Complex ◽

Small Nuclear Rna ◽

Nuclear Rna

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Structures of the Human Spliceosomes Before and After Release of the Ligated Exon

10.1101/496695 ◽

2018 ◽

Author(s):

Xiaofeng Zhang ◽

Xiechao Zhan ◽

Chuangye Yan ◽

Wenyu Zhang ◽

Dongliang Liu ◽

...

Keyword(s):

Electron Microscopy ◽

Splice Site ◽

Mrna Splicing ◽

Small Nuclear Rna ◽

Exon Ligation ◽

Cryo Electron Microscopy ◽

Branch Point Sequence ◽

Nuclear Rna ◽

Before And After ◽

Functional States

Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 Å, respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3'-splice site is recognized by the junction between the 5'-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

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Evidence for a base-pairing interaction between U6 small nuclear RNA and 5' splice site during the splicing reaction in yeast.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.23.11269 ◽

1992 ◽

Vol 89 (23) ◽

pp. 11269-11273 ◽

Cited By ~ 51

Author(s):

H. Sawa ◽

J. Abelson

Keyword(s):

Splice Site ◽

Small Nuclear Rna ◽

Base Pairing ◽

Pairing Interaction ◽

Nuclear Rna ◽

Base Pairing Interaction

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Structure of the activated human minor spliceosome

Science ◽

10.1126/science.abg0879 ◽

2021 ◽

pp. eabg0879

Author(s):

Rui Bai ◽

Ruixue Wan ◽

Lin Wang ◽

Kui Xu ◽

Qiangfeng Zhang ◽

...

Keyword(s):

Electron Microscopy ◽

Splice Site ◽

Zinc Finger Protein ◽

Small Nuclear Rna ◽

Catalytic Center ◽

Cryo Electron Microscopy ◽

Finger Protein ◽

Branch Point Sequence ◽

Nuclear Rna ◽

U5 Snrna

The minor spliceosome mediates splicing of the rare but essential U12-type pre-mRNA. Here we report the atomic features of the activated human minor spliceosome determined by cryo-electron microscopy at 2.9-Å resolution. The 5′-splice site and branch point sequence of the U12-type intron are recognized by U6atac and U12 small nuclear RNA (snRNA), respectively. Five newly identified proteins stabilize the conformation of the catalytic center. The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome. The RBM48/ARMC7 complex binds the γ-monomethyl phosphate cap at the 5′-end of U6atac snRNA. The U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 snRNP. CRIPT stabilizes U12 snRNP. Our study provides a framework for mechanistic understanding of the function of the minor spliceosome.

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Detection of a Novel ATP-Dependent Cross-Linked Protein at the 5′ Splice Site-U1 Small Nuclear RNA Duplex by Methylene Blue-Mediated Photo-Cross-Linking

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.6910 ◽

1998 ◽

Vol 18 (12) ◽

pp. 6910-6920 ◽

Cited By ~ 15

Author(s):

Zhi-Ren Liu ◽

Bruno Sargueil ◽

Christopher W. J. Smith

Keyword(s):

Methylene Blue ◽

Splice Site ◽

Atp Hydrolysis ◽

Cross Linking ◽

Small Nuclear Rna ◽

Rna Structures ◽

Small Nuclear Ribonucleoprotein ◽

Nuclear Rna ◽

Rna Duplexes ◽

Site Recognition

ABSTRACT Assembly of spliceosomes involves a number of sequential steps in which small nuclear ribonucleoprotein particles (snRNPs) and some non-snRNP proteins recognize the splice site sequences and undergo various conformational rearrangements. A number of important intermolecular RNA-RNA duplexes are formed transiently during the process of splice site recognition. Various steps in the assembly pathway are dependent upon ATP hydrolysis, either for protein phosphorylation or for the activity of helicases, which may modulate the RNA structures. Major efforts have been made to identify proteins that interact with specific regions of the pre-mRNA during the stages of spliceosome assembly and catalysis by site-specific UV cross-linking. However, UV cross-linking is often inefficient for the detection of proteins that interact with base-paired RNA. Here we have used the complementary approach of methylene blue-mediated photo-cross-linking to detect specifically proteins that interact with the duplexes formed between pre-mRNA and small nuclear RNA (snRNA). We have detected a novel cross-link between a 65-kDa protein (p65) and the 5′ splice site. A range of data suggest that p65 cross-links to the transient duplex formed by U1 snRNA and the 5′ splice site. Moreover, although p65 cross-linking requires only a 5′ splice site within the pre-mRNA, it also requires ATP hydrolysis, suggesting that its detection reflects a very early ATP-dependent event during splicing.

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Animal, Fungi, and Plant Genome Sequences Harbor Different Non-Canonical Splice Sites

Cells ◽

10.3390/cells9020458 ◽

2020 ◽

Vol 9 (2) ◽

pp. 458 ◽

Cited By ~ 3

Author(s):

Katharina Frey ◽

Boas Pucker

Keyword(s):

Splice Site ◽

Messenger Rna ◽

Plant Genome ◽

Fungal Genome ◽

Small Nuclear Rna ◽

Splice Sites ◽

Genome Sequences ◽

Oikopleura Dioica ◽

Protein Encoding ◽

Encoding Genes

Most protein-encoding genes in eukaryotes contain introns, which are interwoven with exons. Introns need to be removed from initial transcripts in order to generate the final messenger RNA (mRNA), which can be translated into an amino acid sequence. Precise excision of introns by the spliceosome requires conserved dinucleotides, which mark the splice sites. However, there are variations of the highly conserved combination of GT at the 5′ end and AG at the 3′ end of an intron in the genome. GC-AG and AT-AC are two major non-canonical splice site combinations, which have been known for years. Recently, various minor non-canonical splice site combinations were detected with numerous dinucleotide permutations. Here, we expand systematic investigations of non-canonical splice site combinations in plants across eukaryotes by analyzing fungal and animal genome sequences. Comparisons of splice site combinations between these three kingdoms revealed several differences, such as an apparently increased CT-AC frequency in fungal genome sequences. Canonical GT-AG splice site combinations in antisense transcripts are a likely explanation for this observation, thus indicating annotation errors. In addition, high numbers of GA-AG splice site combinations were observed in Eurytemora affinis and Oikopleura dioica. A variant in one U1 small nuclear RNA (snRNA) isoform might allow the recognition of GA as a 5′ splice site. In depth investigation of splice site usage based on RNA-Seq read mappings indicates a generally higher flexibility of the 3′ splice site compared to the 5′ splice site across animals, fungi, and plants.

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