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: bioRxiv

Article Title: Application of AI-Designed OpenCRISPR-1 for Highly Efficient Gene Editing in Soybean and Nicotiana benthamiana

doi: 10.64898/2026.08.27.747635

Figure Lengend Snippet: (A) Schematic representation of the T-DNA region of the pBSE-OpenCRISPR-1 binary vector. The openCRISPR-1 coding sequence is flanked by an N-terminal 3×FLAG tag and nuclear localization signals (NLS) at both the N- and C-termini. Nuclease expression is driven by the soybean ubiquitin promoter ( pGmUBQ ) and terminated by the rbcs E9 terminator ( rbsS-E9t ). The single guide RNA (sgRNA) expression cassette is driven by the Arabidopsis U6-26 promoter ( pAtU6 ), with Bsa I restriction sites (red lines) utilized for the rapid cloning of target-specific protospacers. (B) Schematic representation of the GmFAD2-1B gene structure and the designated OpenCRISPR-1 target site. The target sequence is located within Exon 2. The protospacer sequence is shown with the NGG protospacer adjacent motif (PAM) underlined in orange. An endogenous Nco I restriction site (CCATGG) overlapping the expected Cas cleavage site (dashed red line) was utilized for the cleaved amplified polymorphic sequence (CAPS) assay. Red arrows indicate the binding sites for the PCR genotyping primers. (C) Representative photograph of transgenic soybean hairy roots induced by Agrobacterium rhizogenes strain K599 harboring the pBSE-OC1-GmFAD vector, taken two weeks post-infection. Scale bar = 10 mm. (D) CAPS assay of the wild-type (WT) and six independent pools of transgenic soybean hairy roots. PCR amplicons spanning the target site were digested with Nco I. The WT amplicon is completely digested into two smaller fragments, whereas edited hairy root pools exhibit a resistant (undigested) upper band, indicating the disruption of the Nco I recognition site by OpenCRISPR-1-mediated mutagenesis. (E) Representative mutant alleles identified via deep amplicon sequencing at the GmFAD2-1B target locus. The wild-type reference sequence is shown at the top. Induced deletions are represented by black dashes, and an insertion is highlighted within a red box. The PAM sequence and expected cleavage site are indicated below the alignment.

Article Snippet: For genotyping by amplicon sequencing, PCR products were purified using a DNA extraction kit (K0691, Thermo Fisher Scientific) and subsequently submitted to Plasmidsaurus for Genotyping Analysis (Oxford Nanopore, R10.4.1).

Techniques: Plasmid Preparation, Sequencing, Expressing, Ubiquitin Proteomics, Cloning, Amplification, Binding Assay, Transgenic Assay, Infection, Disruption, Mutagenesis