Post-transcriptional control of mRNA permeates biology. RNA-binding proteins control every aspect of mRNA function including processing, localization, stability, and translational status. These factors serve pivotal roles in memory, nociception, and early development (Crittenden et al., 2002; Dubnau et al., 2003; Barragán-Iglesias et al., 2018). RNA-binding proteins associate with sequences and structures typically situated in untranslated regions (UTRs) of an mRNA. Understanding the specificity of proteins for their regulatory motifs is crucial as these liaisons govern mRNA fate.

RNA-binding proteins comprise 4% and 10% of the human and yeast proteomes, respectively (Castello et al., 2012; Beckmann et al., 2015). A driver of this expansion is gene duplication. For example, RNA recognition motifs (RRMs), a large RNA-binding protein family, have proliferated throughout evolution (Dreyfuss et al., 1993; Ray et al., 2013) and have diverged to acquire distinct RNA recognition properties and biological functions (Chaudhury et al., 2010; Zaharieva et al., 2015). The molecular and structural underpinnings that enable diversification of RNA recognition are fundamental, as they dictate which mRNAs are subject to regulation and are a key source of evolutionary plasticity in the configuration of mRNA regulatory networks.

PUF proteins, named for PUM (Pumilio) and FBF (fem-3 binding factor), are an exemplary system for understanding the divergence of RNA recognition within eukaryotic RNA-binding protein families (Neeb et al., 2017; Wilinski et al., 2017). PUF proteins are present in multiple copies ranging between 1 and 26 different proteins expressed per eukaryotic organism (Wickens et al., 2002). Classical PUF proteins recognize single-stranded RNA sequences. Eight PUM repeats are arranged in a crescent shape with the RNA bound on the concave face of the protein (Edwards et al., 2001; Wang et al., 2001; Wang et al., 2002; Wang et al., 2009b; Zhu et al., 2009). Each repeat contributes a trio of amino acid residues, termed a tripartite recognition motif (TRM), that directly contact the opposing RNA base (Wang et al., 2002; Campbell et al., 2014). The TRM determines the specific RNA base recognized through a combination of edge-on and stacking interactions (Cheong and Hall, 2006; Koh et al., 2011; Valley et al., 2012; Campbell et al., 2014). However, some PUF proteins contain an additional source of specificity, a binding pocket situated within the C-terminal region that accommodates a 5´ nucleotide upstream of the core RNA recognition sequence (Zhu et al., 2009; Qiu et al., 2012).

A striking source of variation among classical PUF proteins is the length of the RNA sequence motif (Campbell et al., 2012a). The prototypical PUF protein, PUM1, uses its eight PUM repeats to recognize an 8-nt motif called the PUM binding element (PBE), 5´-UGUANAUA-3´ (by IUPAC naming convention N is any nucleotide, H is A or C or U, W is A or U, D is A or G or U, and R is A or G), with each PUM repeat engaging one RNA base in a 1:1 recognition pattern. In contrast, other PUF proteins recognize longer RNA sequences with their eight PUM repeats. For instance, yeast PUF proteins preferentially bind core RNA elements with lengths of 8-nt (Puf3p – UGUAHAUA), 9-nt (Puf4p – UGUAHAHUA), and 10-nt (Puf5p – UGUAWYWDUA) (Gerber et al., 2004; Lapointe et al., 2015; Lapointe et al., 2017). All three motifs begin with a 5´ UGUR and end with a 3´ UA dinucleotide, yet the spacing and recognition of bases between these elements varies depending on the PUF protein (Miller et al., 2008; Zhu et al., 2009; Wilinski et al., 2015). PUF motifs correlate with distinct biological processes. In yeast, mRNA targets with different lengths are associated with mitochondrial function (8-nt), ribosome biogenesis (9-nt), and regulation of gene expression (10 nt) (Gerber et al., 2004; Wilinski et al., 2015). This suggests post-transcriptional regulatory networks of PUF proteins are defined by a combination of recognition pattern and motif length.

To understand the relevance of motif length to biological function, we focused on two PUF proteins expressed in the germline of C. elegans that recognize distinct regulatory elements - FBF and PUF-8. FBF recognizes a 9-nt motif, 5´-UGURNNAUA-3´, whereas PUF-8 binds an 8-nt sequence, 5´-UGUANAUA-3´ (Bernstein, 2005; Opperman et al., 2005). One mechanistic model to account for this key change in binding element length is curvature of the RNA-binding surface (Wang et al., 2009b). FBF’s RNA-binding surface is flatter than the 8-nt binding PUM1, and this change in curvature appears to accommodate a longer RNA motif. Other PUF proteins that bind to motifs ≥ 9 nts also have correspondingly flatter surfaces (Miller et al., 2008 and Wilinski et al., 2015). PUF-8 was engineered to have 9-nt specificity by substituting a 45-aa region of FBF-2 containing portions of repeats 4 and 5. Curvature change of FBF-2 is focused in this central region, suggesting that conferring a flattened architecture to PUF-8 produced 9-nt specificity. However, in the absence of a crystal structure of PUF-8, its degree of curvature is unknown, and the curvature of the chimeric protein could not be determined. As a result, the validity of this model is difficult to assess fully. A second mechanistic model to account for differences in preferred motif length is the identity of the TRM RNA-binding residues. In crystal structures of FBF-2 bound to RNA, repeat R5 lies opposite the site where an additional nucleotide is accommodated, but its TRM does not form typical 1:1 base-stacking and edge-interacting contacts with the RNA (Wang et al., 2009b). Conversely, repeat R5 of the 8-nt binding PUM1 forms specific contacts between its TRMs and the base at position 4 (Wang et al., 2002). PUF-8 appears to use a 1:1 recognition pattern like PUM1, binding 8-nt sequences with its eight PUM repeats. The relationship of these characteristic motif lengths to biological function is not well established.

Here, we provide evidence that both PUF protein curvature and TRM interactions can specify binding element length of FBF-2 and PUF-8. We report a crystal structure of PUF-8 in complex with an 8-nt RNA element. PUF-8’s curvature is similar to that of PUM1 and therefore appears to enable its binding to an 8-nt RNA. To explore the role of TRMs in RNA length specificity, we reasoned that substitutions in the TRM of FBF-2 repeat R5 might enable a metamorphic change converting its specificity from a 9-nt to an 8-nt element. Using a modified yeast 3-hybrid screen, we identified and analyzed TRM mutants that allow FBF-2 to favor binding to an 8-nt element. Structural analysis reveals that the repeat R5 TRM mutations allow FBF-2 to bind to an 8-nt element in a 1 repeat:1 RNA base pattern. The curvature of FBF-2 is unaltered by the TRM mutations, suggesting that these mutations alter the length specificity of FBF-2 through a distinct mechanism. Using this information, we used genome engineering to test whether a variant FBF-2 with PUF-8 motif length recognition can function in place of PUF-8 in vivo. Loss of PUF-8 in C. elegans causes tumor formation in a sensitized genetic background (Racher and Hansen, 2012). We generated a strain with a variation in FBF-2 that confers recognition of an 8-nt site. In this strain, tumor formation upon loss of PUF-8 is largely reversed, indicating that the FBF-2 variant substitutes effectively for PUF-8 to suppress overproliferation in the germline. Collectively, these data highlight the critical importance of RNA target recognition, including binding element length, in determining the biological function of PUF proteins. This work has broad implications beyond PUF proteins for understanding the evolution of functional divergence and modulation of consensus binding element features in nucleic acid binding proteins.

Our experiments reveal three insights into RNA-PUF protein interactions. First, PUF-8 adopts a structure reminiscent of human PUM1 and forms base-specific contacts to seven of the eight nucleotides that comprise the binding element. Second, mutations in repeat 5 of FBF-2 can introduce new contacts to the RNA that switch preferential binding to an 8-nt PBE sequence motif over a 9-nt FBE motif. The amino acid residue combinations we obtained through random mutagenesis occur in nature. Third and finally, a variation in FBF-2 that enables it to recognize the consensus binding element of PUF-8 rescues a germline tumor phenotype caused by loss of function of puf-8 in a sensitized background.

Prior results suggest that the degree of curvature of the RNA-binding surface of PUF proteins corresponds to the length of the RNA element that is recognized, but our results here indicate that the identities of the TRMs also contribute to the RNA motif length of PUF proteins. In the case of changing specificity of FBF-2 to that of PUF-8, we have demonstrated that a minimum of two amino acid changes (CQ/R to AQ/Y) can swap binding element length preference, but they do not impact curvature. This finding is consistent with the notion that changes in curvature are remarkably well correlated with, but not necessarily required for alterations in binding element length preference. Crystal structures of different PUF proteins in complex with their RNA targets indicate that PUF proteins with flatter RNA-binding surfaces recognize longer RNA elements (Wang et al., 2001; Miller et al., 2008; Wang et al., 2009b; Wilinski et al., 2015). In a few instances, crystal structures have been determined with and without RNA, and the degree of curvature of these PUF proteins does not change upon RNA binding (Edwards et al., 2001; Wang et al., 2001, Wang et al., 2002, Miller et al., 2008; Weidmann et al., 2016). Chimeras that transfer FBF-2 binding specificity to PUF-8 by substituting central segments of FBF-2 have also argued in favor of curvature as a major driver of motif length (Opperman et al., 2005; Wilinski et al., 2015). Our crystal structure of PUF-8 confirms the difference in its curvature from that of FBF-2. Although the FBF-2-to-PUF-8 specificity switch could be engineered without curvature change, a PUF-8-to-FBF-2 specificity switch might require a curvature change. The degree of curvature of the RNA-binding surface of the chimeric protein is unknown, but the segments of FBF-2 that can transfer its specificity to PUF-8 occur at the region where curvature change originates. The distinct curvature among different PUF proteins in an organism raises the question as to whether topological differences provide additional functional benefits beyond binding element length specificity. Perhaps changes in the convex surface of the protein foster or diminish specific protein partnerships and this impacts RNA regulatory mechanism (Campbell et al., 2012b; Menichelli et al., 2013; Wu et al., 2013).

Our findings directly impact the growing field of synthetic biology applications of engineered PUF proteins with novel specificities (Wang et al., 2013). The specificity of individual TRMs has been established for several dozen combinations (Campbell et al., 2014). Two component systems that join programmable RNA recognition with optical tags or effector domains have broad applications for understanding and manipulating RNA function (Ozawa et al., 2007; Tilsner et al., 2009). For instance, covalent fusion of FBF-2 to a poly(A) polymerase prevented deadenylation of a targeted mRNA (Cooke et al., 2011). Conceptually similar approaches with PUM1 have been used to modify splicing, RNA stability, and localization (Wang et al., 2009a; Dong et al., 2011; Choudhury et al., 2012; Abil et al., 2017). One way to encode recognition of different length RNA elements is through engineering PUF proteins with additional or fewer PUM repeats (Filipovska et al., 2011; Zhao et al., 2018). Our work provides another potential means to reduce the length of the binding element, thus increasing the range of targeting modalities available for tailored RNA recognition by the PUF scaffold.

The finding that FBF-2 bearing mutations that alter motif length recognition can rescue knockdown of PUF-8 provides additional insight into PUF-8 and FBF-2 biological functions. PUF-8 and FBF-2 produce dissimilar cellular outcomes. PUF-8 restricts overproliferation: single puf-8 mutants display proximal gonad tumors due to dedifferentiation of sperm (Subramaniam and Seydoux, 2003). In contrast, FBF-2 (together with FBF-1) promotes mitosis: double fbf-1/2 mutants display profound developmental defects in cellular proliferation (Crittenden et al., 2002). Our experiment capitalized on the fact that PUF-8 and FBF-2 are both expressed in the mitotic region of the germline and recognize similar but non-identical motifs (Campbell et al., 2012a), and the activities of FBF-1 and FBF-2 are largely redundant, which allowed engineering of FBF-2 (Lamont et al., 2004). The ability of the FBF-2 AQ/Y variant to rescue PUF-8 function suggests that the downstream molecular consequences of PUF-8 and FBF-2 RNA recognition are similar. Their different effects on cellular proliferation are therefore directed by the functions of their respective mRNA targets.

Why modulate motif length recognition as a means of differentiating homologous RNA-binding proteins? This is a broadly conserved phenomenon in organisms with multiple PUF genes (Wilinski et al., 2017). Following a gene duplication event, diversification of binding specificity provides a means to regulate a unique set of targets with the potential to engender new and advantageous biological functions. One possible reason why motif length would be an attractive parameter is that the use of a conserved handle (e.g. 5´UGUA) enables evolution of the downstream sequence to sample different RNA regulons through subtle changes to the existing motif. This plasticity could enable more rapid transitions as both the RNA motif and regulators diverge and co-evolve. This principle may also apply to other RNA-binding proteins, which often comprise multiple RNA-binding modules akin to the multiple PUM repeats. For example, the RNA-binding properties of RRM proteins expand with tandem domains. A gene duplication introducing an additional RNA-binding domain maintains recognition of the original RNA motif while specificity of the new domain evolves to produce a distinct RNA motif.

All data associated with the manuscript are present in the source data file. Data have also been deposited to PDB under the accession numbers 6NOD, 6NOH, 6NOF, and 6NOC.

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Author details

1. Vandita D Bhat

   Department of Biological Sciences, University of Texas Dallas, Richardson, United States

   Contribution

   Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   Contributed equally with

   Kathleen L McCann

   Competing interests

   No competing interests declared

2. Kathleen L McCann

   Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, United States

   Contribution

   Investigation, Writing—original draft, Writing—review and editing

   Contributed equally with

   Vandita D Bhat

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7144-4851

3. Yeming Wang

   Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, United States

   Contribution

   Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

4. Dallas R Fonseca

   Department of Biology, Ithaca College, Ithaca, United States

   Contribution

   Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

5. Tarjani Shukla

   Department of Biological Sciences, University of Texas Dallas, Richardson, United States

   Contribution

   Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

6. Jacqueline C Alexander

   Department of Biology, Ithaca College, Ithaca, United States

   Contribution

   Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

7. Chen Qiu

   Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, United States

   Contribution

   Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

8. Marv Wickens

   Department of Biochemistry, University of Wisconsin-Madison, Madison, United States

   Contribution

   Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

9. Te-Wen Lo

   Department of Biology, Ithaca College, Ithaca, United States

   Contribution

   Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

10. Traci M Tanaka Hall

    Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, United States

    Contribution

    Supervision, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    hall4@niehs.nih.gov

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6166-3009

11. Zachary T Campbell

    Department of Biological Sciences, University of Texas Dallas, Richardson, United States

    Contribution

    Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    zachary.campbell@utdallas.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-3768-6996

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