DHX16

DHX16 is a DEAH-box RNA helicase that functions as a core spliceosomal ATPase required for pre-mRNA splicing after assembly of the pre-catalytic spliceosome (complex B), where its activity drives entry into the catalytic phase of splicing (PMID:20423332, PMID:20841358). Inhibition or dominant-negative mutation of its helicase domain blocks splicing in vitro and causes genome-wide nuclear retention of unspliced, intron-containing transcripts that are not cleared by nonsense-mediated decay (PMID:20423332, PMID:20841358). The G-patch protein GPKOW binds DHX16 directly through its G-patch domain and RNA through its KOW1 domain, acting as an essential co-activator: GPKOW depletion inactivates DHX16-bound spliceosomes and recombinant GPKOW restores activity (PMID:25296192). Beyond splicing, DHX16 acts as a viral RNA pattern recognition receptor, binding specific spliced influenza RNA segments via its helicase motif and triggering type I interferon responses that require RIG-I and TRIM6-synthesized unanchored K48-polyubiquitin, with broad antiviral relevance to influenza, Zika, and SARS-CoV-2 (PMID:35263596). The physiological consequence of its splicing function is illustrated in hematopoietic stem cells, where Dhx16 loss causes Emg1 intron retention, impaired ribosome assembly, nucleolar stress, and p53 activation leading to stem cell depletion and bone marrow failure (PMID:39333759).

1. 2010 High

   Established that DHX16 acts at a defined late step of spliceosome activation rather than during early assembly, placing it at the transition into catalysis.

   Evidence In vitro splicing antibody inhibition, immunoprecipitation, and dominant-negative helicase mutants in HeLa nuclear extracts; genomic tiling microarray with nuclear/cytoplasmic fractionation

   PMID:20423332 PMID:20841358

   Open questions at the time

   • No structural model of the DHX16-spliceosome interface
   • Direct RNA substrate specificity within the spliceosome not defined
2. 2014 High

   Identified GPKOW as the direct G-patch co-activator that licenses DHX16 catalytic function, answering how the helicase is functionally coupled to the spliceosome and RNA.

   Evidence Direct protein-protein interaction assays, immunodepletion/add-back in vitro splicing, in vivo overexpression rescue, and domain mutation analysis

   PMID:25296192

   Open questions at the time

   • Mechanism by which GPKOW stimulates DHX16 ATPase/helicase activity not resolved
   • No structure of the DHX16-GPKOW complex
3. 2022 High

   Revealed a second, non-splicing role for DHX16 as a viral RNA sensor feeding into innate antiviral signaling, repurposing its RNA-binding capacity for pattern recognition.

   Evidence Unanchored poly-Ub affinity purification from lung, siRNA silencing in cells and in vivo, RNA binding assays, helicase-motif mutants, and genetic epistasis with RIG-I and TRIM6

   PMID:35263596

   Open questions at the time

   • How DHX16 discriminates viral from cellular spliced RNAs is unclear
   • Molecular link between RNA binding and unanchored K48-polyubiquitin recruitment not detailed
4. 2024 High

   Connected DHX16 splicing fidelity to a physiological output by showing that loss disrupts Emg1 splicing, ribosome assembly, and HSC maintenance through nucleolar stress and p53.

   Evidence Conditional knockout mice, multi-omics including ribosome profiling, Emg1 overexpression rescue, and p53 pathway activation assays

   PMID:39333759

   Open questions at the time

   • Whether other retained-intron transcripts contribute to the HSC phenotype is not established
   • Tissue-specificity of the Emg1 dependency not explored
5. 2011 Medium

   Demonstrated an in vivo developmental requirement for Dhx16 in vertebrate gastrulation and endoderm specification, linking the splicing factor to embryonic patterning.

   Evidence Zebrafish forward genetic screen, positional cloning, genetic rescue, cell transplantation, and blastoderm explant epistasis analysis

   PMID:21396359

   Open questions at the time

   • Whether the developmental defect reflects general splicing loss or specific target mis-splicing is undefined
   • Pathway placement relies on genetic epistasis in a non-mammalian model
6. 2025 Low

   Implicated DHX16 in regulated sex-specific alternative splicing via a scaffolding partner, extending its role to developmental splice-isoform control.

   Evidence Co-immunoprecipitation in 293T cells, RNAi knockdown, and RT-PCR for sex-specific doublesex isoforms in an insect

   PMID:40329630

   Open questions at the time

   • Single Co-IP without reciprocal validation in an invertebrate context
   • Direct role of DHX16 in dsx splice-site selection not biochemically demonstrated

• How DHX16 toggles between its core constitutive splicing role and its antiviral RNA-sensing role, and how each is regulated, remains unresolved.
• No structural basis for substrate or partner selectivity
• Regulation governing splicing versus PRR function unknown