Protein-mRNA interactome capture: cartography of the mRNP landscape

Abstract

RNA-binding proteins play a variety of roles in cellular physiology. Some regulate mRNA processing, mRNA abundance, and translation efficiency. Some fight off invader RNA through small RNA-driven silencing pathways. Others sense foreign sequences in the form of double-stranded RNA and activate the innate immune response. Yet others, for example cytoplasmic aconitase, act as bi-functional proteins, processing metabolites in one conformation and regulating metabolic gene expression in another. Not all are involved in gene regulation. Some play structural roles, for example, connecting the translational machinery to the endoplasmic reticulum outer membrane. Despite their pervasive role and relative importance, it has remained difficult to identify new RNA-binding proteins in a systematic, unbiased way. A recent body of literature from several independent labs has defined robust, easily adaptable protocols for mRNA interactome discovery. In this review, I summarize the methods and review some of the intriguing findings from their application to a wide variety of biological systems.

Keywords: RBPs; RNA-binding proteins; mRNA interactome discovery; messenger RNA.

Figure 1.. mRNA interactome capture.

Schematic of mRNA interactome capture experiments. A. Samples are treated with ultraviolet (UV) light, either 254 nm to crosslink endogenous nucleotides to interacting proteins or 365 nm to crosslink exogenously added 4-thiouridine (4SU) to interacting proteins. B. Total mRNA is recovered from the sample using oligo-deoxythymidine (dT) resin, which base pairs with the polyadenosine (polyA) tail of mRNA. C. Following ribonuclease digestion, the proteins are separated and identified using quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS). D. Summary of the outcomes. Some RNA-binding proteins (RBPs) are found in several samples, across diverse species. These proteins likely have housekeeping functions. Others are specific to unique cell types or are unique to a given species. Many well-studied RBPs are recovered by this approach, as well as many predicted RBPs with canonical domains such as RNA-recognition motif (RRM), KH, etc. New RBPs are also identified, which can be classified into groups that are entirely new or well-studied proteins where RBP-binding activity was unexpected (i.e. EnigmRBPs). Abbreviations: mEF, mouse embryonic fibroblast; mESC, mouse embryonic stem cells; RBD, RNA-binding domain.

Figure 2.. Comparison of multiple interactomes.

Four tables are presented, comparing three human cell lines, three mouse cell lines, three yeast data sets, and two fly data sets, respectively. Below the tables is a key that identifies the source of the given interactome data set. The values in the tables represent the number of shared proteins in a pairwise comparison between the interactomes that intersect in the table. The diagonals (brown squares) show the total number of proteins collected in each given interactome. The orange squares show the number in common between two interactomes. The values in green to the left of each table are the number of proteins in common among the three interactomes compared, and the values in red are the number of proteins that are reported only in the specific interactome. Abbreviations: mEF, mouse embryonic fibroblast; mESC, mouse embryonic stem cell.