Investigation of the role of cytoskeleton protein KATNAL2 in ribosomal gene transcription in mouse cells

Abstract

The discovery that katanin-like cytoskeleton protein KATNAL2, in addition to its intracellular localization at MTs and sites associated with MTs (centrioles, cilia), also, unexpectedly, resides in the nucleolus, suggests an additional putative function to its role in the cytoskeleton. KATNAL2 is highly enriched in the nucleolus, particularly in the chromatin fraction, similar to nucleolar proteins UBF, fibrillarin, treacle and RNA Pol.I, suggesting that KATNAL2 is bound to chromosomal rDNA. Here we investigate the quantification of rRNA expression and explore the role of KATNAL2 in ribosomal gene transcription. As a complement to other approaches, such as Northern blot and quantitative RT-PCR, standard methods used by members of the host laboratory to measure rRNA expression levels, here brief labeling of cells with 5-fluorouridine (FUrd) incorporated into nascent RNA, as a nucleoside analog, was used to quantify total rRNA synthesis. Wild-type NHI 3T3 mouse fibroblast cell line and 2 derivative silenced-KATNAL2 clones, clone 2.43 and clone 8-8, were cultured in three different serum concentrations of 0.5% (restrictive growth media), 10% (normal growth media), and 20% (enriched growth media) and exposed to FUrd. A dataset of 1200 cells in total was analysed (600 cells in total for WT, 300 cells in total for clone 2.43 shKATNAL2 cells and 300 cells in total for clone 8-8shKATNAL2 cells, i.e. 100 cells per growth condition per cell line).The quantitative assessment and statistical evaluation included analysis of FUrd intensity as a proxy for rRNA transcription, nucleolar and nuclear size, number of nucleoli per cell, and FUrd intensity/size of the nucleolus. The IMARIS software was utilized for extracting measurements from microscope images (previously acquired by PhD student Andria Theophanous). Quantification and statistical evaluation were performed with GraphPad Prism. The findings revealed increased rRNA levels in silenced KATNAL2 cells, compared to WT cells, in step with serum concentration increases and at all serum concentrations. Additionally, there was a remarkable increase in the size and number of nucleoli in the absence of KATNAL2 protein, in relation to wild-type cells. Changes in nuclear size were also observed in silenced KATNAL2 cells compared to wild-type cells. Overall, these results are consistent with a role of KATNAL2 in rRNA transcription.