4 RNA-BINDING PROTEINS
Interactions between proteins and RNA are indispensable for virtually all processes in any living cell. From the biosynthesis of RNA molecules to their degradation, the entire life cycle of RNA is associated to RNA-binding proteins (RBP). RBPs are significantly more conserved across evolution than non-RNA-binding proteins and comprise about 3-11% of the proteome in all domains of life (Gerstberger et al., 2014). The interplay of protein and RNA has traditionally been viewed as the formation of highly dynamic ribonucleoprotein complexes that facilitate multiple regulatory functions including RNA processing, modification, translation or regulation (Dreyfuss et al., 2002; Babitzke et al., 2019). Such ‘canonical’ RBPs bind RNA specifically via structurally defined RNA-binding domains such as hnRNP homology (KH) domains (Valverde et al., 2008), the S1 domain (Suryanarayana & Subramanian, 1984), RNA recognition motifs (RRM) (Cléry et al., 2008), the RNA-binding domain of transcriptional antiterminators (Stülke, 2002) or DEAD box helicase domains (Linder and Jankowsky, 2011).
The investigation of protein-RNA interactions is typically based on the premise that specific protein domains can bind RNA. This view, however, neglects the ability of RNA adopt highly diverse structures that in principle can be able to bind any molecule. Strikingly, a study aimed at identifying novel RBPs by crosslinking coupled to high resolution mass spectrometry identified proteins crosslinked to RNA that lack any known canonical RNA-binding domain. The discovery of those non-canonical RNA binders such as the phosphoglycerate kinase, revealed a fundamentally new group of proteins of both known and unknown function to be potential RBPs (Schmidt et al., 2012; Kramer et al., 2014). The existence and identification of those unconventional RBPs harboring unconventional RBDs proposes novel mechanisms of protein-RNA interaction with new biological functions (Hentze et al., 2018). Interestingly, several of the poorly studied but highly expressed proteins of B. subtilisthat are thought to bind to RNA and/ or the ribosome do not possess known RNA-binding domains. These proteins are YlbN, YlxR, YrzB as well as the poorly studied RNA-binding SpoVG protein (see Table 1, Burke & Portnoy, 2016). It is tempting to speculate that these proteins will define novel RNA-binding domains.
One particularly important open question in the investigation of RNA-binding proteins in B. subtilis and other Gram-positive bacteria is related to the base-pairing of small regulatory RNAs with mRNAs. As in other bacteria, small cis- and trans-acting RNA molecules bind to partially complementary mRNAs to control their stability and translation (Ul Haq et al., 2020; Mars et al., 2016). In E. coli , the Hfq protein acts as a chaperone that facilitates the base-pairing between only rather short complementary sequences in the two RNA molecules (Vogel and Luisi, 2011). In B. subtilis , a shorter Hfq protein is present; however, despite all attempts so far, there is no indication that Hfq also has this function in B. subtilis(Dambach et al., 2013; Hämmerle et al., 2014; Rochat et al., 2015). It has recently been proposed that the lack of 5’-3’ exonucleolytic RNases in E. coli and related bacteria results in the accumulation of mRNA 3’ UTR. This may have provided a larger pool of unconstrained RNA sequences that has stimulated the evolution of Hfq function and small RNA (sRNA) regulation. In contrast, the presence of the 5’-3’ exoribonuclease RNase J prevents the accumulation of 3’ UTR sRNAs inB. subtilis . As a consequence, there was no selective pressure on Hfq to evolve to become a mediator of RNA-RNA interactions (Mediati et al., 2021). Since the physical conditions in the B. subtilis cell are certainly similar to those in cells of E. coli , it seems unlikely that regulation exerted by base-pairing of RNA molecules works without a chaperone. The identification of the protein that takes over this function is a major challenge in the future research on RNA-binding proteins in B. subtilis .
Taken together, to understand both basic and complex processes of the cell, extensive research on RNA-protein interactions is required. Even though numerous conventional RBPs have been identified over the past decades, some still remain to be characterized. With the discovery of unconventional RBPs harboring non-canonical RNA-binding domains, the field of understudied RBPs has dramatically increased.