Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo

Waldsich, Christina; Semrad, Katharina; Schroeder, Renée

doi:10.1017/s1355838298981444

Cited by 29 publications

(28 citation statements)

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“…In contrast to splicing at the 5Ј splice site, splicing at this cryptic splice site is not dependent on translation. As has been shown previously, inhibition of translation by the antibiotic neomycin B results in accumulation of cryptic splice product and inhibition of splicing at the 5Ј splice site (25) (Fig. 6B).…”

Section: Folding Of the Td P1 Stem In Vitro And In Vivosupporting

confidence: 84%

“…We have shown that in vivo splicing of this intron requires translation (23) and that inhibition of translation results in splicing deficiency and in increased mis-splicing due to alternative base pairing of the IGS, resulting in a so-called cryptic P1 stem (24,25). To gain a better understanding of td intron folding and substrate binding, basic kinetic parameters were established for the first step of splicing in a trans-cleaving ribozyme.…”

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confidence: 99%

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Folding Problems of the 5′ Splice Site Containing the P1 Stem of the Group I Thymidylate Synthase Intron

Pichler

Schroeder

2002

Journal of Biological Chemistry

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We developed an in vitro cleaving assay for the thymidylate synthase (td) group I intron and observed that the off-rate of the substrate is faster than cleavage. From the sequence stems P1 and P2 can vary from 4 to 8 and from 6 to 10 base pairs, respectively, with folding of a long P1 stem being in competition with folding of a long P2 stem. Shorter substrates, which cannot compete with the formation of an extended P2, result in faster cleavage, suggesting that binding of the substrate indeed interferes with folding of stem P2. In vivo splicing analyses of mutants containing alterations in stems P1 and P2 indicate that the wild-type exon sequence of P1 is suboptimal for splicing. Furthermore, folding of P1 in vivo is in competition with an alternative cryptic P1 stem resulting in mis-splicing. Translation promotes splicing at the correct 5 splice site, whereas in the absence of translation, mis-splicing is favored. The combination of the in vitro and in vivo assays clearly displays the folding problems for correct splice site selection in this group I intron.Group I introns are found in rRNAs, tRNAs, or mRNAs (1, 2). The structure of the flanking exons influences intron folding and contributes significantly to the energy landscape for folding both in vitro and in vivo (3,4). For example, the exon 1 structure of the Tetrahymena rRNA intron affects folding of the 5Ј splice site, reducing misfolding and thereby facilitating splicing (5-7). Although formation of the secondary structure in the core of group I introns is an intrinsic property of the RNA, assembly of the tertiary structure and docking of the splice sites often require additional splicing factors (8 -11).For the Tetrahymena rRNA intron, folding of the 5Ј splice site containing P1 stem has been extensively characterized in vitro using a trans-cleaving ribozyme and providing the 5Ј splice site as a substrate in trans (12, 13). Substrate binding is a two-step process (14, 15). The internal guide sequence (IGS) 1 defines the 5Ј splice sites (SS) by base pairing to the exon sequences, forming a stem named P1. In the first step of binding, the substrate base pairs with the IGS, and in the second step, the P1 stem is docked into the catalytic core of the intron via several hydrogen bond interactions (14, 16 -21). The structural domains after P1 vary substantially between group I introns; some introns contain no P2 stem, some contain a P2 stem alone, and others contain P2 and P2.1 stems (2,22).For the T4 phage-derived td intron, which is inserted into the mainly unstructured coding region of the thymidylate synthase gene, no specific splicing factor has been identified until now. We have shown that in vivo splicing of this intron requires translation (23) and that inhibition of translation results in splicing deficiency and in increased mis-splicing due to alternative base pairing of the IGS, resulting in a so-called cryptic P1 stem (24,25). To gain a better understanding of td intron folding and substrate binding, basic kinetic parameters were established for ...

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Section: Folding Of the Td P1 Stem In Vitro And In Vivosupporting

confidence: 84%

mentioning

confidence: 99%

Folding Problems of the 5′ Splice Site Containing the P1 Stem of the Group I Thymidylate Synthase Intron

Pichler

Schroeder

2002

Journal of Biological Chemistry

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“…Figure 6C depicts our current view of how StpA and Cyt-18 influence the folding pathway of the td intron. At this time, it is unknown which intermediates occur during in vivo folding, but it is known, however, that misfolding of the td pre-mRNA interferes with splicing Waldsich et al 1998). We propose that StpA resolves the tertiary fold of the intron, giving the molecule another chance to fold into the native splicing competent structure.…”

Section: Opposite Mechanisms Of Action: Stpa Versus Cyt-18mentioning

confidence: 95%

“…Efficient splicing of the td intron in vivo is dependent on the ribosome Waldsich et al 1998). Uncoupling splicing from translation by introducing nonsense codons in the upstream exon reduces splicing of these mutant pre-mRNAs compared to the wild type.…”

Section: Stpa Rescues Splicing Of Exonic Td Mutants Containing Nonsenmentioning

confidence: 99%

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RNA chaperone StpA loosens interactions of the tertiary structure in the td group I intron in vivo

Waldsich¹,

Grossberger²,

Schroeder³

2002

Genes Dev.

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Efficient splicing of the td group I intron in vivo is dependent on the ribosome. In the absence of translation, the pre-mRNA is trapped in nonnative-splicing-incompetent conformations. Alternatively, folding of the pre-mRNA can be promoted by the RNA chaperone StpA or by the group I intron-specific splicing factor Cyt-18. To understand the mechanism of action of RNA chaperones, we probed the impact of StpA on the structure of the td intron in vivo. Our data suggest that StpA loosens tertiary interactions. The most prominent structural change was the opening of the base triples, which are involved in the correct orientation of the two major intron core domains. In line with the destabilizing activity of StpA, splicing of mutant introns with a reduced structural stability is sensitive to StpA. In contrast, Cyt-18 strengthens tertiary contacts, thereby rescuing splicing of structurally compromised td mutants in vivo. Our data provide direct evidence for protein-induced conformational changes within catalytic RNA in vivo. Whereas StpA resolves tertiary contacts enabling the RNA to refold, Cyt-18 contributes to the overall compactness of the td intron in vivo. Most RNAs have to fold into a defined native structure in order to fulfill their function. Because RNA supports a variety of non-Watson-Crick base pairs, it becomes difficult for a population of RNA molecules to adopt a single structure. As a consequence, RNAs tend to misfold in vitro and in vivo (Lindahl and Adams 1966;Walstrum and Uhlenbeck 1990;Woodson and Cech 1991;Pan and Woodson 1998;Semrad and Schroeder 1998;Treiber et al. 1998;Nikolcheva and Woodson 1999;Treiber and Williamson 2001). A variety of methodshydroxyl-radical footprinting, oligonucleotide hybridization, UV melting, native gel electrophoresis, X-ray synchrotron analysis, and single molecule fluorescence measurements-were used to study the folding pathway of the Tetrahymena group I intron in

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Molecular Complementarity of Proteomimetic Materials for Target‐Specific Recognition and Recognition‐Mediated Complex Functions

Kim

Jung

et al. 2023

Advanced Materials

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transduction to enable survival and adaption. They are composed of long folded chains made up of a variety of 20 different α-amino acids, which form elaborate molecular structures. These structures can then fit counterpart molecules via molecular complementarity, thus enabling them to perform their specific cellular functions (Figure 1). For instance, membrane receptors specifically recognize extracellular ligand molecules and transfer desired molecular signals into the cell across the ligand-impermeable membrane. [1][2][3] Moreover, different membrane channels exist at the cell surface; in response to external stimuli such as pH, temperature, and action potentials, they undergo conformational changes to modulate the permeability of specific ions and metabolites. [4][5][6] The received extracellular signals are then converted into appropriate cellular responses; this process involves the intracellular increase of certain molecules to control transcription factors and regulate gene expression. [7,8] Even within the cell, there are various proteinligand interactions; for example, some metabolites can bind transcription factors, allosterically regulating subsequent transcription by RNA polymerases. [9,10] To manage cellular metabolism, enzymes catalyze more than 5000 biochemical reactions under highly limited physiological conditions (e.g., mild temperature, neutral pH, and low salt concentrations), attributed to their specific substrate binding abilities and effective collaboration with cofactors or coenzymes. [11] The availability of proteins that interact with many different targets benefits a broad range of biological and biotechnological research. Due to the functional diversity and specificity of the proteins, synthetic biology has successfully refined and rewired various cellular functions to solve numerous issues in a diverse range of fields such as agriculture, [12] manufacturing, [13] pharmaceutics, [14] and therapeutics. [15,16] For example, the use of proteins that enable gene recognition and editing has contributed to the emergence of important metabolic and genetic engineering techniques, allowing for the modulation of microbial cell factories that can efficiently produce high-value products, including biofuels, [17] medicines, [14] and biomolecules for industrial use. [18] Moreover, genetically encoded signal transducers As biomolecules essential for sustaining life, proteins are generated from long chains of 20 different α-amino acids that are folded into unique 3D structures. In particular, many proteins have molecular recognition functions owing to their binding pockets, which have complementary shapes, charges, and polarities for specific targets, making these biopolymers unique and highly valuable for biomedical and biocatalytic applications. Based on the understanding of protein structures and microenvironments, molecular complementarity can be exhibited by synthesizable and modifiable materials. This has prompted researchers to explore the proteomimetic potentials of a diverse range of material...

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Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo

Cited by 29 publications

References 41 publications

Folding Problems of the 5′ Splice Site Containing the P1 Stem of the Group I Thymidylate Synthase Intron

Folding Problems of the 5′ Splice Site Containing the P1 Stem of the Group I Thymidylate Synthase Intron

RNA chaperone StpA loosens interactions of the tertiary structure in the td group I intron in vivo

Molecular Complementarity of Proteomimetic Materials for Target‐Specific Recognition and Recognition‐Mediated Complex Functions

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