Highly ABA-Induced 1 (HAI1)-Interacting protein HIN1 and drought acclimation-enhanced splicing efficiency at intron retention sites.

The Highly ABA-Induced 1 (HAI1) protein phosphatase is a central component of drought-related signaling. A screen for HAI1-interacting proteins identified HAI1-Interactor 1 (HIN1), a nuclear protein of unknown function which could be dephosphorylated by HAI1 in vitro. HIN1 colocalization and interaction with serine-arginine rich (SR) splicing factors and appearance of nuclear speckle-localized HIN1 during low water potential (ψw) stress suggested a pre-mRNA splicing-related function. RNA sequencing of Arabidopsis Col-0 wild type identified more than 500 introns where moderate severity low ψw altered intron retention (IR) frequency. Surprisingly, nearly 90% of these had increased splicing efficiency (decreased IR) during stress. For one-third of these introns, ectopic HIN1 expression (35S:HIN1) in unstressed plants mimicked the increased splicing efficiency seen in stress-treated wild type. HIN1 bound to a GAA-repeat, Exonic Splicing Enhancer-like RNA motif enriched in flanking sequence around HIN1-regulated introns. Genes with stress and HIN1-affected splicing efficiency were enriched for abiotic stress and signaling-related functions. The 35S:HIN1 plants had enhanced growth maintenance during low ψw, while hin1 mutants had reduced growth, further indicating the role of HIN1 in drought response. HIN1 is annotated as an MYB/SANT domain protein but has limited homology to other MYB/SANT proteins and is not related to known yeast or metazoan RNA-binding proteins or splicing regulators. Together these data identify HIN1 as a plant-specific RNA-binding protein, show a specific effect of drought acclimation to promote splicing efficiency of IR-prone introns, and also discover HAI1-HIN1 interaction and dephosphorylation that connects stress signaling to splicing regulation.

A Conserved Helix in the C-Terminal Region of Watermelon Silver Mottle Virus Nonstructural Protein S Is Imperative For Protein Stability Affecting Self-Interaction, RNA Silencing Suppression, and Pathogenicity.

In orthotospovirus, the nonstructural protein S (NSs) is the RNA-silencing suppressor (RSS) and pathogenicity determinant. Here, we demonstrate that a putative α-helix, designated H8, spanning amino acids 338 to 369 of the C-terminal region of the NSs protein, is crucial for self-interaction of watermelon silver mottle virus NSs protein and that the H8 affects RSS function. Co-immunoprecipitation, yeast two-hybrid, and bimolecular fluorescence complementation analyses revealed that the triple point mutation (TPM) of H8 amino acids Y338A, H350A, and F353A resulted in NSs protein self-interaction dysfunction. Transient expression of H8-deleted (ΔH8) and TPM NSs proteins in Nicotiana benthamiana plants by agroinfitration indicated that these proteins have weaker RSS activity and are far less stable than wild-type (WT) NSs. However, an electrophoretic mobility assay revealed that small interfering RNA (siRNA) binding ability of TPM NSs protein is not compromised. The pathogenicity assay of WT NSs protein expressed by the attenuated turnip mosaic virus vector restored severe symptoms in recombinant-infected N. benthamiana plants but not for ΔH8 or TPM proteins. Taken together, we conclude that the H8 helix in the C-terminal region of NSs protein is crucial for stabilizing NSs protein through self-interaction to maintain normal functions of RSS and pathogenicity, but not for NSs-siRNA binding activity.