A major challenge in biological research is to determine how primary transcripts are physically organized in the cell 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 structural characterization of stable hnRNP assemblies sedimenting at 40S. Remarkably, we find that these particles preferentially bind intronic sequences of pre-mRNA, suggesting that they have important roles in controlling the processing and expression of primary transcripts. Supporting this proposal, our initial analyses suggest that the 40S particles have widespread roles in cellular organization and splicing of primary transcripts. Accordingly, 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 expression. We will use cryo-EM and NMR to investigate structure and assembly mechanism of ribonucleosome. Molecular and cell-based assays, combined with mass spectrometry, high-throughput RNA sequencing and computational analyses, will be used to determine cell/tissue-dependent and -independent functions of ribonucleosomes, as well as how protein modifications 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 ribonucleosomes and how these complexes critically function in the regulated biogenesis of mRNA. Much like how the discovery and characterization of nucleosomes advanced our understanding of chromatin, transcriptional regulation and epigenetics, we anticipate our proposed research will be similarly transformative and provide unprecedented insight into the long-standing question of how the packaging of nuclear primary transcripts occurs and relates to the regulation of biogenesis and expression of mRNA.