Repurposed hnRNPC binds mature mRNAs and safeguards the mitotic transcriptome

Abstract

Upon entry to mitosis, RNA metabolism is broadly suppressed. While mechanistic understanding is limited, the mitotic transcriptome is known to be generally preserved to allow the daughter cells an economical and efficient start. We used highly effective cell cycle synchronization to specifically characterize mitotic hnRNPC, an abundant nuclear RNA-binding protein known for intron binding and splicing regulation during interphase. Two density-distinct hnRNPC-RNP populations (low-and high-density; LD and HD) were identified in mitotic cells. RNA-seq analysis combined with fluorescent Cross Linking and Immuno-Precipitation (fCLIP) revealed hnRNPC binding to 17.1% and 8.7% of expressed genes in LD- and HD-complexes, respectively, with most sites mapping to protein-coding genes (77% and 68%, respectively). Mediated by its known cooperative interaction with U-rich motifs, mitotic hnRNPC acquired prevalent interactions with exons, predominantly within the 3 untranslated region of mature mRNAs. Mitotic hnRNPC also retained intron interactions, predominantly in LD-hnRNPC RNPs, which comigrated with both the spliceosome and mono-ribosomes through a density gradient. Interestingly, LD-hnRNPC also interacted with mature mRNAs characterized by short coding sequences, in agreement with mono-ribosome loading. Conversely, HD-hnRNPC, which co-migrated with poly-ribosomes, predominantly interacted with mature mRNA complexes. Downregulation of hnRNPC elicited a global negative effect on the abundance of its mitotic targets. The data points to the global role of mitotic hnRNPC as a stabilizer of pre-mRNA and mRNA. Future studies should provide additional insights related to its multifunctional roles during mitosis.