bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.08.31.555766

Optimization of RNAi efficiency in PVD neuron of C. elegans

Singh, P.·Selvarasu, K.·Ghosh-Roy, A.
View original source ↗Export metadata ↓

Abstract

PVD neuron of C. elegans has become an attractive model for the study of dendrite development and regeneration due to the elaborate and stereotype dendrite morphology in this neuron. The molecular basis for dendrite maintenance and regeneration is poorly understood. RNA interference (RNAi) by feeding E. coli expressing dsRNA has been the basis of several genome wide screens performed using C. elegans. However, the feeding method often fails when it comes to nervous system. Using an optimal induction condition for the dsRNA expression in E coli, we fed the worm strains with HT115 bacteria expressing dsRNA against genes like mec-3, hpo-30, and tiam-1, whose loss of function are known to show dendrite morphology defects in PVD neuron. We found that RNAi of these genes in the strains such as nre-1(-) lin-15b(-), lin-15b(-) and sid-1(-); lin-15b(-); Punc-119::sid-1[+] resulted in significant reduction of dendrite branching. However, the phenotypes were significantly modest compared to the respective loss of function mutants. To obtain stronger phenotype for PVD specific genes, we have made a strain, which strongly expresses sid-1 under mec-3 promoter specific for PVD. When Pmec-3::sid-1 is expressed in either nre-1(-);lin-15b(-) or lin-15b(-) background, the higher order branching phenotype after RNAi of mec-3, hpo-30, and tiam-1 was significantly enhanced as compared to nre-1(-);lin-15b(-) and lin-15b(-) background alone. Next we tested the nre-1(-) lin-15b(-), Pmec-3-sid-1[+] strain for the knockdown of genes playing role in dendrite regeneration process. We found that when aff-1 and ced-10 genes were knocked down in the nre-1(-) lin-15b(-), Pmec-3-sid-1[+] background, the dendrite regeneration was significantly reduced and the extent of reduction was comparable to that of the mutants of aff-1 and ced-10. Essentially, our strain expressing sid-1 in PVD neuron optimizes the condition for RNAi for high throughput screening for PVD development, maintenance and regeneration.

Explore related subjects

geneticsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Singh, P., Selvarasu, K., Ghosh-Roy, A.. 2023-09-01. Optimization of RNAi efficiency in PVD neuron of C. elegans. https://doi.org/10.1101/2023.08.31.555766

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

The histone demethylase Kdm5 and the ARGONAUTE proteins Piwi and Aubergine regulate female abdominal pigmentation in Drosophila melanogaster

Insect pigmentation is an ecologically critical trait influencing many physiological processes. In Drosophila melanogaster, abdominal pigmentation is sexually dimorphic: males have fully pigmented posterior segments, while females exhibit a posterior melanin stripe. Pigmentation relies on the expression of pigmentation genes that encode enzymes involved in pigment synthesis. These genes are tightly regulated during pupal and young adult stages. To expand the gene regulatory network of pigmentation genes, we conducted an RNAi screen using the yellow-Gal4 driver, expressed during the pupal stage in abdominal epidermis. One of the candidates from this screen, Kdm5, encodes a histone demethylase erasing the H3K4me3 histone mark catalyzed by the histone methyl-transferase Trithorax (Trx). We show that Kdm5 down-regulation reduces abdominal pigmentation, mimicking trx down-regulation. Kdm5 activates melanin production through regulation of the pigmentation gene tan. Transcriptomic analyses reveal that Kdm5 and Trx share many targets in pupal abdominal epidermis, including piRNA pathway components such as piwi and aubergine. These piRNA components, originally associated with transposon silencing in the germline, also function in some somatic tissues such as the nervous system, the fat body or the gut. We demonstrate that Piwi and Aubergine participate in female abdominal pigmentation establishment, without evident piRNA production. We also show that Kdm5 and Piwi act not only in pupal abdominal epidermis but also in pupal fat body. This study therefore expands the regulatory network of pigmentation genes. It identifies a new somatic function for Kdm5 and Piwi and reveals a role for pupal fat body in female abdominal pigmentation regulation.

genetics↗
bioRxiv2026

Genetic diversity within and between polyploid sugarcane (Saccharum spp.) families obtained via caryopsis using microsatellite markers and multicategory model

Genetic diversity analyses are essential for sugarcane (Saccharum spp.) breeding programs. Crossbreeding, based on genetic distances between parental plants, is a tool used to increase genetic variability and enhance plant selection; however, quantifying variation in highly polyploid species remains a challenge. The present study aimed to evaluate the diversity within and between 12 families of sugarcane derived from caryopses, analyzing 120 individual seedlings arranged in an augmented block design. Genotyping was performed using primers for 16 microsatellite loci, five simple sequence repeat (SSR) loci, and 11 expressed sequence tag-SSR (EST-SSR) loci. To accurately account for polyploidy, similarity calculations were performed using Bruvos distances among individuals and RST distances among the families. Analysis of molecular variance (AMOVA) indicated that most of the genetic variability was within families (72%), with only 28% found between them. This high level of intra-family variation demonstrates that a significant reservoir of genetic diversity remains available within the crosses. The highest genetic similarity was observed between the families RB986952 x RB986960 and RB036122 x RB03611, whereas the lowest genetic similarity was observed between the families RB97319 x RB966928 and RB106802 x RB855036. Although the evaluated families shared high genetic similarity, the pronounced genetic variation within them demonstrates a robust recombination potential, indicating that the genetic basis of sugarcane can be better explored using the high variability that already exists in the selection of desirable morpho-agronomic characteristics within the families. Furthermore, this study highlights the importance of using appropriate distances for diversity studies with codominant markers, such as microsatellites, in polyploid species.

genetics↗
bioRxiv2026

Optimizing DNA extraction from environmentally degraded bone samples for molecular identification of cetacean species

Molecular identification of cetacean bone remains can be limited by DNA degradation and the presence of PCR inhibitors. Here, we present an optimized DNA extraction protocol based on a total demineralization method for environmentally exposed cetacean bones. The protocol uses 100 mg of bone powder, 24 h digestion with EDTA, N-lauroylsarcosine, and proteinase K, followed by a modified silica-column purification. Nine environmentally degraded bone samples representing eight individuals were processed. DNA concentrations ranged from 7.3 to 57.1 ng/uL (mean SD = 25.91- 13.91 ng/uL). The mitochondrial cytochrome b gene was successfully amplified from all samples using conventional PCR, and five samples (55.6%) yielded sequences suitable for downstream analysis. BLASTn identified Balaenoptera physalus as the closest database match for all recovered sequences, and phylogenetic analysis further supported their association with B. physalus reference sequences. These results demonstrate that the proposed protocol provides a practical approach for recovering amplifiable and molecularly informative mitochondrial DNA from environmentally degraded cetacean bone material, facilitating molecular identification from challenging skeletal remains.

genetics↗