Subcellular mRNA localization regulates ribosome biogenesis in migrating cells

Abstract

Translation of Ribosomal Protein coding mRNAs (RP-mRNAs) constitutes a key step in regulation of ribosome biogenesis, but the mechanisms which modulate RP-mRNAs 20 translation under various cellular and environmental conditions are poorly understood. Here we show that the subcellular localization of RP-mRNAs acts as a key regulator of their translation in migrating cells. As cells migrate into their surroundings, RP-mRNAs localize to the actin-rich protrusions at the front the cells. This localization is mediated by La related protein-6 (LARP6), an RNA Binding Protein that is enriched in protrusions. Protrusions act as 25 hotspots of translation for localized RP-mRNAs, resulting in enhancement of RP synthesis, upregulation of ribosome biogenesis, and increased overall protein synthesis of migrating cells in a LARP6 dependent manner. In human breast carcinomas, Epithelial to Mesenchymal Transition (EMT) upregulates LARP6 expression to enhance protein synthesis, and can be targeted using a small molecule inhibitor that interferes with LARP6 RNA binding. Our findings 30 reveal LARP6 mediated mRNA localization as a key regulator of ribosome biogenesis during cell-migration, and demonstrate a role for this process in cancer progression downstream of EMT.35 65 mRNAs to promote their enrichment in protrusions. Protrusions are also highly enriched in translation initiation and elongation factors, acting as hotspots for translation of localized RP-mRNAs. LARP6 dependent localization of RP-mRNAs results in upregulation of RP levels, leading to enhancement of ribosome biogenesis and global protein synthesis in migrating cells. In human breast carcinomas, higher LARP6 protein expression is associated with the 70 invasive mesenchymal-like subtypes. EMT induces LARP6 protein expression, which acts to promote malignant growth and invasion. Crucially, LARP6 regulation of RP-mRNAs can be therapeutically targeted using small molecule inhibitors which specifically interfere with its RNA binding. Collectively, our findings reveal a new mechanism that governs ribosome biogenesis in mesenchymal-like migratory cells via subcellular localization of RP-mRNAs, and 75 demonstrate a therapeutically targetable role for this process in cancer downstream of EMT. Results 80 RP-mRNAs localize to protrusions of all migratory cellsWe utilized a micro-porous transwell filter based method (Mardakheh et al., 2015;Mili et al., 2008) to assess if RP-mRNAs localization to actin-rich protrusions was a conserved feature of all migratory cells. We modified the procedure to allow cells to adhere to the top of the filter first, followed by synchronized induction of protrusion formation through the pores (Figure 85 1A). Importantly, the small (3µm) size of the pores enables protrusions to form but prevents the cell bodies from passing through, thus resulting in separation of the protrusions and the cell bodies on opposite sides of the filter, which can be independently imaged or purified for multi-omics analysis ( Figure 1A). Using this method, we profiled ...

Mechanisms and consequences of subcellularRNAlocalization across diverse cell types

“…Furthermore, mRNAs encoding ribosome subunits have been shown to localize to protrusions of migrating cells in a LARP6 dependent manner. The local translation of ribosomal proteins at these protrusions enhances ribosome biogenesis and local protein production 58 . In each of these cases, the localized mRNA helps determine the localization of the resulting proteins which aid critical cell functions.…”

RNA-Seq Analysis Reveals Localization-Associated Alternative Splicing across 13 Cell Lines

“…In other words, the subcellular location of an RNA is strongly related to its biological function [ 9 ]. For example, the asymmetric distribution of RNA in cells can influence the expression of genes [ 9 ], formation and interaction of protein complexes [ 11 ], biosynthesis of ribosomes [ 12 ], and development of cells [ 13 , 14 ], among other functions. Many techniques have been developed to investigate the subcellular localization of RNAs.…”

Differential contribution of transcriptomic regulatory layers in the definition of neuronal identity

“…3e, f). These results indicate that changes in the length of 3′ UTRs between the proximal-distal axis, relative to other hippocampal axes, are likely coupled to transcript regulation through differential localization 47 .…”