ARL16

ARL16 is an ARF-family GTPase that operates as a GTP-dependent regulatory switch in two distinct cellular contexts (PMID:21233210, PMID:35196065). In innate immunity, GTP-loaded ARL16 binds the C-terminal domain of RIG-I and suppresses RIG-I association with viral RNA, thereby negatively regulating type I interferon signaling; GDP-restricted mutants (T37N, Δ45-54) fail to bind RIG-I or inhibit its signaling, establishing that nucleotide-dependent activation is required for both the interaction and its inhibitory function (PMID:21233210). In ciliated cells, ARL16 is required for a Golgi-to-cilia trafficking pathway that specifically exports IFT140 and INPP5E to cilia: its loss reduces ciliogenesis, depletes ARL13B, ARL3, INPP5E, and IFT140 from cilia, and causes INPP5E and IFT140 to accumulate at the Golgi (PMID:35196065). In this pathway ARL16 acts downstream of or in parallel with the ARF GAPs ELMOD1 and ELMOD3, since an activating ARL16 mutant rescues the ciliary defects of ELMOD1 or ELMOD3 deletion (PMID:34818063). The biochemical mechanism linking these two roles and the direct effectors of ARL16 in cilia have not been characterized in the available corpus.

1. 2011 High

   Established the first molecular function for ARL16 by showing it is a GTP-dependent negative regulator of RIG-I antiviral signaling, answering how a small GTPase restrains the interferon response.

   Evidence Co-IP, RNA-binding suppression assay, RNAi knockdown with IFN-β reporter and VSV readout, and GDP-locked dominant-negative mutants in cells

   PMID:21233210

   Open questions at the time

   • Structural basis of the GTP-dependent ARL16–RIG-I CTD interaction not resolved
   • Upstream signals controlling ARL16 nucleotide state during infection unknown
   • GEF/GAP regulating ARL16 in this context not identified
2. 2021 Medium

   Placed ARL16 within a defined ciliary trafficking pathway by genetic epistasis, showing it acts downstream of or in parallel with the ARF GAPs ELMOD1/3.

   Evidence Activating-mutant rescue in Elmod1 and Elmod3 KO MEFs with ciliogenesis and ciliary protein localization readouts

   PMID:34818063

   Open questions at the time

   • Direct biochemical relationship between ELMOD1/3 GAP activity and ARL16 nucleotide cycling not demonstrated
   • Single-lab genetic epistasis only
   • Whether ELMOD1/3 act directly on ARL16 not shown
3. 2021 Low

   Phylogenetic analysis placed ARL16 in the last eukaryotic common ancestor, establishing it as an ancient and broadly conserved member of the ARF GTPase family.

   Evidence Molecular phylogenetic analysis of >2,000 curated ARF family genes across 114 eukaryotic species

   PMID:34247240

   Open questions at the time

   • Computational inference only with no experimental test of ARL16 function
   • Conservation of specific roles across species not addressed
4. 2022 High

   Defined the cell-biological role of ARL16 in ciliogenesis, showing it is specifically required for Golgi-to-cilia export of IFT140 and INPP5E.

   Evidence CRISPR/Cas9 ARL16 knockout in MEFs with immunofluorescence of ciliary protein content and Golgi accumulation assays

   PMID:35196065

   Open questions at the time

   • Direct effectors or cargo adaptors recruited by ARL16 not identified
   • Mechanism by which ARL16 selects IFT140/INPP5E cargo unknown
   • Increase in ciliary length despite reduced ciliogenesis unexplained

• Whether and how the immune-regulatory and ciliary-trafficking functions of ARL16 are mechanistically related, and what GEFs/GAPs and direct effectors govern its nucleotide cycle in each context, remains unresolved.
• No structural model of ARL16 in either context
• Direct ciliary effectors unidentified
• Regulators of ARL16 GTP loading unknown