RESUMO
We generate high-precision measurements of the in vivo rates of both chemical steps of pre-mRNA splicing across the genome-wide complement of substrates in yeast by coupling metabolic labeling, multiplexed primer-extension sequencing, and kinetic modeling. We demonstrate that the rates of intron removal vary widely, splice-site sequences are primary determinants of 1st step but have little apparent impact on 2nd step rates, and the 2nd step is generally faster than the 1st step. Ribosomal protein genes (RPGs) are spliced faster than non-RPGs at each step, and RPGs share evolutionarily conserved properties that may contribute to their faster splicing. A genetic variant defective in the 1st step of the pathway reveals a genome-wide defect in the 1st step but an unexpected, transcript-specific change in the 2nd step. Our work demonstrates that extended co-transcriptional association is an important determinant of splicing rate, a conclusion at odds with recent claims of ultra-fast splicing.
Assuntos
Precursores de RNA , Splicing de RNA , Íntrons/genética , Cinética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Targeted RNA sequencing (RNA-seq) aims to focus coverage on areas of interest that are inadequately sampled in standard RNA-seq experiments. Here we present multiplexed primer extension sequencing (MPE-seq), an approach for targeted RNA-seq that uses complex pools of reverse-transcription primers to enable sequencing enrichment at user-selected locations across the genome. We targeted hundreds to thousands of pre-mRNA splice junctions and obtained high-precision detection of splice isoforms, including rare pre-mRNA splicing intermediates.
Assuntos
Primers do DNA , Genes Fúngicos , Splicing de RNA , Saccharomyces cerevisiae/genética , Sequenciamento de Nucleotídeos em Larga Escala , Transcrição ReversaRESUMO
Abnormal metabolism and sustained proliferation are hallmarks of cancer. Pyruvate kinase M2 (PKM2) is a metabolic enzyme that plays important roles in both processes. Recently, PKM2 was shown to have protein kinase activity phosphorylating histone H3 and promoting cancer cell proliferation. However, the mechanism and extent of this protein kinase in cancer cells remain unclear. Here, we report that binding of succinyl-5-aminoimidazole-4-carboxamide-1-ribose-5'-phosphate (SAICAR), a metabolite abundant in proliferating cells, induces PKM2's protein kinase activity in vitro and in cells. Protein microarray experiments revealed that more than 100 human proteins, mostly protein kinases, are phosphorylated by PKM2-SAICAR. In particular, PKM2-SAICAR phosphorylates and activates Erk1/2, which in turn sensitizes PKM2 for SAICAR binding through phosphorylation. Additionally, PKM2-SAICAR was necessary to induce sustained Erk1/2 activation and mitogen-induced cell proliferation. Thus, the ligand-induced protein kinase activity from PKM2 is a mechanism that directly couples cell proliferation with intracellular metabolic status.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Proteínas de Transporte/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/metabolismo , Inibidores de Proteínas Quinases/química , Ribonucleotídeos/química , Transdução de Sinais , Hormônios Tireóideos/metabolismo , Difosfato de Adenosina/química , Aminoimidazol Carboxamida/química , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Células HeLa , Humanos , Isoenzimas/metabolismo , Ligantes , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Análise Serial de Proteínas , Ligação Proteica , Proteínas Recombinantes/metabolismo , Xenopus laevis , Proteínas de Ligação a Hormônio da TireoideRESUMO
The study of pre-mRNA splicing has been greatly aided by the advent of RNA sequencing (RNA-seq), which enables the genome-wide detection of discrete splice isoforms. Quantification of these splice isoforms requires analysis of splicing informative sequencing reads, those that unambiguously map to a single splice isoform, including exon-intron spanning alignments corresponding to retained introns, as well as exon-exon junction spanning alignments corresponding to either canonically- or alternatively-spliced isoforms. Because most RNA-seq experiments are designed to produce sequencing alignments that uniformly cover the entirety of transcripts, only a comparatively small number of splicing informative alignments are generated for any given splice site, leading to a decreased ability to detect and/or robustly quantify many splice isoforms. To address this problem, we have recently described a method termed Multiplexed Primer Extension sequencing, or MPE-seq, which uses pools of reverse transcription primers to target sequencing to user selected loci. By targeting reverse transcription to pre-mRNA splice junctions, this approach enables a dramatic enrichment in the fraction of splicing informative alignments generated per splicing event, yielding an increase in both the precision with which splicing efficiency can be measured, and in the detection of splice isoforms including rare splicing intermediates. Here we provide a brief review of the shortcomings associated with RNA-seq that drove our development of MPE-seq, as well as a detailed protocol for implementation of MPE-seq.
Assuntos
Isoformas de RNA/genética , RNA Mensageiro/genética , RNA-Seq/métodos , Processamento Alternativo , Biologia Computacional/métodos , Loci Gênicos , Precursores de RNA/genética , Sítios de Splice de RNA/genéticaRESUMO
To cause disease, Salmonella must invade the intestinal epithelium employing genes encoded within Salmonella Pathogenicity Island 1 (SPI1). We show here that propionate, a fatty acid abundant in the intestine of animals, repressed SPI1 at physiologically relevant concentration and pH, reducing expression of SPI1 transcriptional regulators and consequently decreasing expression and secretion of effector proteins, leading to reduced bacterial penetration of cultured epithelial cells. Essential to repression was hilD, which occupies the apex of the regulatory cascade within SPI1, as loss of only this gene among those of the regulon prevented repression of SPI1 transcription by propionate. Regulation through hilD, however, was achieved through the control of neither transcription nor translation. Instead, growth of Salmonella in propionate significantly reduced the stability of HilD. Extending protein half-life using a Lon protease mutant demonstrated that protein stability itself did not dictate the effects of propionate and suggested modification of HilD with subsequent degradation as the means of action. Furthermore, repression was significantly lessened in a mutant unable to produce propionyl-CoA, while further metabolism of propionyl-CoA appeared not to be required. These results suggest a mechanism of control of Salmonella virulence in which HilD is post-translationally modified using the high-energy intermediate propionyl-CoA.