A major and fundamental challenge in biological research is to determine how primary transcripts are organized in the nucleus and the functional consequences of this organization for gene expression. More than 50 years ago, a family of heterogenous nuclear ribonucleoproteins (hnRNPs) was detected and proposed to package and regulate transcript expression. Recently, we have initiated a characterization of structures of stable hnRNP assemblies sedimenting at 40S.
Remarkably, these particles preferentially bind precursor (pre)-mRNA intronic RNA sequences, suggesting that they have important roles in controlling the processing of primary transcripts. We hypothesize that the 40S particles represent the RNA functional analogue of nucleosomes, and as such refer to them as “ribonucleosomes”. Here, we propose an in-depth, multidisciplinary approach to elucidate the structures of ribonucleosomes and their functions in the biogenesis of mRNA. We will employ state-of-the-art structural, functional and genome-wide approaches to understand the roles of ribonucleosomes
in the regulation of pre-mRNA processing and mRNA expression. We will use cryo-EM and NMR to determine the structures of different variants of ribonuclesomes in vitro and in situ, and investigate their assembly. Molecular and cell-based assays, combined with mass spectrometry, high-throughput sequencing and computational analyses, will be used to determine cell/
tissue-dependent and -independent functions of ribonucleosomes, as well as how protein, RNA modifications, and evolutionary change, impact their assembly and activities. With the combined and internationally-leading expertise of our team, we are in a unique position to address how transcripts are organized in the nucleus by hnRNP ribonucleosomes and how these complexes critically function in the regulated biogenesis of mRNA.