Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins abundant in a nucleus. They are known to coat the pre-mRNA and to be involved in many aspects of nucleic acid metabolism, like mRNA stabilization, alternative splicing, regulation of transcription and translation, RNA export, and degradation [1]. The early studies (in the 1970s) have shown, that in nuclei lysed without the RNase inhibitors, a large fraction of pre-mRNA was in a defined protein-RNA complex sedimenting at 40S [2]. It was shown that the core of this particle is formed by hnRNP C1/C2, hnRNP A1/B2, and hnRNP A2/B1 [1]. It has led to an intriguing suggestion, that the 40S hnRNP particle could represent a counterpart to the DNA nucleosome [3].
Our goal is to describe the biogenesis of the 40S particle, provide a structural description of the 40S particle using cryo-electron microscopy (cryo-EM), and visualize it in situ. We have established TRex-293 lines expressing FLAG-tagged hnRNPC1/C2 proteins and were able to pull down the 40S particles. We have prepared negatively stained grids with the particle and are now optimizing the preparation of vitrified grids for the cryo-EM to structurally characterize these particles using single particle analysis. To study ribonuclosome in intact cells we have prepared thin lamellae by cryo-FIB/SEM and collected the data using cryo-EM.