Biology subjects

Song, Y. J.

Publications and source records attributed to Song, Y. J..

4 recordsLinked to original sources

bioRxiv2022

CRISPR/Cas-based precision gene replacement in plants via homologous recombination-independent approaches

Precise gene or allele replacement is a desirable technology, but implementing it in plants remains challenging. CRISPR-Cas-based approaches, such as gene targeting (GT) and prime editing (PE), have opened up new possibilities for precise gene replacement in plants. However, their editing size and efficiency still need improvement. Recently, strategies using canonical nonhomologous end-joining (cNHEJ) and microhomology-mediated end joining (MMEJ) have been considered promising alternatives for precise gene replacement in yeast and mammals. However, these approaches have not been extensively explored and applied to plants. Here, we proposed and tested a CRISPR-Cas-based tool, termed PREMJ (precision gene replacement via microhomology-mediated end joining), for precision gene replacement in plants. The PREMJ strategy employing 20-bp microhomology MMEJ donors ([~]100 bp lengths) and a canonical nonhomologous end-joining (cNHEJ) inhibitor, NU7441, produced high targeted gene replacement efficiencies, up to 1.60 {+/-} 0.14, 4.47 {+/-} 1.98, and 8.98 {+/-} 4.73 % in protoplasts of tomato, lettuce, and cabbage, respectively. Our data also revealed the critical impacts of the microhomology length and NU7441 concentration on PREMJ-based precision gene editing in plants. Although obtaining edited plants remains challenging due to inefficient protoplast regenerations and Agrobacterium-mediated delivery, PREMJ may significantly contribute to precision gene editing in plants competent to PREMJ complex delivery and plant regeneration. Key MessageWe designed and tested a method, termed PREMJ, for precise gene replacement using the CRISPR-Cas-mediated double-stranded break (DSB) formation and repairing the DSBs via microhomology-mediated end joining (MMEJ) with MMEJ donor template carrying desired base changes and microhomologies flanking the DSB ends. PREMJ showed feasibility in precise gene replacement in protoplasts of tomatoes, lettuce, and cabbage, albeit its efficacy in Agrobacterium-mediated plant transformation requires further optimization.

synthetic biology↗

bioRxiv2022

Monoallelically-expressed Noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression

Out of the several hundred copies of rRNA genes that are arranged in the nucleolar organizing regions (NOR) of the five human acrocentric chromosomes, [~]50% remain transcriptionally inactive. NOR-associated sequences and epigenetic modifications contribute to differential expression of rRNAs. However, the mechanism(s), controlling the dosage of active versus inactive rRNA genes in mammals is yet to be determined. We have discovered a family of ncRNAs, SNULs (Single NUcleolus Localized RNA), which form constrained sub-nucleolar territories on individual NORs and influences rRNA expression. Individual members of the SNULs monoallelically associate with specific NOR-containing chromosome. SNULs share sequence similarity to pre-rRNA and localize in the sub-nucleolar compartment with pre-rRNA. Finally, SNULs control rRNA expression by influencing pre-rRNA sorting to the DFC compartment and pre-rRNA processing. Our study discovered a novel class of ncRNAs that by forming constrained nucleolar territories on individual NORs contribute to rRNA expression.

cell biology↗

bioRxiv2020

Cocaine receptor identified as BASP1

Cocaine is a behavioral stimulant with substantial abuse potential related to its positively rewarding actions 1,2. Cocaine inhibits the reuptake inactivation of neurotransmitters such as dopamine, serotonin, and norepinephrine at high nanomolar to low micromolar concentrations 2. There is evidence for substantially more potent influences of cocaine. For instance, Calligaro and Eldefrawi reported binding of [3H]cocaine to brain membranes with a dissociation constant of about 16 nM 3. At 10 nM concentration, cocaine elicits environmental place conditioning in planarians 4. Furthermore, 1nM cocaine enhances dopamine D2 receptor agonist-mediated signaling 5. Inhibition of amine reuptake by cocaine is substantially less potent than some of these high affinity actions. Thus, evidence for a specific, high affinity receptor for cocaine that mediates its behavioral actions has been lacking. We now report high affinity binding of cocaine to the membrane-associated brain acid soluble protein-1 (BASP1) with a Kd of 7 nM. Knocking down BASP1 in the striatum inhibits [3H]cocaine binding to striatal synaptosomes. Depletion of BASP1 in the nucleus accumbens diminishes locomotor stimulation, acquisition, and expression of locomotor sensitization to cocaine. Our findings indicate that BASP1 is a pharmacologically relevant receptor for cocaine and a putative therapeutic target for psychostimulant addiction.

pharmacology and toxicology↗

bioRxiv2020

Mechanistic analysis of enhancer sequences in the Estrogen Receptor transcriptional program

BackgroundEstrogen Receptor (ER) is a major lineage determining transcription factor (TF) in mammary gland development, orchestrating the expression of thousands of genes. Dysregulation of ER-mediated transcriptional program results in abnormal cell proliferation and cancer. Transcriptomic and epigenomic profiling of breast cancer cell lines has revealed large numbers of enhancers involved in this regulatory program, but how these enhancers encode function in their sequence remains poorly understood. ResultsA subset of ER-bound enhancers are transcribed into short bidirectional RNA (enhancer RNA or eRNA), and this property is believed to be a reliable marker of active enhancers. We therefore analyze thousands of ER-bound enhancers and build quantitative, mechanism-aware models to discriminate eRNAs from non-transcribing enhancers based on their sequence. Our thermodynamics-based models provide insights into the roles of specific TFs in ER-mediated transcriptional program, many of which are supported by the literature. We use in silico perturbations to predict TF-enhancer regulatory relationships and integrate these findings with experimentally determined enhancer-promoter interactions to construct a gene regulatory network. We also demonstrate that the model can prioritize breast cancer-related sequence variants while providing mechanistic explanations for their function. Finally, we experimentally validate the model-proposed mechanisms underlying three such variants. ConclusionsWe modeled the sequence-to-expression relationship in ER-driven enhancers and gained mechanistic insights into the workings of a major transcriptional program. Our model is consistent with the current body of knowledge and its predictions are confirmed by experimental observations. We believe this to be a promising approach to analysis of regulatory sequences and variants.

bioinformatics↗