The role of hnRNPC in translation during mitosis

Mitosis is a complex cellular process controlled at multiple levels of gene expression. While global protein synthesis decreases during mitosis, translation of some mRNAs escapes the attenuation. Quantitative proteomic analysis of ribosomes purified from cells at G1 vs cells at mitosis, revealed enrichment of nuclear mRNA binding proteins, including hnRNPC, specifically on mitotic ribosomes. Polysomes profiling confirmed the association of hnRNPC with mitotic ribosomes. Subsets of mRNA, encoding ribosomal proteins and other components of the translation machinery itself, showed dependency to hnRNPC for their translation during mitosis.

Immunostaining experiments exposed the unique mitotic phenotype of hnRNPC, not shared with other nuclear markers, as it is localized predominantly to the cell periphery during metaphase. Puromycin labeling confirmed that this region is translationally active. In agreement with active translation, we observed the presence of translation elongation factors at the cell periphery. Surprisingly, ~70% of elongation factor eEF1D is located to the cell periphery during metaphase. Using optimized synchronization procedures to achieve a clear-cut separation between G2 and metaphase, we revealed the preferential association of hnRNPC with polysomes in metaphase, but not with polysomes in G2. Enrichment of specific sub-classes of mRNAs on G2 polysomes vs metaphasic polysomes will be discussed. To identify hnRNPC-bound mRNAs engaged with metaphasic polysomes and reveal the hnRNPC binding sites, we preformed fluorescence-based cross-linking-immuno-precipitation (fCLIP) using polysomal vs sub-polysomal fractions. The data exposes novel roles of hnRNPC in post-transcription regulation of specific subclasses of mRNAs, specifically during a critical step of mitosis.