Multi-disciplinary methods to define RNA-protein interactions and regulatory networks

Abstract

The advent of high-throughput technologies including deep-sequencing and protein mass spectrometry is facilitating the acquisition of large and precise data sets toward the definition of post-transcriptional regulatory networks. While early studies that investigated specific RNA-protein interactions in isolation laid the foundation for our understanding of the existence of molecular machines to assemble and process RNAs, there is a more recent appreciation of the importance of individual RNA-protein interactions that contribute to post-transcriptional gene regulation. The multitude of RNA-binding proteins (RBPs) and their many RNA targets has only been captured experimentally in recent times. In this review, we will examine current multidisciplinary approaches toward elucidating RNA-protein networks and their regulation.

Figure 1. The stoichiometric challenge of post-transcriptional regulatory factors

Regulation of gene expression at the transcriptional level is limited to the number of encoded target sites and their accessibility in the genome. In contrast, regulation of gene expression at the posttranscriptional level is dependent on the number of possible target sites across the transcriptome and their respective stoichiometric concentrations. Hence posttranscriptional gene regulation is highly dependent on the gene expression program of particular cell types and tissues and regulatory effects variably change with concentration of target isoforms and other RBPs and RNA regulatory factors.

Figure 2. Schematic of current RNA-protein investigation methodologies

(A) and (B) Schematic protocols for the RNA crosslinking followed by mass spectrometry for the genome wide identification of RBPs by Castello et al. (A) and Baltz et al. (B). In vivo UV crosslinking captures proteins directly bound to RNA in their native conformation. After UV crosslinking, mRNAs are enriched by polyA purification under denaturing conditions to remove proteins not directly bound to RNA. Washes and nuclease treatment prepares the samples for mass spectrometry. Castello et al. use LC-MS/MS whereas Baltz et al. use quantitative SILAC mass spectrometry. (C) Venn diagrams analyzing the two studies for the overlap of total proteins, canonical RBPs, and novel RBPs. A selection of the novel RBPs validated by secondary biochemical assays are highlighted in the boxes, validated by either Castello et al. (purple) or Baltz et al. (gold). (D) Experimental scheme for ribosome profiling. After cell lysis and DNase digest, RNA digest yields RNA fragments protected by ribosomes. rRNAs are removed by hybridization to rRNA probes, mRNA fragments are size fragmented and prepared for deep-sequencing.