Biology subjects

Polash, A. H.

Publications and source records attributed to Polash, A. H..

4 recordsLinked to original sources

bioRxiv2026

The human DEAD-box protein DDX3X regulates host and viral mRNA translation during Sendai Virus infection

DDX3X is a multifunctional DEAD-box RNA helicase with important roles in translation initiation and antiviral innate immune signaling, yet it is currently unknown whether viral infection affects its interactions with host RNAs. Here, we define the transcriptome-wide binding landscape of endogenous DDX3X in Sendai virus-infected human cells using PAR-CLIP. We show that DDX3X maintains its preference for GC-rich, highly structured 5'UTR regions during infection, but acquires a distinct set of infection-induced targets, including IFNB1 and multiple interferon-stimulated genes. We demonstrate that DDX3X directly binds the IFNB1 5'UTR and promotes its translation, establishing a previously unrecognized post-transcriptional mechanism contributing to DDX3X-dependent IFN-{beta} production. We also evaluated DDX3Xs binding to SeV RNAs and concluded that DDX3X is likely not actively recruited by SeV or has a significant effect on its viral life cycle. Our findings add a novel dimension to DDX3Xs involvement in anti-viral immunity with implications for further therapeutic development of DDX3X inhibitors.

molecular biology↗

bioRxiv2025

RBProximity-CLIP Enables Subcellular Mapping of RNA-Binding Protein Interactions at Nucleotide Resolution

RNA-binding proteins (RBPs) enable post-transcriptional gene regulation (PTGR) through specific interactions with RNA molecules, influencing processes ranging from nuclear processing and export to cytoplasmic localization, translation, storage and degradation. A key determinant of PTGR processes is the subcellular compartmentalization of RBPs, which dictates RNA targets they can access and the regulation performed in that environment. To characterize RBP-RNA interactions at subcellular resolution, we developed RBProximity-CLIP. RBProximity-CLIP enables compartment-specific isolation and profiling of individual RBP-RNA interactions by combining APEX2-based proximity labeling and 4-thiouridine-enhanced RNA-protein crosslinking, with sequential RBP- and biotin-affinity purifications. Using this approach, we profiled the RNA targets of three RBPs, AGO2, YBX1, and ELAVL1, across the cytoplasmic, nuclear, and nucleolar compartments, revealing nucleus-specific miRNA-mediated AGO2 targets, as well as subsets of YBX1 and ELAVL1 targets that differ by compartment, yet share identical binding motifs. RBProximity-CLIP enables specific and sensitive detection of compartment-specific RBP-RNA interactomes, thereby providing new insight into spatial gene regulation by RBPs.

cell biology↗

bioRxiv2025

PCLIPtools: A Robust Framework for Identifying RNA-Protein Interaction Sites from PAR-CLIP experiments.

PAR-CLIP is a widely used method for identifying binding sites of RNA-binding proteins (RBPs) transcriptome-wide. A characteristic T-to-C transition in the sequenced cDNA pinpoints the site of RBP-RNA crosslinking and is induced by the use of a photoreactive uridine analogue, 4-thiouridine (4SU). As with other systems-wide methods, PAR-CLIP, too, is prone to false discoveries as the T-to-C signal might result from systematic noise, pre- existing SNPs, and PCR errors. Therefore, rigorous statistical methods are required for analyzing PAR-CLIP data. The few existing tools to analyze PAR-CLIP data lack updates and sufficient documentation, and often fail to process current higher-depth sequencing data. Here we report PCLIPtools, a lightweight, customizable suite for analyzing PAR-CLIP data. PCLIPtools considers the read depth, T-to-C transitions, and the other mutations to statistically estimate high-confidence interaction sites. Benchmarking shows that PCLIPtools identifies more functionally significant targets than the current standard tool, PARalyzer, without losing high-confidence sites and outperforming it in runtime. Exploratory analyses show PCLIPtools specific targets are enriched for read depth and T-to-C conversion, supporting their validity. With simplicity, robustness, and speed, PCLIPtools improves the precision of PAR-CLIP data analysis while being accessible to experimental RNA biologists. PCLIPtools can be found on github (https://github.com/paulahsan/pcliptools). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/688892v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@19b32d5org.highwire.dtl.DTLVardef@267a45org.highwire.dtl.DTLVardef@255880org.highwire.dtl.DTLVardef@1d55b60_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

The DND1-NANOS3 complex shapes the primordial germ cell transcriptome via a heptanucleotide sequence in mRNA 3'UTRs

The RNA-binding proteins DND1 and NANOS3 are essential for primordial germ cell survival1-5. Their co-immunoprecipitation and overlapping loss-of-function phenotypes suggest joint function6-8, yet how they co-regulate target mRNAs remains unclear. Here, we developed Tandem PAR-CLIP and identified a DND1-NANOS3 ribonucleoprotein that specifically recognizes an AUGAAUU heptanucleotide on target mRNAs, termed the NANOS3-dependent DND1 Recognition Element (N3-DRE). mRNAs containing 3'-UTR N3-DREs are aberrantly upregulated in DND1- or NANOS3-deficient germ cells and encode key cell-cycle and epigenome regulators, such as CDK1. Genome editing showed that the N3-DRE is essential for Cdk1 repression in mouse PGCs in vivo. A 1.7-[A] crystal structure of the ternary complex of DND1, NANOS3, and CDK1- N3-DRE RNA revealed a continuous RNA-binding surface that confers high-affinity, sequence- specific recognition. Together, these findings define the molecular and functional basis of N3-DRE-mediated mRNA regulation in germ cell development. Moreover, we provide a paradigm of two RNA-binding proteins with low (DND1) or no (NANOS3) intrinsic sequence-specificity, jointly building a high-information-content RNA sequence motif that is different from the sum of their individual preferences. Because RNA-binding protein specificities are typically studied individually9-13, rather than in the context of ribonucleoproteins, this type of "two-factor authorization" may be an underappreciated mechanism to protect posttranscriptional gene regulatory networks from aberrant expression of an individual ribonucleoprotein component.

molecular biology↗