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.