RESUMO
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.
Assuntos
Sítios de Splice de RNA , Saccharomyces cerevisiae , Processamento Alternativo , Splicing de RNA , Fatores de Processamento de RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Spliceossomos/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The fidelity of splice site selection is thought to be critical for proper gene expression and cellular fitness. In particular, proper recognition of 3'-splice site (3'SS) sequences by the spliceosome is a daunting task considering the low complexity of the 3'SS consensus sequence YAG. Here we show that inactivating the near-essential splicing factor Prp18p results in a global activation of alternative 3'SS, many of which harbor sequences that highly diverge from the YAG consensus, including some highly unusual non-AG 3'SS. We show that the role of Prp18p in 3'SS fidelity is promoted by physical interactions with the essential splicing factors Slu7p and Prp8p and synergized by the proofreading activity of the Prp22p helicase. Strikingly, structure-guided point mutations that disrupt Prp18p-Slu7p and Prp18p-Prp8p interactions mimic the loss of 3'SS fidelity without any impact on cellular growth, suggesting that accumulation of incorrectly spliced transcripts does not have a major deleterious effect on cellular viability. These results show that spliceosomes exhibit remarkably relaxed fidelity in the absence of Prp18p, and that new 3'SS sampling can be achieved genome-wide without a major negative impact on cellular fitness, a feature that could be used during evolution to explore new productive alternative splice sites.
RESUMO
We describe an RT-PCR protocol that allows high-resolution mapping of splicing products and isoforms using fluorescently labeled primers. Each species contains one fluorescent group allowing a direct comparison of the different isoforms despite size differences. A custom-size ladder enables the precise determination of cDNA lengths and discrimination of isoforms differing by less than five nucleotides on polyacrylamide gels. This protocol also allows the detection of products from in vitro splicing reactions, circumventing the need to use radiolabeled transcripts. For complete details on the use and execution of this protocol, please refer to Gabunilas and Chanfreau (2016).
Assuntos
Isoformas de Proteínas/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Processamento Alternativo/genética , Sequência de Bases/genética , Primers do DNA , DNA Complementar/genética , Humanos , Nucleotídeos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Splicing de RNA/genética , RNA-Seq/métodos , Análise de Sequência de RNA/métodosRESUMO
Multiplexed assays allow functional testing of large synthetic libraries of genetic elements, but are limited by the designability, length, fidelity and scale of the input DNA. Here, we improve DropSynth, a low-cost, multiplexed method that builds gene libraries by compartmentalizing and assembling microarray-derived oligonucleotides in vortexed emulsions. By optimizing enzyme choice, adding enzymatic error correction and increasing scale, we show that DropSynth can build thousands of gene-length fragments at >20% fidelity.
