Abstract 2815: Analyzing nuclear paraspeckle-dependent protection of unphosphorylated MicroRNA
Authors: Read Graham H. , Salzman David W. , Whitelegge Julian , Cohn Whitaker , Yang Tiffany , Tsang Cynthia , Turk Ihsan A. , Weidhaas Joanne B. 
Published: April 2023

Abstract
MicroRNA-34 (miR-34) is widely studied for its role in tumor suppression, DNA regulation, and cell cycle regulation. During DNA damage, miR-34 is directly upregulated by p53, inhibiting proliferation and promoting DNA repair. Accordingly, radiation therapy induces upregulation of miR-34 expression, coordinating several downstream radiation-induced stress responses. Recent work has demonstrated the existence of a stable, unphosphorylated pool of miR-34, which is rapidly phosphorylated and activated via loading onto Argonaute after ionizing radiation, independent of de novo transcription. Since Argonaute binding to the 5’ phosphate is also important for miRNA stability, we hypothesize that a separate mechanism must exist to stabilize the unphosphorylated pool of miR-34. Mass spectrometry performed on co-precipitates of biotinylated mimics of unphosphorylated miR-34 identified predominantly nuclear proteins, with a notable enrichment for proteins localized in nuclear paraspeckles. Nuclear paraspeckles are a phase-separated nuclear subcompartment comprised of several key structural proteins and a long noncoding RNA NEAT1_2 that prevents mixing of internal contents with the rest of the nucleoplasm and are canonically implicated in regulating gene expression by sequestering inosine-modified mRNA. To further investigate the role of nuclear paraspeckles in the stability or sequestering of unphosphorylated miR-34, stable cell lines with deletion of a a motif required for stability of paraspeckles through disruption of the lncRNA NEAT1_2 were created (A549 NEAT1 dTH). Interestingly, luciferase reporter assays for miR-34 activity in the NEAT1 dTH lines showed a general loss of miR-34 activity after radiation compared to wildtype cells which exhibit radiation induced activation of miR-34. This loss of activity occurred both immediately after radiation, which is attributable to phosphorylation of the unphosphorylated pool, and at later time points after radiation, which has been attributed to de novo miRNA production. Ongoing research will monitor colocalization of proteins known to activate the unphosphorylated pool with paraspeckles to determine if paraspeckles coordinate activation of unphosphorylated miR-34, as well as will investigate the potential impact of disruption of paraspeckles on de novo miR-34 production. A better understanding of the interplay between nuclear paraspeckles and stress-responsive miRNA production may provide a means to selectively modify cancer stress responses and uncover novel mechanisms in miRNA production and coordination.
Citation Format: Graham H. Read, David W. Salzman, Julian Whitelegge, Whitaker Cohn, Tiffany Yang, Cynthia Tsang, Ihsan A. Turk, Joanne B. Weidhaas. Analyzing nuclear paraspeckle-dependent protection of unphosphorylated MicroRNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2815.