Biology subjects

Aleman, M. M.

Publications and source records attributed to Aleman, M. M..

7 recordsLinked to original sources

bioRxiv2025

Charting the Multi-level Molecular Response to Palbociclib in ER-Positive Breast Cancer

The addition of CDK4/6 inhibitors to endocrine therapy has significantly improved outcomes in HR+/HER2-breast cancer. However, variable patient responses and acquired resistance remain a clinical challenge. We therefore defined the comprehensive molecular response to palbociclib, the most clinically used CDK4/6 inhibitor. Global analyses of gene expression, protein abundance, splicing, and chromatin accessibility revealed broad patterns and specific changes that result from CDK4/6-inhibition in breast cancer cells. We uncovered unexpected feedback between CDK4/6 and estrogen-response signaling, which has clear clinical implications. We also revealed a widespread alternative splicing program that partially overlapped with genes whose expression is regulated, and which is expected to impact protein function. These molecular changes nominated combination therapies that interfere with the activation of CDKs or ER. Accordingly, co-targeting CDK7, which regulates CDK2, CDK4/6 and ER, additively impacted cell fitness. Collectively, these data reveal a complex, multi-tiered response to CDK4/6 inhibition, with implications for therapeutic efficacy.

molecular biology↗

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↗

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

Iron-sensitive RNA regulation by poly C binding proteins

Iron is essential for normal cellular function. Homeostatic responses to low iron availability have long been known to rely on posttranscriptional mechanisms. Poly C binding proteins (PCBPs) are essential RNA binding proteins that regulate alternative splicing, translation and RNA stability. They also serve as critical iron chaperones that manage intracellular iron flux. However, the impact of cellular iron levels on the PCBP-directed transcriptome has not been globally evaluated. We have found broad transcriptome changes in response to low iron availability consistent with numerous operant posttranscriptional mechanisms that sense iron. By comparing gene expression and alternative splicing directed by PCBPs to the iron-sensitive transcriptome, we found genes with iron-sensitive PCBP RNA regulation. Further, we demonstrate that cellular iron levels impact PCBP1 RNA binding. Our data show a large-scale RNA regulatory response to cellular iron levels and open the possibility for other iron-sensitive RNA binding proteins.

molecular biology↗

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↗

bioRxiv2021

Quantitative Profiling of Adaptation to Cyclin E Overproduction

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Cyclin E overproduction is toxic to mammalian cells, although the gene encoding cyclin E (CCNE1) is overexpressed in some cancers. To gain insight into how cancer cells tolerate high cyclin E, we extensively characterized non-transformed epithelial cells throughout a time course of chronic cyclin E overproduction. Cells overproducing human cyclin E, but not cyclin D or cyclin A, briefly experienced truncated G1 phases, then endured a transient period of DNA replication origin underlicensing, replication stress, and severely impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite maintaining high cyclin E-associated activity. Transcriptome analysis revealed that adapted cells downregulated a cohort of G1-regulated genes. Withdrawing cyclin E induction only partially reversed the intermediate licensing phenotype of adapted cells indicating that adaptation is at least partly independent of mutations. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing by an indirect mechanism through premature S phase onset and provides further insight into the relationship between CDK activity and licensing in mammals. It serves as an example of specific oncogene adaptation that may recapitulate molecular changes during tumorigenesis.

cell biology↗