Biology subjects

Guzman, B. B.

Publications and source records attributed to Guzman, B. B..

10 recordsLinked to original sources

bioRxiv2026

Sequence and structure of protein binding sites in RNA impact biomolecular condensates

Biomolecular condensates are central to subcellular compartmentalization. Although many condensates contain and regulate RNA, research has primarily focused on protein interactions. Here, we investigate RNA-protein interactions underlying cell cycle-regulating condensates in the multinucleate fungus Ashbya gossypii. These condensates form through interactions between G1 cyclin mRNA CLN3 and RNA-binding protein Whi3, which was predicted by homology to recognize a five-nucleotide motif repeated within the transcript. Natural variation in motif number in Ashbya strains led us to hypothesize that binding site valence may influence condensate properties. Using unbiased binding assays, we determined the preference of Ashbya Whi3 protein for specific primary RNA sequence and mutated individual Whi3-binding sites within CLN3 mRNA. Mutants exhibited distinct condensate properties despite having the same valence in terms of binding site number. Mutations altered the saturation concentration (Csat) and dense phase concentration of RNA and protein in cell-free reconstitution experiments. A subset of mutants, showed reduced number of condensates and deregulation of the cell cycle in cells. We also find that enhanced availability of single-stranded RNA can compensate for loss of binding sites. Together, these data indicate that differences in RNA protein binding-site context and not simply valence plays a critical role in determining condensate properties.

cell biology↗

bioRxiv2025

The role of the L421P mutation in Penicillin-Binding Protein 1 (PBP1) in the evolution of chromosomally mediated penicillin resistance in Neisseria gonorrhoeae

ponAL421P encodes a mutated variant of penicillin-binding protein 1 (PBP1) and is a key resistance determinant that increases the penicillin MIC (MICPEN) above the clinical breakpoint in Neisseria gonorrhoeae. Despite the removal of penicillin from treatment guidelines for gonococcal infections in the 1980s, ponAL421P is present in nearly 50% of current N. gonorrhoeae isolates in the PubMLST database. Bioinformatic analysis indicates that ponAL421P is exclusive to N. gonorrhoeae isolates, whereas Leu-421 is 100% conserved in other Neisseria species. To understand the involvement of ponAL421P in antibiotic resistance, we introduced ponA variants encoding 16 different amino acids at position-421 into FA6140, a penicillin-resistant gonococcal isolate that naturally harbors ponAL421P. Proline-421 was the only mutation that increased the MICPEN to the same level as FA6140. We also assessed the fitness of strains with the 16 mutant ponA alleles over multiple serial passages, both with and without sub-MIC levels of penicillin. There was no fitness defect attributed to ponAL421P under these experimental conditions; instead, our analyses suggest that the widespread occurrence of ponAL421P is driven by its capacity to increase the MICpen above the clinical breakpoint. In FA6140 transformed with the mosaic penA allele from strain H041, a ceftriaxone-resistant isolate, ponAL421P increased the MIC of ceftriaxone, suggesting that ceftriaxone targets PBP1 in this strain. We conclude that the ponAL421P allele emerged in gonococcal isolates, increasing the MICPEN above the clinical breakpoint, and has remained in the population even after the removal of penicillin from treatment guidelines. ImportanceThe emergence of antibiotic-resistant Neisseria gonorrhoeae threatens effective treatment of gonorrhea, one of the most common sexually transmitted infections worldwide. Understanding the genetic changes that drive and maintain resistance is crucial for anticipating future resistance trends. Here, we investigated the impact of a key resistance mutation in PBP1 (encoded by ponAL421P). Although penicillin has not been used to treat gonorrhea for decades, this mutation remains widespread even in recent N. gonorrhoeae isolates. ponAL421P confers clinically relevant penicillin resistance without imposing an in vitro fitness cost. ponAL421P also increases resistance to ceftriaxone in strains with penA alleles that are associated with ceftriaxone resistance. This work highlights the role of the ponAL421P allele in shaping the current antibiotic resistance landscape and supports the need for ongoing surveillance and evolutionary studies of such mutations in the gonococcal population.

microbiology↗

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↗

bioRxiv2025

Massively parallel characterization of RNA G-quadruplex stability and molecular recognition

RNA G-quadruplexes (rG4s) have been implicated as important regulators of RNA metabolism and are promising targets for RNA-targeted therapeutics. rG4s typically require a canonical (G[≥]2N1-7)4 motif, but the sequence features that affect rG4 stability and recognition by RNA-binding proteins (RBPs) and rG4-binding ligands are not fully understood. To interrogate sequence-level drivers of rG4 folding, we applied a reverse-transcriptase stop sequencing strategy to a library of [~]3,000 synthetic rG4s with varied G-tract lengths, loop lengths, and loop compositions, permitting massively parallel quantification of rG4 stability. Our data confirm known sequence-level features and characterize novel combinatorial impacts of these features. We also assessed systematically mutagenized natural rG4s, revealing unexpected mutations that significantly affect rG4 stability, including contributions from flanking sequences outside of the rG4. We further used our strategy to assess rG4 recognition preferences of the model rG4- ligand pyridostatin, revealing a preferential stabilization of rG4s containing mixed-length G-tracts. We further demonstrated the potential for large-scale protein binding assays with our library to reveal rG4 features recognized by RBPs, specifically G3BP1 and FMRP. Our approach and data provide a generalizable framework to study sequence-level drivers of rG4 stability, binding by RBPs, and ligand interactions, defining basic principles of rG4 formation and downstream biology.

biochemistry↗

bioRxiv2025

TDP-43 directly inhibits RNA accumulation in neurites through modulation of RNA stability

The subcellular localization of hundreds of RNAs to neuronal projections allows neurons to efficiently and rapidly react to spatially restricted external cues. However, for the vast majority of these RNAs, the mechanisms that govern their localization are unknown. Here we demonstrate that the ALS-associated RNA binding protein TDP-43 primarily acts to keep RNAs out of neuronal projections. Using subcellular fractionation and single molecule RNA FISH we find that TDP-43 loss results in the increased neurite accumulation of hundreds of RNAs. These RNAs are highly enriched for known TDP-43 binding sites, suggesting that TDP-43 directly binds them to regulate their localization. We then identified precise regions within RNAs that mediate their TDP-43-dependent localization and interaction with TDP-43 using high-throughput functional assays in cells and high-throughput binding assays in vitro. We found that these regions also mediated TDP-43-dependent RNA instability, identifying the mechanism by which TDP-43 regulates RNA localization. ALS-associated mutations in TDP-43 resulted in similar RNA mislocalization phenotypes as did TDP-43 loss in human iPS-derived motor neurons. These findings establish TDP-43 as a direct negative regulator of RNA abundance in neurites and suggest that mislocalization of specific transcripts may occur in ALS patients.

molecular biology↗

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

G4mer: An RNA language model for transcriptome-wide identification of G-quadruplexes and disease variants from population-scale genetic data

RNA G-quadruplexes (rG4s) are key regulatory elements in gene expression, yet the effects of genetic variants on rG4 formation remain underexplored. Here, we introduce G4mer, an RNA language model that predicts rG4 formation and evaluates the effects of genetic variants across the transcriptome. G4mer significantly improves accuracy over existing methods, highlighting sequence length and flanking motifs as important rG4 features. Applying G4mer to 5 untranslated region (UTR) variations, we identify variants in breast cancer-associated genes that alter rG4 formation and validate their impact on structure and gene expression. These results demonstrate the potential of integrating computational models with experimental approaches to study rG4 function, especially in diseases where non-coding variants are often overlooked. To support broader applications, G4mer is available as both a web tool and a downloadable model.

genetics↗

bioRxiv2023

SERF2, an RNA G-quadruplex Binding Protein, promotes stress granule formation

Liquid-liquid phase transitions govern a wide range of protein-protein and protein-RNA interactions. Although the importance of multivalency and protein disorder in driving these transitions is clear, there is limited knowledge concerning the structural basis of phase transitions or the conformational changes that accompany this process. In this work, we found that a small human protein, SERF2, is important for the formation of stress granules. We determined the solution NMR structure ensemble of SERF2. We show that SERF2 specifically interacts with non-canonical tetrahelical RNA structures called G-quadruplexes, structures linked to stress granule formation. The biophysical amenability of both SERF2 and RNA G4 quadruplexes have allowed us to characterize the multivalent protein-RNA interactions involved in liquid-liquid phase transitions, the role that protein disorder plays in these transitions, identify the specific contacts involved, and describe how these interactions impact the structural dynamics of the components enabling a detailed understanding of the structural transitions involved in early stages of ribonucleoprotein condensate formation.

cell biology↗

bioRxiv2023

Effects of protein G-quadruplex interactions on phase transitions and protein aggregation

The SERF family of proteins were originally discovered for their ability to accelerate amyloid formation. Znf706 is an uncharacterized protein whose N-terminus is homologous to SERF proteins. We show here that human Znf706 can promote protein aggregation and amyloid formation. Unexpectedly, Znf706 specifically interacts with stable, non-canonical nucleic acid structures known as G-quadruplexes. G-quadruplexes can affect gene regulation and suppress protein aggregation; however, it is unknown if and how these two activities are linked. We find Znf706 binds preferentially to parallel G-quadruplexes with low micromolar affinity, primarily using its N-terminus, and upon interaction, its dynamics are constrained. G-quadruplex binding suppresses Znf706s ability to promote protein aggregation. Znf706 in conjunction with G-quadruplexes therefore may play a role in regulating protein folding. RNAseq analysis shows that Znf706 depletion specifically impacts the mRNA abundance of genes that are predicted to contain high G-quadruplex density. Our studies give insight into how proteins and G-quadruplexes interact, and how these interactions affect both partners and lead to the modulation of protein aggregation and cellular mRNA levels. These observations suggest that the SERF family of proteins, in conjunction with G-quadruplexes, may have a broader role in regulating protein folding and gene expression than previously appreciated.

biochemistry↗

bioRxiv2019

FMRP promotes RNA localization to neuronal projections through interactions between its RGG domain and G-quadruplex sequences

The sorting of RNA molecules to distinct subcellular locations facilitates the activity of spatially restricted processes through local protein synthesis. This process affects thousands of transcripts yet precisely how these RNAs are trafficked to their destinations remains generally unclear. Here we have analyzed subcellular transcriptomes of FMRP-null mouse neuronal cells to identify transcripts that depend on FMRP for efficient transport to neurites. We found that these FMRP RNA localization targets contain a large enrichment of G-quadruplex sequences, particularly in their 3' UTRs, suggesting that FMRP recognizes these sequences to promote the localization of transcripts that contain them. Fractionation of neurons derived from human Fragile X Syndrome patients revealed a high degree of conservation in the identity of FMRP localization targets between human and mouse as well as an enrichment of G-quadruplex sequences in human FMRP RNA localization targets. Using high-throughput RNA/protein interaction assays and single-molecule RNA FISH, we identified the RGG domain of FMRP as important for both interaction with G-quadruplex RNA sequences and the neuronal transport of G-quadruplex-containing transcripts. Finally, we used ribosome footprinting to identify translational regulatory targets of FMRP. The translational regulatory targets were not enriched for G-quadruplex sequences and were largely distinct from the RNA localization targets of FMRP, indicating that the two functions can be biochemically separated and are mediated through different target recognition mechanisms. These results establish a molecular mechanism underlying FMRP-mediated neuronal RNA localization and provide a framework for the elucidation of similar mechanisms governed by other RNA-binding proteins.

molecular biology↗