Determinants of affinity and specificity in RNA-binding proteins

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Highlights

  • Interactions between RNA and RNA-binding proteins (RBPs) are typified by diversity.

  • Many enigmatic RBPs have been reported, but their interactions are poorly understood.

  • Interactions between long non-coding RNAs and putative partners are also mysterious.

  • Low-complexity regions in RBPs can form droplets/gels. A new era for cell biology?

Emerging data suggest that the mechanisms by which RNA-binding proteins (RBPs) interact with RNA and the rules governing specificity might be substantially more complex than those underlying their DNA-binding counterparts. Even our knowledge of what constitutes the RNA-bound proteome is contentious; recent studies suggest that 10–30% of RBPs contain no known RNA-binding domain. Adding to this situation is a growing disconnect between the avalanche of identified interactions between proteins and long noncoding RNAs and the absence of biophysical data on these interactions. RNA–protein interactions are also at the centre of what might emerge as one of the biggest shifts in thinking about cell and molecular biology this century, following from recent reports of ribonucleoprotein complexes that drive reversible membrane-free phase separation events within the cell. Unexpectedly, low-complexity motifs are important in the formation of these structures. Here we briefly survey recent advances in our understanding of the specificity of RBPs.

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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