Orb-dependent polyadenylation contributes to PLP expression and centrosome scaffold assembly

Abstract

As the microtubule-organizing centers (MTOCs) of most cells, centrosomes engineer the bipolar mitotic spindle required for error-free mitosis. Drosophila Pericentrin (PCNT)-like protein (PLP) directs formation of a pericentriolar material (PCM) scaffold required for PCM organization and MTOC function. Here, we investigate the post-transcriptional regulation of plp mRNA. We identify conserved binding sites for cytoplasmic polyadenylation element binding (CPEB) proteins within the plp 3’-untranslated region and examine the role of the CPEB ortholog, oo18 RNA-binding protein (Orb), in plp mRNA regulation. Our data show Orb biochemically interacts with plp mRNA to promote polyadenylation and PLP protein expression. Loss of orb, but not orb2, diminishes PLP levels in embryonic extracts. Consequently, PLP localization to centrosomes and function in PCM scaffolding is compromised in orb mutant embryos, resulting in genome instability and embryonic lethality. Moreover, we find PLP over-expression restores centrosome scaffolding and rescues the cell division defects caused by orb depletion. Our data suggest Orb modulates PLP expression at the level of plp mRNA polyadenylation and showcases the post-transcriptional regulation of core, conserved centrosomal mRNAs as critical for centrosome function.

The PCM scaffold enables RNA localization to centrosomes

“…We previously showed some Plp mRNA colocalizes with Plp protein at centrosomes [18]. Consistent with these observations, recent work highlights cotranslational transport as a major paradigm for RNA localization to centrosomes [16,15,20].…”

“…smFISH probes were dissolved in nuclease-free water at 25 μM and stored at -20°C before use. smFISH experiments were performed as previously described using RNase-free solutions [17,71,18]. Fixed embryos were rehydrated and washed first in 0.1% PBST 1 3…”

“…Raw images were processed with Airyscan joint deconvolution in Zen Blue with varying iterations per channel (15 iterations for Plp or Cen mRNA, 15 iterations for Cnn, 20 iterations for Asl). smFISH signals were detected and single molecule normalization was performed as described [17,71,18]. Briefly, single-channel .tif raw images were segmented in three dimensions using Python scripts adapted from the Allen Institute for Cell Science Cell Segmenter [74].…”

“…Among the most conserved mRNAs localizing to centrosomes is Pericentrin (PCNT) mRNA, as observed in cell culture, zebrafish, and Drosophila models [16][17][18].…”

Reflections on mentorship as an early career researcher

“…Our work demanded a computational pipeline to quantitatively define RNA distributions at single molecule resolution, an effort spearheaded by my first postdoctoral fellow, Dr. Pearl Ryder ( Ryder and Lerit, 2020 ). This open-source pipeline was adapted by others in the laboratory, including Dr. Junnan Fang, to examine other RNA distributions and contributions of conserved RNA-binding proteins to centrosome regulation and function ( Fang and Lerit, 2022 ). In many respects, I feel that our work is only just begun.…”