Discusión de los resultados.

2.4.1

Towards an RNA processing parts list of RNA elements and

RBPs

A substantial body of work has aimed to catalog functional RNA elements and their inter- acting proteins to gain a more complete and mechanistic understanding of RNA processing in cells. For example, analysis of 𝑘mer frequency and evolutionary conservation in specific regions of the genome has led to the appreciation that many cis- sequences are associated with specific types of regulation (Fairbrother et al. [2002], Ke et al. [2011]), even when the

trans-acting RBPs that bind them are not always known. Here, we characterized 78 hu- man RBPs using a high-throughput version of RBNS, a one-step in vitrobinding assay that assesses the spectrum of RBP primary sequence binding specificities along with contextual features that influence binding such as secondary structure, bipartite motifs, and flanking nucleotide content.

2.4.2

RBPs recognize a small subset of the available sequence space

Considering a diverse set of 78 proteins, we found that many RBPs bind a relatively small, defined subset of primary RNA sequence space rich in low-complexity motifs primarily com- posed of just one or two bases. This trend was seen independently for RRM and KH domain proteins that do not share common ancestry, suggesting convergent evolution toward recogni- tion of these types of motifs. These findings are consistent with previous studies implicating AU-, U-, and G-rich sequences as functional elements that regulate stability and splicing (Fu and Ares Jr [2014],Wu and Brewer[2012]). Previous studies have shown certain mono- and dinucleotide-rich sequences occur in clusters to mediate their effects on RNA processing (Barreau et al.[2006],Cereda et al.[2014]). These motifs also have lower propensity to form secondary structures that might block RBP binding, both in random sequences (Fig. 2-4H) and in sequences from the human transcriptome (Fig. 2-S6E). Thus, greater average acces- sibility for binding may have favored evolution of RBP specificity towards a set of motifs. Binding modes involving sliding of RBPs along RNA may also favor low complexity motifs.

For example, HNRNPC was shown to bind runs of uridines with a potential to slide among registers within an RNA (Cieniková et al. [2014]). Such a sliding model would be most feasi- ble for mono- or dinucleotide repeat motifs, while more complex motifs would likely require the RBDs to completely dissociate from the RNA between specific binding interactions.

A similar analysis of DNA binding proteins and transcription factors (not shown) revealed that they do not show the same inherent propensity to target low complexity sequences, per- haps due to differences in the size of the search space and differences in DNA and RNA structure and the biochemical mechanism of binding (Jolma et al. [2013]). While the over- lapping specificity across the 78 RBPs is high, we found that 18 of the 78 profiled RBPs have motifs dissimilar from that of any other RBP assayed (‘Unclustered’ inFig. 2-2A), suggest- ing that a subset of RBPs may have evolved to recognize more distinct sets of RNA targets. These 18 RBPs tended to have broader expression profiles across human tissues than did RBPs within motif clusters, while RBPs with motifs shared by other RBPs were more often expressed tissue-specifically (Fig. 2-S6F). Thus, some members of the same clusters may serve redundant functions in different cell or tissue types. Even so, coexpression of many members of a cluster appears widespread, and this is likely to provide versatile opportunities for post-transcriptional gene regulatory networks and evolution (Lapointe et al. [2017]).

2.4.3

RBP binding specificities harbor hidden complexity

Closer analysis of the complexity of RBNS data revealed that linear sequence motifs are often insufficient to fully capture RBP binding specificities, with sequence features beyond short motifs contributing to the specificity of most RBPs (Fig. 2-S6C). We find that nearly all RBPs have strong preferences for or more often against RNA secondary structure, and about half of RBPs were identified as favoring noncontiguous ‘bipartite’ motifs. While the most common representation of RBP binding sites is a single position weight matrix (PWM), our data suggest that in many cases RBP specificity may be better described by pairs of short motifs with variable spacing, and sometimes by representations describing structural preferences. We hypothesize that these context preferences may be general features that allow RBPs with similar motifs to select distinct targets in cells, a paradigm that has been put forth for pairs of RBPs (Smith et al. [2013]).

In assaying a number of RBPs containing RRM, KH, and zinc finger domains, we noted several commonalities across RBPs with similar RBDs. For example, RRMs and ZNFs were identified that bound to A-, C-, G- and U-rich motifs, while KH domains bound A-, C- and U-rich but not G-rich motifs, in agreement with previous structural studies showing that G-recognition by KH domains is rare (reviewed by Nicastro et al. [2015]). Further, while many motif clusters included RBPs from all three RBD types analyzed (Fig. 2-2A), 5mer enrichments were more highly correlated among RBPs with the same RBD type relative to RBPs with different RBD types, even after excluding paralogs (Fig. 2-S6G), suggesting greater residual similarity in binding preferences.

We also noted domain-specific trends among preferences for contextual features. For ex- ample, while RBPs overwhelmingly preferred unstructured motifs bothin vitro(Fig. 2-4A) andin vivo(Fig. 2-S4C), ZNFs bound motifs encompassing a wide range of secondary struc- ture contexts including those with greater stem content (Fig. 2-S6H), and RBPs containing ZNFs showed greater variability in their 𝑃paired preferences than did other RBPs (Fig. 2-

S6I). These observations are consistent with a recent study finding that more than twenty ZNF-containing proteins selectively bound highly-structured pre-microRNAs (Treiber et al.

[2017]). Proteins with KH domains also shared numerous properties, including a preference for binding to large hairpin loops, flanking nucleotide compositional preferences, and com- mon preference for bipartite motifs. Notably, all but one KH-containing RBP assayed by RBNS (SF1) had either more than one KH domain or a known homodimerization domain, suggesting that binding of RNA by pairs of KH domains is very common.

Together, our findings emphasize the complexity of the interactions between proteins and RNA and motivate future studies of the structural basis of the dependence on the various contextual features documented here.