Isomer‐Specific Effect of miR‐29b on Nuclear Morphology
Authors: Kriegel Alison J. , Terhune Scott S. , Greene Andrew S. , Noon Kathleen R. , Pereckas Michael S. , Liang Mingyu 
Published: November 2023

Targeting mRNA via seed region pairing is the canonical mechanism by which microRNAs (miRNAs) regulate cellular functions and disease processes. Emerging evidence suggests miRNAs might act through unconventional mechanisms. miRNA isomers that contain identical seed region sequences, such as miR‐29a and miR‐29b, provide natural, informative models for identifying miRNA effects independent of seed region pairing. It has been previously shown that miR‐29a and miR‐29b are both endogenously expressed in HeLa cells, however miR‐29b is upregulated and localized to the nucleus only in mitosis and miR‐29a is abundant in the cytoplasm throughout the cell cycle. We examined the effect of inhibiting miR‐29a or miR‐29b on nuclear morphology in HeLa cells 24 hours after release from a double synchronization. Anti‐miR‐29b transfection, not miR‐29a, resulted in a significantly increased percentage of large irregularly shaped nuclei, including multinucleated cells, nuclear lobulation or rings of small amounts of nuclear material. We hypothesized that unique interactions with microribonucleoproteins (miRNPs), rather than conventional mRNA silencing mechanisms, may determine the unique phenotypes observed. We developed an approach that utilized tagged, synthetic miRNA duplexes, rapid cryopreservation, and proteomics to identify miRNPs that interacted specifically with miR‐29a or miR‐29b in HeLa cells. Targeted proteomic analysis identified 38 proteins that formed complexes with miR‐29b, but not miR‐29a. One of these proteins, ADP/ATP translocase 2 (ANT2), known to be involved in the formation of mitotic spindles, was colocalized with native miR‐29b in perinuclear clusters in fluorescence microscopy studies. We used two pull‐out approaches to validate ANT2 and miR‐29b were interacting in a molecular complex, using miR‐29a as a stringent control. First, Western blot analysis confirmed the presence of ANT2 in protein samples affinity‐purified from cells transfected with biotinylated miR‐29b, but not biotinylated miR‐29a or scrambled miR‐29b oligonucleotides. Second, ANT2 and its interacting partners were isolated from untransfected HeLa cells by immunoprecipitation (IP) using an ANT2 antibody. The IP complexes obtained contained an enrichment of endogenous, native miR‐29b relative to miR‐29a, compared to whole cell lysate or IP complexes obtained using a control antibody. While nuclear localization of miR‐29b was observed in many cells, clustering of ANT2 just outside the nucleus was only observed when ANT2 is co‐localized with miR‐29b in early mitosis when the nucleus is still intact. Knockdown of ANT2 resulted in nuclear irregularities similar to those observed following miR‐29b blockade and inhibited nuclear localization of miR‐29b. The study demonstrates an effect of miR‐29 on nuclear morphology during mitosis that is unique to the miR‐29b isoform and may be mediated by an unconventional mechanism for miRNA action.Support or Funding InformationHL111580, HL082798, HL121233, Innovation Center (MCW)