Determinants of affinity and specificity in RNA-binding proteins
Introduction
In contrast to the situation for DNA-binding proteins, the rules governing functional specificity for RNA-binding proteins (RBPs) have proven hard to define, because of diversity in the structures of both RNA and RBPs themselves. A recent census [1•], for example, predicted approximately 1500 human RBPs in ∼600 distinct structural classes — many of which contain only a single member!
Although SELEX (Systematic Evolution of Ligands by Exponential enrichment — a method in which a protein is incubated with a random library of RNA oligonucleotides and the tightest binding sequences identified) and CLIP (Cross Linking ImmunoPrecipitation — in which proteins are cross-linked in live cells to their RNA targets and purified by immune-affinity; the associated RNA is then sequenced) approaches have delineated the sequence specificity of dozens of RBPs, the motifs that have been identified are often surprisingly short and degenerate. Furthermore, the RNA-binding affinities often appear to be significantly lower than those exhibited by typical DNA-binding proteins (micromolar rather than nanomolar for individual domains). An important question is whether this situation offers sufficient specificity for proper function. If not, then either first, the methods used to assess sequence specificity are not reflecting the biology or second, specificity is also effectively provided by other elements of the protein, perhaps through contacts made to other RBPs. In this review, we will discuss recently reported data relating to the specificity — or lack thereof — of RBPs.
Section snippets
Specificity and affinity in canonical RNA-binding domains
Numerous studies indicate that RNA-binding domains (RBDs) exhibit substantially more diversity in their interactions with RNA than do their DNA-binding counterparts. For example, C2H2 zinc fingers (ZFs) and homeodomains always recognise their DNA targets using the same surface and in the same orientation, whereas the most common RBD, the RNA-recognition motif (RRM), binds target single-stranded (ss) RNAs in a wide variety of relative orientations [2] (Figure 1a). Likewise, the translational
More complications: abundance, shape, avidity, accessory proteins and cellular context
In any case, the physiologically relevant RNA targets of an RBP might not always be the highest affinity interactions. For example, a high-throughput kinetics approach has shown that the biological substrates of C5 (a component of the tRNA processing RNAse P in Escherichia coli) are not the tRNA sequences it binds tightest but rather those near the median of the affinity distribution [10]. Similarly, the inhibition of PRC2 histone methyltransferase activity by both forward and reverse noncoding
Specificity in non-traditional RBPs
A growing body of work has identified RNA-binding activity in proteins with no prior connection to RNA biology (recently termed enigmRBPs [22•]). TFIIIA is a DNA-binding transcription factor that binds the promoter of the 5S rRNA gene — and also the 5S rRNA transcript itself [23], creating a negative feedback loop that represses further transcription of the gene. The functional sidestep from DNA- to RNA-binding activity is easy to envisage [24], but much more surprising is the iron responsive
HOTAIR? The uncertain specificity of lncRNA-binding proteins
Numerous recent papers describe an emerging role for lncRNAs such as HOTAIR [36] in gene regulation and disease pathogenesis. Functionally, lncRNA–protein interactions can be loosely classified as: first, guiding recruitment of protein complexes to target genes; second, fulfilling an architectural role in ribonucleoprotein (RNP) complexes; and third, sequestering regulatory proteins away from target genes [37]. However, at the mechanistic and molecular level, we remain largely in the dark.
It's just a phase: the role of low-complexity domains in functional cellular structures
Sixty or more years of protein and nucleic acid research has suggested that the properties and activities of biomacromolecules observed in dilute aqueous solution by and large recapitulate in vivo biochemistry. Despite these data enabling prediction of mutant phenotypes and allowing the development of effective small molecule inhibitors, recent discoveries centred on RBPs and their participation in membrane-free cellular bodies open the door to what might be a radically different view of cell
Future directions
The last few years have seen the emergence of a substantial number of new ideas that, individually and collectively, have the prospect of radically changing the way we view protein–RNA interactions — and more broadly cell biology.
Some of the biggest mountains that must be scaled to see these views include: first, the development of tools to permit the structural analysis of both lncRNAs and membrane-less cellular structures; second, parsing ncRNAs and ncRNA–protein interactions into ‘important’
Conflict of interest
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was funded by a Senior Research Fellowship (1058916; to JPM) and project grants (1048659 to CSB and 1063188 to JPM) from the National Health and Medical Research Council of Australia.
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2022, Computational and Structural Biotechnology JournalCitation Excerpt :Real-life RNA-binding strategies, while based on the same fundamental principles, have many more facets and nuances, sometimes resulting in behaviour significantly deviating from the one described here [3]. For example, many RBPs, such as Hfq, possess multiple RNA-binding sites, which enable them to exploit semi-competitive, or even non-competitive, interactions with more than one transcript at a time, with different functional outcomes [50,81–83]. Several RBPs can additively or cooperatively interact with one RNA, with a wide variety of consequences: from ‘silent’ binding, which only titrates an RBP out without affecting the target, to complex synergistic outputs, which cannot be achieved through isolated binding by individual RBPs [1,18,67,84–90].
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These two authors contributed equally.