Journal: Human Reproduction Update

Article Title: The intricate dance of RNA-binding proteins: unveiling the mechanisms behind male infertility

doi: 10.1093/humupd/dmaf023

Figure Lengend Snippet: Workflow for identifying 38 candidate RNA-binding protein (RBP) genes potentially implicated in male infertility. This flowchart illustrates the stepwise strategy used to prioritize 38 candidate RBP genes for further investigation in male reproductive biology. The process began with mining the ‘male mouse germ cell RBPome’ database, comprising 408 RBPs enriched (n=168) and specific (n=240) to mouse testes. Human orthologs were identified through homology mapping and cross-referenced with expression data from the Genotype-Tissue Expression and Human Protein Atlas databases. A total of 339 RBPs were found to be expressed in human testicular tissue. Applying a selection criterion of testis-specific enrichment (≥5-fold higher mRNA expression in testis compared to other tissues), 163 testis-enriched human-mouse homologous RBP genes were retained. A comprehensive literature search (PubMed) was conducted to evaluate the functional relevance of these genes in male fertility. Of the 163 RBPs, 125 had previously been associated with reproductive phenotypes in mouse models. The remaining 38 genes (comprising 3 classical, 3 non-classical, and 32 novel RBPs) lacked knockout mouse models, representing a prioritized subset for future functional validation. GTEx, Genotype-Tissue Expression； HPA, Human Protein Atlas; KO, knockout; mMGC, male mouse germ cell.

Article Snippet: To identify candidate RBPs lacking knockout mouse models, we mined the RBP atlas and integrated transcriptomic and proteomic evidence from the Genotype-Tissue Expression (GTEx), Human Protein Atlas (HPA), and UniProt databases.

Techniques: RNA Binding Assay, Expressing, Selection, Functional Assay, Knock-Out, Biomarker Discovery

Journal: Human Reproduction Update

Article Title: The intricate dance of RNA-binding proteins: unveiling the mechanisms behind male infertility

doi: 10.1093/humupd/dmaf023

Figure Lengend Snippet: Integrated analysis of 163 testis-enriched RNA-binding proteins (RBPs): gene expression, functional enrichment, and protein-protein interaction (PPI). (A) Heatmap of RBP Gene Expression across Human Tissues (GTEx). Normalized expression levels of 163 testis-enriched RBP genes are shown across multiple human tissues using data from the GTEx database. Genes are arranged on the y -axis and tissues on the x -axis. Color intensity reflects expression levels (low: light yellow; high: dark blue). Genes with testis-specific enrichment (>5-fold higher expression in testes relative to other tissues) are highlighted by RBP classification: classical (blue, n=17), non-classical (green, n=26), and novel (red, n=120). This heatmap illustrates the tissue-specific expression landscape of RBPs, with a particular emphasis on testis-predominant expression. (B) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Enrichment Analyses. GO terms and KEGG pathways enriched among the 163 testis-enriched RBPs are depicted. The GO analysis includes biological process (BP), cellular component (CC), and molecular function (MF) categories, highlighting roles in spermatogenesis, RNA processing, and subcellular localization. KEGG analysis identifies key signaling and metabolic pathways relevant to testicular function and male fertility. (C) PPI network. A PPI network of testis-enriched RBPs was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins database. Nodes represent individual RBPs, and edges indicate predicted or known interactions, weighted by confidence scores. This network provides insight into the potential cooperative functions and regulatory hubs of RBPs involved in spermatogenesis and testicular physiology.

Article Snippet: To identify candidate RBPs lacking knockout mouse models, we mined the RBP atlas and integrated transcriptomic and proteomic evidence from the Genotype-Tissue Expression (GTEx), Human Protein Atlas (HPA), and UniProt databases.

Techniques: RNA Binding Assay, Gene Expression, Functional Assay, Expressing, Construct

Journal: Science Advances

Article Title: A forward genetics platform for papillomavirus research

doi: 10.1126/sciadv.aei3417

Figure Lengend Snippet: ( A ) Production of a discordant virus stock expressing wild-type and FLAG-tagged L1 at a 100:1 ratio. A discordant virus stock (DVS-L1WF100:1) was prepared from cells cotransfected with a 100:1 ratio of p16Y-L1 (blue) to p16Y-L1FLAG (purple), together with p16NP-L2HA. 293TT cells were then infected with DVS-L1WF100:1 at low MOI and coinfected with PVS-L2HA at high MOI, and a concordant virus stock expressing wild-type and FLAG-tagged L1 in a 100:1 ratio (CVS-L1WF100:1) was prepared and treated with L1.V5. The packaged p16Y-L2HA expression plasmid in CVS-L1WF1:100 is indicated in red. ( B ) Selection of mutant L1 from a concordant virus stock expressing wild-type and FLAG-tagged L1 at a 100:1 ratio. 293TT cells were mock-infected or infected with CVS-L1WF100:1, and flow cytometry was performed 3 days postinfection as described in the legend to . As indicated, CVS-L1WF100:1 was treated with L1.V5 antibody before infection. ( C ) DNA recovered from cells after selection is greatly enriched for plasmids encoding mutant L1. LMW DNA prepared from 293TT cells infected with CVS-L1WF100:1 or CVS-L1WF100:1(V5) (see fig. S5) was used to transform bacteria. PCR was performed on individual ampicillin-resistant colonies using primers that generate a 125 bp product from the wild-type L1 gene and a 149 bp product from the mutant L1 gene. Amplification products were separated by polyacrylamide gel electrophoresis and stained with ethidium bromide. The gel shows amplification products from representative colonies isolated from cells infected with CVS-L1WF100:1 (unselected clones) or CVS-L1WF100:1(V5) (selected clones). M, markers; the next three lanes show amplification products using as template p16Y-L2HA, which lacks the L1 gene (N, negative control), p16Y-L1 (W, wild-type), or p16Y-L1FLAG (F, FLAG-tagged). The table shows the results for all analyzed plasmids.

Article Snippet: Samples were then treated with proteinase K (1.15 μg/ml), and DNA was isolated using DNeasy Blood and Tissue Kit (Qiagen, 69506).

Techniques: Virus, Expressing, Infection, Plasmid Preparation, Selection, Mutagenesis, Flow Cytometry, Bacteria, Amplification, Polyacrylamide Gel Electrophoresis, Staining, Isolation, Clone Assay, Negative Control