Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes

Black, Corbin; Whelan, Thomas A.; Garside, E.L.; MacMillan, Andrew M.; Fast, Naomi M.; Rader, Stephen D.

doi:10.1261/rna.079273.122

Cited by 10 publications

(5 citation statements)

References 154 publications

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“…The splicing of human 5′ss is not always dependent on the presence of U1 snRNP and in some cases, the depletion of U1 snRNP can be overcome by the addition of high concentrations of SR proteins ( 33–37 ). Interestingly, Cyanidioschyzon merolae might even lack the U1 snRNP and associated proteins completely and the spliceosome assembly might begin with the recognition of 5′ss by tri-snRNP ( 38 ). Therefore, it is theoretically possible that splicing of human STAT3 tandem 5′ss is U1-independent.…”

Section: Resultsmentioning

confidence: 99%

Splicing analysis of STAT3 tandem donor suggests non-canonical binding registers for U1 and U6 snRNAs

Kramárek,

Souček,

Réblová

et al. 2024

Nucleic Acids Research

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Tandem donor splice sites (5′ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Δ3 tandem 5′ss are a specific subclass of 5′ss separated by 3 nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5′ss is typically preferred, yet factors governing particular 5′ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Δ3 tandem 5′ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5′ss. However, the downstream 5′ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5′ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5′ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5′ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson–Crick base pairing with the downstream regulatory region. This study suggests that U6:5′ss interaction is more flexible than previously thought.

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Section: Resultsmentioning

confidence: 99%

Splicing analysis of STAT3 tandem donor suggests non-canonical binding registers for U1 and U6 snRNAs

Kramárek,

Souček,

Réblová

et al. 2024

Nucleic Acids Research

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“…In intracellular eukaryotes, the trend can also extend to introns, either due to loss, length reduction, or both. Microsporidians generally have few introns that are relatively short and retain a higher-than-average sequence similarity to one another [ 18 , 21 , 41 , 42 ], as well as a reduced spliceosomal machinery [ 41 , 43 , 44 ]. A few microsporidians have independently lost introns altogether [ 18 , 45 , 46 ].…”

Section: Resultsmentioning

confidence: 99%

Contrasting outcomes of genome reduction in mikrocytids and microsporidians

Žárský,

Karnkowska,

Boscaro

et al. 2023

BMC Biol

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Background Intracellular symbionts often undergo genome reduction, losing both coding and non-coding DNA in a process that ultimately produces small, gene-dense genomes with few genes. Among eukaryotes, an extreme example is found in microsporidians, which are anaerobic, obligate intracellular parasites related to fungi that have the smallest nuclear genomes known (except for the relic nucleomorphs of some secondary plastids). Mikrocytids are superficially similar to microsporidians: they are also small, reduced, obligate parasites; however, as they belong to a very different branch of the tree of eukaryotes, the rhizarians, such similarities must have evolved in parallel. Since little genomic data are available from mikrocytids, we assembled a draft genome of the type species, Mikrocytos mackini, and compared the genomic architecture and content of microsporidians and mikrocytids to identify common characteristics of reduction and possible convergent evolution. Results At the coarsest level, the genome of M. mackini does not exhibit signs of extreme genome reduction; at 49.7 Mbp with 14,372 genes, the assembly is much larger and gene-rich than those of microsporidians. However, much of the genomic sequence and most (8075) of the protein-coding genes code for transposons, and may not contribute much of functional relevance to the parasite. Indeed, the energy and carbon metabolism of M. mackini share several similarities with those of microsporidians. Overall, the predicted proteome involved in cellular functions is quite reduced and gene sequences are extremely divergent. Microsporidians and mikrocytids also share highly reduced spliceosomes that have retained a strikingly similar subset of proteins despite having reduced independently. In contrast, the spliceosomal introns in mikrocytids are very different from those of microsporidians in that they are numerous, conserved in sequence, and constrained to an exceptionally narrow size range (all 16 or 17 nucleotides long) at the shortest extreme of known intron lengths. Conclusions Nuclear genome reduction has taken place many times and has proceeded along different routes in different lineages. Mikrocytids show a mix of similarities and differences with other extreme cases, including uncoupling the actual size of a genome with its functional reduction.

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“…Because this type of substrate was shown not to require Prp18p for efficient splicing, it is possible that the effect of inactivating Prp18p in this organism might be less severe than in S. cerevisiae . In addition, Prp18p (and Slu7p) are not universally present in eukaryotes, as shown by the reduced spliceosome of the red algae Cyanidioschyzon merolae , which lacks these two proteins entirely ( 53 ). This observation begs the question of which proteins might be primarily involved in the mechanism of 3′SS fidelity, and whether the role of Prp18p in this process has been replaced by stronger interactions between Prp8p and the 3′SS, or by a more prominent role for Prp22p.…”

Section: Discussionmentioning

confidence: 99%

Splicing factor Prp18p promotes genome-wide fidelity of consensus 3′-splice sites

Roy,

Gabunilas,

Neutel

et al. 2023

Nucleic Acids Research

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The fidelity of splice site selection is critical for proper gene expression. In particular, proper recognition of 3′-splice site (3′SS) sequences by the spliceosome is challenging considering the low complexity of the 3′SS consensus sequence YAG. Here, we show that absence of the Prp18p splicing factor results in genome-wide activation of alternative 3′SS in S. cerevisiae, including highly unusual non-YAG sequences. Usage of these non-canonical 3′SS in the absence of Prp18p is enhanced by upstream poly(U) tracts and by their potential to interact with the first intronic nucleoside, allowing them to dock in the spliceosome active site instead of the normal 3′SS. The role of Prp18p in 3′SS fidelity is facilitated by interactions with Slu7p and Prp8p, but cannot be fulfilled by Slu7p, identifying a unique role for Prp18p in 3′SS fidelity. This fidelity function is synergized by the downstream proofreading activity of the Prp22p helicase, but is independent from another late splicing helicase, Prp43p. Our results show that spliceosomes exhibit remarkably relaxed 3′SS sequence usage in the absence of Prp18p and identify a network of spliceosomal interactions centered on Prp18p which are required to promote the fidelity of the recognition of consensus 3′SS sequences.

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Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes

Cited by 10 publications

References 154 publications

Splicing analysis of STAT3 tandem donor suggests non-canonical binding registers for U1 and U6 snRNAs

Splicing analysis of STAT3 tandem donor suggests non-canonical binding registers for U1 and U6 snRNAs

Contrasting outcomes of genome reduction in mikrocytids and microsporidians

Splicing factor Prp18p promotes genome-wide fidelity of consensus 3′-splice sites

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