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Detection of Ψ in RNA oligonucleotides and in tRNAs from S. cerevisiae strains by reverse transcription using RT-KTq I614Y

ABSTRACT: We have engineered an RT-active DNA polymerase variant called RT-KTq I614Y that produces error RT‑signatures specific for pseudouridine (Ψ) without prior chemical treatment of the RNA samples. These signatures are amplified during DNA amplicon library preparation and are detected by NGS. This method was applied to distinguish U from Ψ in RNA oligonucleotides (modified or unmodified) used in the previous polymerase screening and oligonucleotides which are designed from the human 18S rRNA at the position around 1445. Finally, RT-KTq I614Y was used to detect Ψ55 in tRNAGly(GCC) from Saccharomyces cerevisiae wildtype compared to data from tRNAGly(GCC) from S. cerevisiae pus4Δ.

ORGANISM(S): synthetic construct Saccharomyces cerevisiae

PROVIDER: GSE217198 | GEO | 2023/03/15

REPOSITORIES: GEO

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Genome-wide translational changes induced by the prion [PSI+]

Project description:Prions are infectious proteins that can adopt a structural conformation different from that of the normal protein. This change of conformation is then propagated among other molecules of the same protein. Prions are associated with neurodegenerative diseases in mammals, but are also found in fungi (in the yeast Saccharomyces cerevisiae and the filamentous fungus Podospora anserina), in which they control heritable traits. They are widespread in wild yeast strains, suggesting a biologically important role. [PSI+] is one of the most widely studied yeast prions. It corresponds to an aggregated conformation of the translational release factor, eRF3, which suppresses nonsense codons. [PSI+] modifies cellular fitness, inducing various phenotypes, depending on the genetic background. However, the genes displaying [PSI+]-controlled expression remain largely unknown. We used the recently described ribosome profiling approach to identify genes displaying changes in expression in the presence of [PSI+]. This made it possible to determine the positions of all active ribosomes within the genome, in both [PSI+] and [PSI-] isogenic strains. Comparisons of the translatomes and transcriptomes of the two strains revealed that the primary effect of [PSI+] was to repress genes involved in the stress response. Thus, we provide the first description of the global translational effect of [PSI+] and a new genetic explanation of the phenotypic differences between [PSI-] and [PSI+] strains under stress conditions.

2014-07-18 | GSE41590 | GEO

Genome-wide translational changes induced by the prion [PSI+]

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5’ half of specific tRNAs feeds back to promote corresponding tRNA gene transcription in vertebrate embryos [small RNA-Seq, ssDRIP-seq]

Project description:5’tRFls are small tRNA fragments derived from 5’ half of mature tRNAs. However, it is unknown whether 5’tRFls could feed back to regulate tRNA biogenesis. Here we show that 5’tRFlGly/GCC and 5’tRFlGlu/CTC function to promote transcription of corresponding tRNA genes and are essential for vertebrate early embryogenesis. During zebrafish embryogenesis, dynamics of 5’tRFlGly/GCC and 5’tRFlGlu/CTC levels correlates with that of tRNAGly/GCC and tRNAGlu/CTC levels. Morpholino-mediated knockdown of 5’tRFlGly/GCC or 5’tRFlGlu/CTC down-regulates tRNAGly/GCC or tRNAGlu/CTC levels respectively, and causes embryonic lethality that is efficiently rescued by co-injection of properly refolded corresponding tRNA. In zebrafish embryos, tRNA:DNA and 5’tRFl:DNA hybrids commonly exist on the template strand of tRNA genes. Mechanistically, unstable 5’tRFl:DNA hybrid may prevent the formation of transcriptionally inhibitory stable tRNA:DNA hybrids on the same tRNA loci so as to facilitate tRNA genes transcription. The uncovered mechanism may be implicated in other physiological and pathological processes.

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5’ half of specific tRNAs feeds back to promote corresponding tRNA gene transcription in vertebrate embryos [tRNA-seq]

2021-08-25 | GSE181683 | GEO

PSI supercomplex copurification using PSI His-tag

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2018-10-03 | PXD008857 | Pride

Identification of genome-wide binding sites of Psi, Myc and Pol in Drosophila imaginal wing discs using Targeted DamID

Project description:Targeted DamID was performed to identify binding targets of P-element somatic inhinitor (Psi), Myc and RNA Polymerase (using the RpII18 subunit common to all 3 polymerases). Scalloped(sd)-GAL4 was used to restrict expression to the wing disc.

2020-06-30 | GSE149718 | GEO

PSI photoinhibition alters iron homeostasis in chloroplasts and initiates retrograde signaling regulating nuclear gene expression

Project description:Photosystem I (PSI) is a critical component of the photosynthetic machinery in plants. Under conditions of environmental stress, PSI becomes photoinhibited, leading to redox imbalance in the chloroplast. PSI photoinhibition is caused by an increase in electron pressure within PSI, which damages the iron-sulfur centers. In this study, we investigated the effect of PSI electron acceptors on the susceptibility of PSI to photoinhibition at different CO2 concentrations in the plant environment. We also analyzed the global gene expression in plants exposed to PSI photoinhibition. PSI was photoinhibited using a specific illumination technique that inhibited PSI with minimal effect on PSII. CO2 levels neither increased nor decreased the likelihood of PSI photodamage. PSI photoinhibition, independent of CO2 levels, upregulated the genes involved in the response to iron excess in plants and downregulated the genes involved in iron deficiency. It also induced the genes of photosynthetic proteins that act as electron acceptors for PSI. We propose that PSI photoinhibition causes a release of iron from iron-sulfur centers, which initiates a retrograde signal from the chloroplast to the nucleus to modify gene expression. In addition, deprivation of CO2 from the air initiated a signal that induced flavonoid biosynthesis genes, probably via jasmonate production.

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Proteomic analysis of [PSI+] yeast strain

Project description:The yeast Sup35 protein is a subunit of the translation termination factor, and its conversion to the [PSI+] prion state leads to more translational read-through. Although extensive studies have been done on [PSI+], changes at the proteomic level have not been performed exhaustively. We therefore used a SILAC-based quantitative mass spectrometry approach and identified 4187 proteins from both [psi-] and [PSI+] strains. Surprisingly, there was very little difference between the two proteomes under standard growth conditions.

2018-10-24 | PXD007168 | Pride

RNA-sequencing following Psi or Myc knockdown in Drosophila larval wing tissue

Project description:RNA-seq analysis of gene expression changes following the depletion of P-element somatic inhibitor (Psi) or Myc via RNAi in wing discs of mid-third instar larvae, compared with control. Differential splicing analysis was also performed on the Psi RNAi dataset.

2020-06-30 | GSE149719 | GEO

The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila

Project description:Alternative pre-mRNA splicing (AS) is a critical regulatory mechanism that operates extensively in the nervous system to produce diverse protein isoforms. Fruitless AS isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown. Using genome-wide approaches and quantitative behavioral assays, we show that the P element somatic inhibitor (PSI) and its interaction with U1 snRNP control male courtship behavior. PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts. The PSIΔAB mutant results in significant changes in the AS patterns of ~1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior. PSI directly regulates the AS of at least a third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior. Importantly, one of these direct targets is fruitless — the master regulator of courtship. Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system. This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior.

2016-04-30 | GSE79916 | GEO

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