Project description:MicroRNAs (miRNAs) are a class of small, endogenous RNAs that regulate mRNA expressions post-transcriptionally. To study their expression, we collected and sequenced small RNA libraries in Drosophila species. To survey their regulation divergence, we dissected testes from miR-983 mutant and control flies in two sibling species and performed an RNA-seq analysis.
Project description:Intervention type:DRUG. Intervention1:Huaier, Dose form:GRANULES, Route of administration:ORAL, intended dose regimen:20 to 60/day by either bulk or split for 3 months to extended term if necessary. Control intervention1:None.
Primary outcome(s): For mRNA libraries, focus on mRNA studies. Data analysis includes sequencing data processing and basic sequencing data quality control, prediction of new transcripts, differential expression analysis of genes. Gene Ontology (GO) and the KEGG pathway database are used for annotation and enrichment analysis of up-regulated genes and down-regulated genes.
For small RNA libraries, data analysis includes sequencing data process and sequencing data process QC, small RNA distribution across the genome, rRNA, tRNA, alignment with snRNA and snoRNA, construction of known miRNA expression pattern, prediction New miRNA and Study of their secondary structure Based on the expression pattern of miRNA, we perform not only GO / KEGG annotation and enrichment, but also different expression analysis.. Timepoint:RNA sequencing of 240 blood samples of 80 cases and its analysis, scheduled from June 30, 2022..
| 2612481 | ecrin-mdr-crc
Project description:Sequencing of small non-coding RNA and mRNA in Drosophila testes
Project description:RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors through nucleolytic processing. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi in cultured cells, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA in vitro, shuttles between nucleus and cytoplasm and the abundance of certain endogenous siRNAs is reduced in blanks mutant testes. Transgenic expression of a Blanks protein variant with increased nuclear retention reduces endogenous siRNA abundance unless the protein also carries a mutation that prevents binding of dsRNA to the second dsRBD. Blanks thus facilitates the export of dsRNA to the cytoplasm for further processing by the canonical RNAi machinery. In contrast, rescue of male fertility in blanks mutant animals was independent of RNA binding to the second dsRBD, consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.
Project description:Adult stem cells support tissue homeostasis and repair throughout the life of an individual. However, numerous intrinsic and extrinsic changes occur with age that result in altered stem cell behavior and reduced tissue maintenance and regeneration. In the Drosophila testis, stem cells surround and contact the apical hub, a cluster of somatic cells that express the self-renewal factor Unpaired (Upd), which activates the JAK-STAT pathway in adjacent stem cells. However, aging results in a dramatic decrease in upd expression, with a concomitant loss of germline stem cells (GSCs). Here we present genetic and biochemical data to demonstrate that IGF-II mRNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd RNA and contribute to maintenance of the niche. However, Imp expression decreases in hub cells of older males, similar to upd, which is due to targeting of Imp by the heterochronic microRNA let-7. Therefore, in the absence of Imp, upd mRNA becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for aging-related changes in stem cell behavior will lead to the development of strategies to treat age-onset diseases and facilitate stem cell based therapies in older individuals. Examination of small RNA levels in testes from young (1day old) and aged (30days old) males of Drosophila melanogaster by deep sequencing (using Illumina GAII).
Project description:Nocte is an RNA binding protein but its functions in Drosophila eye development is unknown. We found that knockdown of nocte by RNAi in Drosophila eyes leads to small and rough eye phenotypes. We detected nocte's functions at mRNA levels by doing RNA-seq with purified polyA mRNA. We detected nocte's functions in mRNA translation by doing Ribo-seq and total RNA-seq to calculate the translation efficiencies.
Project description:The RNA-binding protein LIN28A is required for maintaining tissue homeostasis, including in the reproductive system, but the underlying mechanisms on how LIN28A regulates germline progenitors remain unclear. Here, we dissected LIN28A-binding targets using high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse testes. LIN28A preferentially binds to mRNA coding sequence (CDS) or 3'UTR regions at sites enriched wiGAG(A) sequences. Further investigation of Lin28a null mouse testes indicated that meiosis-associated mRNAs bound by LIN28A were differentially expressed. Next, ribosome profiling revealed that the mRNA levels of these targets were significantly reduced in polysome fractions, and their protein expression levels decreased in the Lin28a null mouse testes, even when meiotic arrest in the null mouse testes was not apparent. Collectively, these findings provide a set of LIN28A-regulated target mRNAs, and show that LIN28A binding might be mechanism through which LIN28A acts to regulate undifferentiated spermatogonia fates and male fertility in mammals.