Biology subjects

Eichhorn, C. D.

Publications and source records attributed to Eichhorn, C. D..

6 recordsLinked to original sources

bioRxiv2026

Quantifying Mg2+ dependence on conformational equilibrium in the two-state 7SK RNA stem-loop 3

RNA structural heterogeneity is increasingly recognized as essential for RNA function, yet quantitative understanding of intrinsic RNA structural dynamics and how cellular conditions modulate these dynamics remains limited. 7SK RNA is an abundant eukaryotic noncoding RNA that assembles with protein cofactors to form the 7SK ribonucleoprotein (RNP), a dynamic complex that regulates transcription elongation. In particular, the 7SK RNA stem-loop 3 (SL3) domain is a critical hub for protein recruitment that is required for 7SK RNP function. We recently discovered that SL3 undergoes exchange between two equally populated yet structurally distinct conformers, named SL3e and SL3a, providing a model system to understand RNA conformational equilibria. Here, we combined quantitative dimethyl sulfate mutational profiling with sequencing (qDMS-MaPseq), solution NMR spectroscopy, and isothermal titration calorimetry (ITC) experiments to gain quantitative insights into SL3 equilibria under varying ionic conditions. We find that Mg2+ shifts the SL3 conformational equilibrium from 52%:48% to 67%:33% populations, with ITC experiments showing [~]2-fold higher affinity for the SL3e conformer, providing the thermodynamic basis for this population shift. NMR 1H-1H NOESY experiments show that Mg2+ stabilizes an A-form helical geometry in the distal end of the SL3e conformer that contains multiple noncanonical base pairs. Validating our combined approach, NMR and DMS-MaPseq Mg2+-induced perturbation data are highly correlated for nucleobase moieties at the Watson-Crick face (R2 = 0.87). From this work, we identify a characteristic DMS reactivity signature for A*C wobble base pairs from DMS-MaPseq MgCl2 titration experiments, expanding the utility of chemical probing for detecting noncanonical base pairs. Our findings demonstrate how modest differences in cation affinity between conformational states can modulate RNA population ensembles. More broadly, this work establishes a generalizable thermodynamic framework to quantitatively dissect ion-dependent conformational equilibria in structurally heterogeneous RNAs and advances structural understanding of the essential SL3 regulatory element in the 7SK RNP. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/717930v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@71bf36org.highwire.dtl.DTLVardef@1c0a7ccorg.highwire.dtl.DTLVardef@1484400org.highwire.dtl.DTLVardef@1688bd8_HPS_FORMAT_FIGEXP M_FIG C_FIG KEY POINTSO_LIAddition of Mg2+ shifts the SL3 two-state conformational equilibrium through preferential association with the SL3e state C_LIO_LIMg2+ association stabilizes an A-form-like geometry in the SL3e upper stem C_LIO_LIDMS reactivity patterns in MgCl2 titration experiments provide a signature for identifying A*C wobble base pairs C_LIO_LIDMS-MaPseq mutation fraction perturbation data are correlated with NMR chemical shift perturbation data, validating chemical probing as a structural reporter C_LI

biophysics↗

bioRxiv2025

Pepper RNA variants reveal decoupling of HBC530 binding thermodynamics and fluorescence activation

Fluorogenic RNA aptamers have emerged as powerful tools for live-cell imaging and synthetic biology applications due to their ability to activate fluorescence upon ligand binding. However, the sequence-structure-function relationships governing ligand recognition and fluorescence activation remain poorly understood, limiting rational aptamer design. The Pepper aptamer binds HBC530 with nanomolar affinity in a magnesium-dependent manner, producing bright fluorescence suitable for cellular applications. Here, we generated a library of 53 Pepper variants containing substitutions, insertions, and/or deletions to quantitatively evaluate the contributions of individual nucleotides to HBC530 binding affinity, magnesium binding affinity, and fluorescence intensity. Our results reveal that the correlation between HBC530 binding affinity and fluorescence intensity is only modest. We identify several variants with binding affinities similar to wild-type Pepper but dramatically reduced fluorescence, indicating that ligand recognition and fluorescence intensity are decoupled. Further, we find that distal structural elements significantly influence both binding thermodynamics and fluorescence intensity. Confirming the critical role of divalent cations in Pepper-HBC530 recognition, we observe a strong correlation between HBC530 and magnesium binding thermodynamics. These findings provide quantitative insights into the molecular mechanisms underlying fluorescence activation, toward developing a framework for the rational design of the next generation of fluorogenic RNA aptamers with enhanced performance. KEY POINTSO_LIThe HBC530 binding affinities of 53 Pepper RNA variants are only moderately correlated with fluorescence intensity C_LIO_LIStructural elements distal to the HBC530 binding pocket influence both binding affinity and fluorescence C_LIO_LIThe binding affinities of Pepper RNA to HBC530 and Mg2+ are highly correlated, and Mg2+ stoichiometry is correlated with binding thermodynamics and fluorescence intensity C_LI

biophysics↗

bioRxiv2024

An evolutionarily conserved tryptophan cage promotes folding of the extended RNA recognition motif in the hnRNPR-like protein family

The heterogeneous nuclear ribonucleoprotein (hnRNP) R-like family is a class of RNA binding proteins in the hnRNP superfamily that has diverse functions in RNA processing. Here, we present the 1.90 [A] X-ray crystal structure and solution NMR studies of the first RNA recognition motif (RRM) of human hnRNPR. We find that this domain adopts an extended RRM (eRRM1) featuring a canonical RRM with a structured N-terminal extension (Next) motif that docks against the RRM and extends the {beta}-sheet surface. The adjoining loop positions the Next for docking to the RRM and forms a tryptophan cage motif, which has only been reported previously in a synthetic peptide. Using a combination of mutagenesis, solution NMR spectroscopy, and thermal denaturation studies we evaluate the importance of residues in the Next-RRM interface and adjoining loop on eRRM folding and conformational dynamics. We find that these sites are essential for protein solubility, thermal stability, and conformational ordering. Consistent with their importance, mutations in the Next-RRM interface and loop are associated with several cancers in a survey of somatic mutations in cancer studies. Sequence and structure comparison of the human hnRNPR eRRM1 to experimentally verified and predicted hnRNPR-like proteins reveal conserved features in the eRRM. Significance statementThis study provides the first structural and thermodynamic analysis of the hnRNPR extended RNA recognition motif (eRRM1). Here an N-terminal extension docks alongside a canonical RRM, stabilized by an intervening loop containing a tryptophan cage motif. Highly conserved among hnRNPR-like proteins, this motif has only been previously observed in a synthetic peptide. Substitutions that disrupt observed interactions reduce thermal stability and are associated with cancers. These findings significantly advance our understanding of atypical RRM folding.

biophysics↗

bioRxiv2023

Visualizing a two-state conformational ensemble in stem-loop 3 of the transcriptional regulator 7SK RNA

Structural plasticity is integral to RNA function; however, there are currently few methods to quantitatively resolve RNAs that have multiple structural states. NMR spectroscopy is a powerful approach for resolving conformational ensembles but is size-limited. Chemical probing is well-suited for large RNAs but provides limited structural and no kinetics information. Here, we integrate the two approaches to visualize a two-state conformational ensemble for the central stem-loop 3 (SL3) of 7SK RNA, a critical element for 7SK RNA function in transcription regulation. We find that the SL3 distal end exchanges between two equally populated yet structurally distinct states in both isolated SL3 constructs and full-length 7SK RNA. We rationally designed constructs that lock SL3 into a single state and demonstrate that both chemical probing and NMR data fit to a linear combination of the two states. Comparison of vertebrate 7SK RNA sequences shows conservation of both states, suggesting functional importance. These results provide new insights into 7SK RNA structural dynamics and demonstrate the utility of integrating chemical probing with NMR spectroscopy to gain quantitative insights into RNA conformational ensembles.

biophysics↗

bioRxiv2022

C-terminal determinants for RNA binding motif 7 protein stability and RNA recognition

The 7SK ribonucleoprotein (RNP) is a critical regulator of eukaryotic transcription. Recently, RNA binding motif 7 (RBM7), which contains an RNA recognition motif (RRM), was reported to associate with 7SK RNA and core 7SK RNP protein components in response to DNA damage. However, little is known about the mode of RBM7-7SK RNA recognition. Here, we recombinantly expressed and purified RBM7 RRM constructs and found that constructs containing extended C-termini have increased solubility and stability compared to shorter constructs. To identify potential RBM7-7SK RNA binding sites, we analyzed deposited data from in cellulo crosslinking experiments and found that RBM7 crosslinks specifically to the distal region of 7SK stem-loop 3 (SL3). Electrophoretic mobility shift assays and NMR chemical shift perturbation experiments showed weak binding to 7SK SL3 constructs in vitro. Together, these results provide new insights into RBM7 RRM folding and recognition of 7SK RNA. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/491737v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@10ac49org.highwire.dtl.DTLVardef@9f1de4org.highwire.dtl.DTLVardef@63985eorg.highwire.dtl.DTLVardef@a870a7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIExtending the RBM7 RRM C-terminus improves protein expression and solubility C_LIO_LIThe RBM7 RRM interacts weakly with 7SK stem-loop 3 single-stranded RNA C_LIO_LISolution state NMR dynamics measurements support presence of additional {beta}4 strand C_LI

biophysics↗

bioRxiv2019

Structure of S. pombe telomerase protein Pof8 C-terminal domain is an xRRM conserved among LARP7 proteins

La related proteins group 7 (LARP7) are a class of RNA chaperones that bind the 3ends of RNA and are constitutively associated with their specific target RNAs. In metazoa, Larp7 binds to the long non-coding 7SK RNA as a core component of the 7SK RNP, a major regulator of eukaryotic transcription. In ciliates, a LARP7 protein (p65 in Tetrahymena) is a core component of telomerase, an essential ribonucleoprotein complex that maintains the DNA length at eukaryotic chromosome ends. p65 is important for the ordered assembly of telomerase RNA (TER) with telomerase reverse transcriptase (TERT). Although a LARP7 as a telomerase holoenzyme component was initially thought to be specific to ciliate telomerases, Schizosaccharomyces pombe Pof8 was recently identified as a LARP7 protein and a core component of fission yeast telomerase essential for biogenesis. There is also evidence that human Larp7 associates with telomerase. LARP7 proteins have conserved N-terminal La motif and RRM1 (La module) and C-terminal RRM2 with specific RNA substrate recognition attributed to RRM2, first structurally characterized in p65 as an atypical RRM named xRRM. Here we present the X-ray crystal structure and NMR studies of S. pombe Pof8 RRM2. Sequence and structure comparison of Pof8 RRM2 to p65 and hLarp7 xRRMs reveals conserved features for RNA binding with the main variability in the length of the non-canonical helix 3. This study shows that Pof8 has conserved xRRM features, providing insight into TER recognition and the defining characteristics of the xRRM.\n\nHighlightsO_LIThe structure of the S. pombe LARP7 Pof8 C-terminal domain is an xRRM.\nC_LIO_LICiliates, human, and fission yeast contain LARP7 proteins with xRRMs involved in telomerase biogenesis.\nC_LIO_LIWith three examples of xRRM structures, we refine the definition of xRRM.\nC_LI

biophysics↗