Introduction
From their synthesis to their degradation, RNAs are escorted by proteins that dictate their fate. In addition to transcription, interactions between RNA and proteins underlie post-transcriptional processes, including maturation, splicing, nuclear export, localisation, stability, translatability, and degradation. RBPs fine-tune gene expression profiles which are essential to maintain cellular homeostasis. Accordingly, disturbance of physiological RNA-protein interactions decreases microbial capacity to rapidly reprogram the transcriptome and adapt to environmental changes 1,2.
Methodological developments during the last decade have vastly increased the technical repertoire to explore RNA-protein interactions in vivo . Most methods to detect RNA-protein interactions are based on UV cross-linking, which entails irradiating cells with short wavelength (254 or 365 nm) UV light to induce the formation of covalent bonds between RBPs and directly bound transcripts (“zero distance”; reviewed in 3). This property makes it possible to isolate RBP-bound RNAs under very stringent and denaturing conditions, greatly reducing noise. Although UV cross-linking is notoriously inefficient and biased towards coupling pyrimidines to a select number of amino acids (reviewed in 3), it has become a hugely popular tool to study protein-nucleic acid interactions in living systems.
UV irradiation approaches used to study protein-RNA interactions can be classified as RNA-centric, which isolate RNA species to identify cross-linked RBPs, or protein-centric, which capture a specific RBP to study its bound RNA targets (Fig. 1) 4. Recently, a variety of UV-based high-throughput RNA-centric strategies have characterised the RNA-binding proteome (RBPome) in eukaryotic and prokaryotic microorganisms, which unearthed many novel RBPs. Surprisingly, many of these newly identified proteins lack hitherto known RNA recognition motifs (RRM) or functional links to RNA metabolism. For instance, in multiple studies, metabolic enzymes constituted a prominent fraction of these putative RBPs5. However, each of these high-throughput RNA-centric approaches has its own technical caveats and noise levels. Therefore, protein-centric procedures are critical to functionally validate recently discovered RBPs. By combining RNA and protein-centric approaches one can shed light on how RNA binding affects (i) the life cycle of target transcripts and (ii) the primary function of the associated protein. For example, individual protein-centric analyses have verified that, indeed, some metabolic enzymes moonlight as post-transcriptional regulators or their enzymatic activity is regulated by RNA 6.
This perspective article aims to offer a selective review of these protein and RNA-centric options, discuss their individual strengths and limitations, consider possible technical improvements, and present a complementary workflow for the identification and functional characterisation of novel RBPs in microorganisms.