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: STAR Protocols

Article Title: Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells

doi: 10.1016/j.xpro.2026.104468

Figure Lengend Snippet: Illustration of the PiggyBac system for exogenous DNA insertion into the human genome After the co-transfection of the PiggyBac transposase expression plasmid and PiggyBac cloning and expression vector, the expressed PiggyBac transposase cut the Ins, Insulator. ITR, inverted terminal repeat. Adapted from the website of System Biosciences.

Article Snippet: Obtain PiggyBac transposase expression plasmid from System Biosciences (Cat# PB210PA-1).

Techniques: Cotransfection, Expressing, Plasmid Preparation, Cloning

Journal: STAR Protocols

Article Title: Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells

doi: 10.1016/j.xpro.2026.104468

Figure Lengend Snippet: The map of PiggyBac cloning and expression plasmid The schematic illustrates a plasmid backbone containing an ampicillin resistance marker, a multiple cloning site (MCS) for insertion of the desired expression cassette, and an SV40 polyadenylation signal to facilitate proper transcriptional termination.

Article Snippet: Obtain PiggyBac transposase expression plasmid from System Biosciences (Cat# PB210PA-1).

Techniques: Cloning, Expressing, Plasmid Preparation, Marker

Journal: STAR Protocols

Article Title: Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells

doi: 10.1016/j.xpro.2026.104468

Figure Lengend Snippet: FACS analysis showed the GFP+ cell percentage after plasmid transfection The transfection of PiggyBac-SVA-GFP-pA and PiggyBac-SVA-AAA-GFP-pA into HEK293T cells can produce a GFP+ cell population, while the transfection of PiggyBac-SVA-A-GFP-pA and PiggyBac-SVA-AA-GFP-pA cannot, indicating the frame shift of translation.

Article Snippet: Obtain PiggyBac transposase expression plasmid from System Biosciences (Cat# PB210PA-1).

Techniques: Plasmid Preparation, Transfection