Biology subjects

Cavazos, F. F.

Publications and source records attributed to Cavazos, F. F..

3 recordsLinked to original sources

bioRxiv2025

Contributions of Folded and Disordered Domains to RNA Binding by HNRNPR

RNA binding proteins (RBPs) interact with and tightly regulate the fate of messenger RNAs but how RNA targets are recognized remains a challenging question. RBPs often contain multiple domains known to directly bind RNA, such as RNA recognition motifs (RRMs), as well as domains whose RNA binding capacity remains incompletely understood, e.g., low complexity domains (LCDs). Here, we dissect HNRNPR, an RBP with three RRMs and an arginine-glycine rich (RG-rich) LCD. We apply unbiased high-throughput biochemical approaches and identify critical RNA binding domains that confer specificity. We show that not all RRMs contribute equally to binding and find that RRM3, along with a downstream C-terminal charged region, are required for RNA binding. We find that HNRNPR also binds RNA G-quadruplexes (rG4s) and map multiple rG4 binding sites including RRM3 with the C-terminal charged region and RG-rich regions within the LCD. We dissect rG4 specificity for the full length HNRNPR and LCD using a newly created RNA pool focused on rG4s and reveal that binding is dependent on RNA folding and find specific rG4 features that enhance HNRNPR-rG4 interactions. Our work highlights the complexity of RBP-RNA interactions and motivates the study of disordered regions as RNA binding domains.

biochemistry↗

bioRxiv2024

Dissecting RNA Selectivity Mediated by Tandem RNA-Binding Domains

RNA-protein interactions are pivotal to proper gene regulation. Many RNA-binding proteins possess multiple RNA-binding domains; however, how these domains interplay to specify and regulate RNA targets remains poorly understood. Here, we investigate three multi-domain proteins, Musashi-1, Musashi-2, and Unkempt, three factors which share a high degree of RNA specificity. We use a combination of massively parallel in vitro assays with random or naturally derived RNA sequences and find that individual domains within a protein can have differing affinities, specificities, and spacing preferences. Further, we emphasize that while all three proteins have overlapping motif specificities, non-overlapping sequences may allow for target discrimination. We carry out large scale competition assays between these proteins and determine how individual protein specificities and affinities influence competitive binding. Integration of in vivo binding and regulation with in vitro specificities shows that target selection involves a combination of the protein intrinsic specificities described here, but cellular context is critical to drive these proteins to motifs in specific transcript regions. Finally, evolutionarily conserved RNA regions display evidence of binding multiple RBPs in vivo, and these RNA regions recapitulate this trend with the highest affinity in vitro. We highlight the importance of understanding features of complex RNA-protein interactions and how protein-target discrimination can be established.

biochemistry↗

bioRxiv2024

Understanding species-specific and conserved RNA-protein interactions in vivo and in vitro

While evolution is often considered from a DNA- and protein-centric view, RNA-based regulation can also impact gene expression and protein sequences. Here we examined interspecies differences in RNA-protein interactions using the conserved neuronal RNA binding protein, Unkempt (UNK) as model. We find that roughly half of mRNAs bound in human are also bound in mouse. Unexpectedly, even when transcript-level binding was conserved across species differential motif usage was prevalent. To understand the biochemical basis of UNK-RNA interactions, we reconstituted the human and mouse UNK-RNA interactomes using a high-throughput biochemical assay. We uncover detailed features driving binding, show that in vivo patterns are captured in vitro, find that highly conserved sites are the strongest bound, and associate binding strength with downstream regulation. Furthermore, subtle sequence differences surrounding motifs are key determinants of species-specific binding. We highlight the complex features driving protein-RNA interactions and how these evolve to confer species-specific regulation.

evolutionary biology↗