Isomer-specific effect of microRNA miR-29b on nuclear morphology

Abstract

Targeting mRNAs via seed region pairing is the canonical mechanism by which microRNAs (miRNAs) regulate cellular functions and disease processes. Emerging evidence suggests miRNAs might also act through other mechanisms. miRNA isomers that contain identical seed region sequences, such as miR-29a and miR-29b, provide naturally occurring, informative models for identifying those miRNA effects that are independent of seed region pairing. miR-29a and miR-29b are both expressed in HeLa cells, and miR-29b has been reported to localize to the nucleus in early mitosis because of unique nucleotide sequences on its 3' end. Here, we sought to better understand the mechanism of miR-29b nuclear localization and its function in cell division. We hypothesized that its nuclear localization may be facilitated by protein-miRNA interactions unique to miR-29b. Specific blockade of miR-29b resulted in striking nuclear irregularities not observed following miR-29a blockade. We also observed that miR-29b, but not miR-29a, is enriched in the nucleus and perinuclear clusters during mitosis. Targeted proteomic analysis of affinity-purified samples identified several proteins interacting with synthetic oligonucleotides mimicking miR-29b, but these proteins did not interact with miR-29a. One of these proteins, ADP/ATP translocase 2 (ANT2), known to be involved in mitotic spindle formation, colocalized with miR-29b in perinuclear clusters independently of Argonaute 2. Of note, ANT2 knockdown resulted in nuclear irregularities similar to those observed following miR-29b blockade and prevented nuclear uptake of endogenous miR-29b. Our findings reveal that miR-29 regulates nuclear morphology during mitosis and that this critical function is unique to the miR-29b isoform.

Chromatin’s physical properties shape the nucleus and its functions

“…This mimics the abnormal nuclear shape seen in more metastatic breast cancers [18]. Depletion of other cancer-relevant molecules, particularly p63 [86], miR29-b [87], and NOP53 [88], can also induce abnormal nuclear shape, although these perturbations may have effects beyond those on chromatin. Even prior to nuclear formation, chromatin mechanics regulates shape; barrier-to-autointegration factor (BAF) stiffens chromatin by bridging chromatin sites, which inhibits the formation of micronuclei [89].…”

Changes in nuclear and cytoplasmic microRNA distribution in response to hypoxic stress

“…As such, RNA binding proteins and complexes like SIRC may regulate the subcellular distribution of miRNAs, as we observe also in response to hypoxic stimulus. Some studies have suggested that short sequence motifs might act as nuclear localization signals, as in the case of the 3ʹ terminal hexanucleotide motif in miR-29b-3p, presumably by recognizing specific RNA binding proteins such as ANT2 (ADP/ATP translocase 2) 58 . However, while we did observe a modest nuclear enrichment of miR-29b-3p, the related miRNAs miR-29a-3p and miR-29c-3p which lack the hexanucleotide motif were similarly nuclear-enriched.…”

Functionally Essential Tubular Proteins Are Lost to Urine-Excreted, Large Extracellular Vesicles during Chronic Renal Insufficiency

“…Imaging was performed on Nikon A1-R laser scanning confocal microscope. Immunohistochemistry for megalin was performed as previously described, 7 with 1:50 megalin antibody (Abcam; AB184676) and 1:100 Texas Red-labeled antimouse (1:100, Invitrogen #T-862). Immunohistochemistry for NHE3 (Slc9a3) was performed in the same manner except with 1:50 NHE3 antibody (Millipore Sigma: MABN1813).…”

“…The unique proteins detected in the experiments were uploaded to Ingenuity Pathway Analysis using the Uniprot Accession number for subsequent categorization of proteins by known cellular location and function/type. 7 The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD019207 and 10.6019/PXD019207. 13 Statistical Analysis.…”