Project description:We applied high throughput sequencing technology to identify microRNA genes in bighead carp and silver carp. We identified 167 conserved miRNAs in bighead carp and 166 in silver carp. By two computational stragegies, we obtained 39 novel miRNAs in bighead carp and 54 in silver carp, for which, no homologs were found in other species. Several miRNA* sequences were found in our dataset as well, some particular ones might have gene regulation function. Gain and loss of family members were observed in several miRNA families, which partially reflected the fate of miRNA gene duplicates.
Project description:Common carp is one of the main commercial fishes captured and cultured worldwide. Although common carp genome is not finished yet, this study provides a first large scale cloning and characterization of common carp miRNAs and their potential targets. These miRNAs add to the growing database of new miRNA and lay the foundation for further understanding of miRNA function in gene regulation of common carp.
Project description:MicroRNA’s function is defined by the targets it regulates. Despite miRNAs’ crucial role in various cellular processes and disease states, the identification of their targets remains challenging. Existing methods suffer from high false-positive rates and are inadequate in differentiating direct targets from downstream regulatory changes. Here, we introduce a simple approach involving combined analysis of RNA-seq and Precise RunOn-seq (PRO-seq) (CARP) to effectively measure post-transcriptional regulation. We apply this approach to seven different miRNAs and robustly distinguish their direct targets from downstream changes. We validate the efficacy of our approach using argonaute-CLIP-seq. We show using CARP and ribosome-profiling analyses that accelerated mRNA decay is the major mode of target regulation by miRNAs. Additionally, our comparative analyses reveal that CARP aids in discovery of complex regulatory mechanisms and in capturing false-negatives of existing approaches. Furthermore, post-transcriptional changes measured using CARP uncover miRNA-dependent regulation of coding sites. Lastly, we demonstrate that PRO-seq provides mechanistic explanation of indirect targeting, unraveling miRNA regulatory networks. Finally, we believe that CARP is particularly suitable to study in vivo functions of miRNAs in primary cells.
Project description:We applied high throughput sequencing technology to identify microRNA genes in bighead carp and silver carp. We identified 167 conserved miRNAs in bighead carp and 166 in silver carp. By two computational stragegies, we obtained 39 novel miRNAs in bighead carp and 54 in silver carp, for which, no homologs were found in other species. Several miRNA* sequences were found in our dataset as well, some particular ones might have gene regulation function. Gain and loss of family members were observed in several miRNA families, which partially reflected the fate of miRNA gene duplicates. Total RNA of juvenile bighead carp and silver carp were sequenced on one Solexa lane, respectively.
Project description:The biological impact of microRNAs is determined by their targets, and robustly identifying direct miRNA targets remains challenging. Existing methods suffer from high false-positive rates and are unable to effectively differentiate direct miRNA targets from downstream regulatory changes. Here, we present a simple approach to deconvolute post-transcriptional and transcriptional changes using PRO-seq with RNA-seq. In combination, these methods allow us to systematically profile the regulatory impact of a miRNA. We refer to this approach as CARP: Combined Analysis of RNA-seq and PRO-seq. We apply CARP to multiple miRNAs and show that it robustly distinguishes direct targets from downstream changes, while greatly reducing false positives. We validate our approach using Argonaute eCLIP-seq and ribosome profiling, demonstrating that CARP defines a comprehensive repertoire of targets. We identify miRNA-specific activity of target sites within the coding region. CARP facilitates the dissection of complex changes in gene regulatory networks triggered by miRNAs and identification of transcription factors that underlie downstream regulatory changes. Given the robustness of the approach, CARP is particularly suitable for dissecting miRNA regulatory networks in vivo.
Project description:Common carp is one of the main commercial fishes captured and cultured worldwide. Although common carp genome is not finished yet, this study provides a first large scale cloning and characterization of common carp miRNAs and their potential targets. These miRNAs add to the growing database of new miRNA and lay the foundation for further understanding of miRNA function in gene regulation of common carp. We constructed a small RNA library from 17 Cyprinus carpio samples.
Project description:Grass carp (Ctenopharyngodon idellus), the world’s largest aquaculture fish species, exhibits superior growth in females compared to males. However, the lengthy sexual maturation period of four to five years poses a significant obstacle to the genetic reproduction and breeding of grass carp. Consequently, classical methods such as gonadogenesis or sex reversal through steroid treatment, employed for breeding all-female grass carp, demand considerable time and effort. In this study, we developed an super-fast breeding strategy for generating all-female grass carp in a total of half a year, using a surrogate production method. We first characterized grass carp female germline stem cells (GSCs) from genetic female juveniles at three months post-fertilization (mpf). The female GSCs with XX chromosomes were then transplanted into germ cell-depleted zebrafish larvae at five days post-fertilization (dpf). The transplanted grass carp XX germ cells underwent rapid spermatogenesis in the zebrafish recipient. At three months after transplantation, all zebrafish recipients had developed into males capable of producing the all-X sperm of the grass carp. By using these sperm to fertilize wildtype grass carp eggs, we successfully produced an all-female grass carp offspring. This groundbreaking achievement highlights the potential of surrogate production in the genetic breeding of valuable fish species, and opens a new avenue for advancing genetic breeding in aquaculture.