Biology subjects

Kremsky, I.

Publications and source records attributed to Kremsky, I..

5 recordsLinked to original sources

bioRxiv2022

Characterization of a strain-specific CD-1 reference genome reveals potential inter- and intra-strain functional variability

BackgroundCD-1 is an outbred mouse stock that is frequently used in toxicology, pharmacology, and fundamental biomedical research. Although inbred strains are typically better suited for such studies due to minimal genetic variability, outbred stocks confer practical advantages over inbred strains, such as improved breeding performance and low cost. Knowledge of the full genetic variability of CD-1 would make it more useful in toxicology, pharmacology, and fundamental biomedical research. ResultsWe performed deep genomic DNA sequencing of CD-1 mice and used the data to identify genome-wide SNPs, indels, and germline transposable elements relative to the mm10 reference genome. We used multiple genome-wide sequencing data types and previously published CD-1 SNPs to validate our called variants. We used the called variants to construct a strain-specific CD-1 reference genome, which we show can improve mappability and reduce experimental biases from genome-wide sequencing data derived from CD-1 mice. Based on previously published ChIP-seq and ATAC-seq data, we find evidence that genetic variation between CD-1 individuals can lead to alterations in transcription factor binding. We also identified a number of variants in the coding region of genes which could have effects on splicing and translation of genes. ConclusionsWe have identified millions of previously unidentified CD-1 variants with the potential to confound studies involving CD-1. We used the identified variants to construct a CD-1-specific reference genome, which can improve accuracy and reduce bias when aligning genomics data derived from CD-1 individuals.

genomics↗

bioRxiv2020

A Budding Yeast Model for Human Disease Mutations in the EXOSC2 Cap Subunit of the RNA Exosome

RNA exosomopathies, a growing family of tissue-specific diseases, are linked to missense mutations in genes encoding the structural subunits of the conserved 10-subunit exoribonuclease complex, the RNA exosome. Such mutations in the cap subunit gene EXOSC2 cause the novel syndrome SHRF (Short stature, Hearing loss, Retinitis pigmentosa and distinctive Facies). In contrast, exosomopathy mutations in the cap subunit gene EXOSC3 cause pontocerebellar hypoplasia type 1b (PCH1b). Though having strikingly different disease pathologies, EXOSC2 and EXOSC3 exosomopathy mutations result in amino acid substitutions in similar, conserved domains of the cap subunits, suggesting that these exosomopathy mutations have distinct consequences for RNA exosome function. We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by introducing the EXOSC2 mutations in the orthologous S. cerevisiae gene RRP4. The resulting rrp4 mutant cells have defects in cell growth and RNA exosome function. We detect significant transcriptomic changes in both coding and non-coding RNAs in the rrp4 variant, rrp4-G226D, which models EXOSC2 p.Gly198Asp. Comparing this rrp4-G226D mutant to the previously studied S. cerevisiae model of EXOSC3 PCH1b mutation, rrp40-W195R, reveals that these mutants have disparate effects on certain RNA targets, providing the first evidence for different mechanistic consequences of these exosomopathy mutations. Congruently, we detect specific negative genetic interactions between RNA exosome cofactor mutants and rrp4-G226D but not rrp40-W195R. These data provide insight into how SHRF mutations could alter the function of the RNA exosome and allow the first direct comparison of exosomopathy mutations that cause distinct pathologies.

genetics↗

bioRxiv2020

The Nab2 RNA binding protein promotes sex-specific splicing of Sex lethal in Drosophila neuronal tissue

The Drosophila polyadenosine RNA binding protein Nab2, which is orthologous to a human protein lost in a form of inherited intellectual disability, controls axon projection, locomotion, and memory. Here we define an unexpectedly specific role for Nab2 in regulating splicing of [~]150 exons/introns in the head transcriptome and link the most prominent of these, female retention of a male-specific exon in the sex determination factor Sex-lethal (Sxl), to a role in m6A-dependent mRNA splicing. Genetic evidence indicates that aberrant Sxl splicing underlies multiple phenotypes in Nab2 mutant females. At a molecular level, Nab2 associates with Sxl pre-mRNA and ensures proper female-specific splicing by preventing m6A hypermethylation by Mettl3 methyltransferase. Consistent with these results, reducing Mettl3 expression rescues developmental, behavioral and neuroanatomical phenotypes in Nab2 mutants. Overall these data identify Nab2 as a required regulator of m6A-regulated Sxl splicing and imply a broader link between Nab2 and Mettl3-regulated brain RNAs.

developmental biology↗

bioRxiv2019

Transcription factors protect from DNA re-methylation during reprograming of primordial germ cells and pre-implantation embryos

A growing body of evidence suggests that certain phenotypic traits of epigenetic origin can be passed across generations via both the male and female germlines of mammals. These observations have been difficult to explain owing to a global loss of the majority of known epigenetic marks present in parental chromosomes during primordial germ cell development and after fertilization. By integrating previously published BS-seq, DNase-seq, ATAC-seq, and RNA-seq data collected during multiple stages of primordial germ cell and preimplantation development, we find that the methylation status of the majority of CpGs genome-wide is restored after global reprogramming, despite the fact that global CpG methylation drops to 10% in primordial germ cells and 20% in the inner cell mass of the blastocyst. We estimate the proportion of such CpGs with preserved methylation status to be 78%. Further, we find that CpGs at sites bound by transcription factors during the global re-methylation phases of germ line and embryonic development remain hypomethylated across all developmental stages observed. On the other hand, CpGs at sites not bound by transcription factors during the global re-methylation phase have high methylation levels prior to global de-methylation, become de-methylated during global de-methylation, and then become re-methylated. The results suggest that transcription factors can act as carriers of epigenetic information during germ cell and pre-implantation development by ensuring that the methylation status of CpGs is maintained after reprogramming of DNA methylation. Based on our findings, we propose a model in which transcription factor binding during the re-methylation phases of primordial germ cell and pre-implantation development allow epigenetic information to be maintained trans-generationally even at sites where DNA methylation is lost during global de-methylation.

genomics↗

bioRxiv2019

A Drosophila Model of Pontocerebellar Hypoplasia Reveals a Critical Role for the RNA Exosome in Neurons

The RNA exosome is an evolutionarily-conserved ribonuclease complex critically important for precise processing and/or complete degradation of a variety of cellular RNAs. The recent discovery that mutations in genes encoding structural RNA exosome subunits cause tissue-specific diseases makes defining the role of this complex within specific tissues critically important. Mutations in the RNA exosome component 3 (EXOSC3) gene cause Pontocerebellar Hypoplasia Type 1b (PCH1b), an autosomal recessive neurologic disorder. The majority of disease-linked mutations are missense mutations that alter evolutionarily-conserved regions of EXOSC3. The tissue-specific defects caused by these amino acid changes in EXOSC3 are challenging to understand based on current models of RNA exosome function with only limited analysis of the complex in any multicellular model in vivo. The goal of this study is to provide insight into how mutations in EXOSC3 impact the function of the RNA exosome. To assess the tissue-specific roles and requirements for the Drosophila ortholog of EXOSC3 termed Rrp40, we utilized tissue-specific RNAi drivers. Depletion of Rrp40 in different tissues reveals a general requirement for Rrp40 in the development of many tissues including the brain, but also highlight an agedependent requirement for Rrp40 in neurons. To assess the functional consequences of the specific amino acid substitutions in EXOSC3 that cause PCH1b, we used CRISPR/Cas9 gene editing technology to generate flies that model this RNA exosome-linked disease. These flies show reduced viability; however, the surviving animals exhibit a spectrum of behavioral and morphological phenotypes. RNA-seq analysis of these Drosophila Rrp40 mutants reveals increases in the steady-state levels of specific mRNAs and ncRNAs, some of which are central to neuronal function. In particular, Arc1 mRNA, which encodes a key regulator of synaptic plasticity, is increased in the Drosophila Rrp40 mutants. Taken together, this study defines a requirement for the RNA exosome in specific tissues/cell types and provides insight into how defects in RNA exosome function caused by specific amino acid substitutions that occur in PCH1b can contribute to neuronal dysfunction. Author SummaryPontocerebellar Hypoplasia Type 1b (PCH1b) is a devastating genetic neurological disorder that preferentially affects specific regions of the brain. Typically, children born with PCH1b have structural defects in regions of the brain including those associated with key autonomic functions. Currently, there is no cure or treatment for the disease. PCH1b is caused by mutations in the RNA exosome component 3 (EXOSC3) gene, which encodes a structural component of the ubiquitous and essential multi-subunit RNA exosome complex. The RNA exosome is critical for both precise processing and turnover of multiple classes of RNAs. To elucidate the functional consequences of amino acid changes in EXOSC3 that cause PCH1b, we exploited well-established genetic approaches in Drosophila melanogaster that model EXOSC3 mutations found in individuals with PCH1b. Using this system, we find that the Drosophila EXOSC3 homolog (termed Rrp40) is essential for normal development and has an important function in neurons. Furthermore, PCH1b missense mutations modeled in Rrp40 cause reduced viability and produce neuronal-specific phenotypes that correlate with altered levels of target RNAs that encode factors with key roles in neurons. These results provide a basis for understanding how amino acid changes that occur in the RNA exosome contribute to neuronal dysfunction and disease.

genetics↗