J Integr Plant Biol.

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NSP2 phosphorylation modulates HYL1 nuclear import via KETCH1 to promote miRNA biogenesis in Arabidopsis

Tingting Zhai1, 2, Mengjie Gu1, Yanjiao Wang1, Ke Qu1, Ping Liu2, Wei Yang1, Shuxin Zhang1*   

  1. 1. State Key Laboratory of Wheat Improvement, College of Life Science, Shandong Agricultural University, Tai'an 271018, China;
    2. Shandong Province Engineering Research Center for Agricultural Equipment Intelligence, College of Mechanical and Electronic Engineering, Shandong Agricultural University, Tai'an 271018, China
    *Correspondence: Shuxin Zhang (sxzhang@sdau.edu.cn)
  • Received:2026-03-16 Revised:2026-09-24 Accepted:2026-10-01 Online:2026-10-30
  • Supported by:
    This work was supported by grants from the National Natural Science Foundation of China (32670762, 31970602 to S.Z.) and the China Postdoctoral Science Foundation (2025M774029 to T.Z.). We are grateful to Yan Zhang for the ProUBQ10::KETCH1-GFP plasmid and the ABRC for nsp2-1 and nsp2-2.

Abstract: MicroRNA (miRNA) biogenesis in plants relies on the precise nuclear-cytoplasmic trafficking of the HYL1 protein. Here, we report that the NITRILE-SPECIFIER PROTEIN 2 (NSP2) is a previously unrecognized component of the Arabidopsis miRNA pathway. Through yeast two-hybrid, BiFC, Co-IP, and genetic analyses, we demonstrate that NSP2 physically interacts with HYL1 and genetically suppresses hyl1-2. NSP2 overexpression increases global miRNA abundance. Mechanistically, NSP2 acts as an adapter that bridges the importin-β family member KETCH1 to HYL1, thereby regulating HYL1 nuclear import and dicing-body (D-body) assembly. Dephosphorylation of NSP2 at a conserved threonine (T314) strengthens KETCH1–HYL1 association and further enhances pri-miRNA processing. Our study establishes NSP2 as a phosphorylation-regulated scaffold that coordinates HYL1 nuclear trafficking and microprocessor assembly, and reveals critical roles for NSP2 in miRNA biogenesis.

Key words: HYL1, KETCH1, miRNA biogenesis, NSP2, nuclear-cytoplasmic trafficking

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