Biology subjects

Wuttke, D. S.

Publications and source records attributed to Wuttke, D. S..

10 recordsLinked to original sources

bioRxiv2026

MECP2 MBD-ID Module: A Unified DNA/RNA Binding Interface Disrupted in Rett Syndrome

Rett syndrome neurodevelopmental disorder is caused by mutations in the gene encoding the epigenetic regulator MECP2. While the MECP2 methyl-CpG binding domain (MBD) is well-characterized, the function of the adjacent intervening domain (ID) remains largely understudied. The ID has been described as a distinct RNA-binding region, yet evidence also suggests RNA competitively displaces MECP2 from DNA. Here, we address these conflicting findings by demonstrating the MBD and ID do not function in isolation but as a synergistic functional unit, establishing a new model for MECP2 function. We show the ID significantly enhances affinity of the MBD for methylated DNA by [~]35-fold. Moreover, together these two subdomains form a high-affinity, promiscuous RNA-binding module, with affinity for structured RNAs increased over 1,000-fold compared to the MBD or ID alone. We find binding to RNA precludes binding to DNA, such that the integrated MBD-ID unit explains the competition phenomenon. Analysis of Rett syndrome-associated ID mutations (R167W, K174Q, and R190H) and a therapeutic MiniGene reveals they do not disrupt methyl-DNA binding but instead selectively weaken RNA and non-methylated DNA binding, thereby disrupting the competitive balance between nucleic acid ligands. Our work establishes the MBD-ID module as MECP2s central nucleic acid interaction hub, whose disruption provides a potential molecular etiology of Rett syndrome due to mutations in the intervening domain. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/706016v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1c60d7dorg.highwire.dtl.DTLVardef@19f94b5org.highwire.dtl.DTLVardef@1dc3781org.highwire.dtl.DTLVardef@c033a1_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

A distinct mechanism of RNA recognition by the transcription factor GATA1

Several human transcription factors (TFs) have been reported to directly bind RNA through non-canonical RNA-binding domains; however, most of these TFs remain to be further validated as bona fide RNA-binding proteins (RBPs). Our systematic analysis of RBP discovery datasets reveals a varied set of candidate TF-RBPs that encompass most TF families. These candidate RBPs include members of the GATA family, which are essential factors in embryonic development. Investigation of the RNA-binding features of GATA1, a major hematopoietic TF, reveals robust non-sequence specific binding to RNAs in vitro. Moreover, RNA binding by GATA1 is competitive with DNA binding, which occurs through a shared binding surface spanning the DNA-binding domain and arginine-rich motif (ARM) like domain. We show that the ARM-like domain contributes both substantially to high-affinity DNA binding and electrostatically to plastic RNA recognition, suggesting that the separable RBD assigned to the ARM-domain in GATA1 is an oversimplification of a more complex recognition network. These biochemical data demonstrate a unified integration of DNA- and RNA-binding surfaces within GATA1, whereby the ARM-like domain provides an electrostatic surface for RNA binding but does not fully dominate GATA1-RNA interactions, which may also apply to other TF-RBPs. This competitive DNA/RNA binding activity using overlapping nucleic acid binding regions points to the possibility of RNA-mediated regulation of GATA1 function during hematopoiesis. Our study highlights the multifunctionality of DNA-binding domains in RNA recognition and supports the need for robust characterization of predicted non-canonical RNA-binding domains such as ARM-like domains.

biochemistry↗

bioRxiv2024

The RNA-binding selectivity of the RGG/RG motifs of hnRNP U is abolished by elements within the intrinsically disordered region

The abundant nuclear protein hnRNP U interacts with a broad array of RNAs along with DNA and protein to regulate nuclear chromatin architecture. The RNA-binding activity is achieved via a disordered [~]100 residue C-terminal RNA-binding domain (RBD) containing two distinct RGG/RG motifs. Although the RNA-binding capabilities of RGG/RG motifs have been widely reported, less is known about hnRNP Us RNA-binding selectivity. Furthermore, while it is well established that hnRNP U binds numerous nuclear RNAs, it remains unknown whether it selectively recognizes sequence or structural motifs in target RNAs. To address this question, we performed equilibrium binding assays using fluorescence anisotropy (FA) and electrophoretic mobility shift assays (EMSAs) to quantitatively assess the ability of human hnRNP U RBD to interact with segments of cellular RNAs identified from eCLIP data. These RNAs often, but not exclusively, contain poly-uridine or 5-AGGGAG sequence motifs. Detailed binding analysis of several target RNAs reveal that the hnRNP U RBD binds RNA in a promiscuous manner with high affinity for a broad range of structured RNAs, but with little preference for any distinct sequence motif. In contrast, the isolated RGG/RG of hnRNP U motif exhibits a strong preference for G-quadruplexes, similar to that observed for other RGG motif bearing peptides. These data reveal that the hnRNP U RBD attenuates the RNA binding selectivity of its core RGG motifs to achieve an extensive RNA interactome. We propose that a critical role of RGG/RG motifs in RNA biology is to alter binding affinity or selectivity of adjacent RNA-binding domains.

biochemistry↗

bioRxiv2024

Transcription factors ERα and Sox2 have differing multiphasic DNA and RNA binding mechanisms

Many transcription factors (TFs) have been shown to bind RNA, leading to open questions regarding the mechanism(s) of this RNA binding and its role in regulating TF activities. Here we use biophysical assays to interrogate the kon, koff, and Kd for DNA and RNA binding of two model human transcription factors, ER and Sox2. Unexpectedly, we found that both proteins exhibited multiphasic nucleic acid binding kinetics. We propose that Sox2 RNA and DNA multiphasic binding kinetics could be explained by a conventional model for sequential Sox2 monomer association and dissociation. In contrast, ER nucleic acid binding exhibited biphasic dissociation paired with novel triphasic association behavior, where two apparent binding transitions are separated by a 10-20 min "lag" phase depending on protein concentration. We considered several conventional models for the observed kinetic behavior, none of which adequately explained all the ER nucleic acid binding data. Instead, simulations with a model incorporating sequential ER monomer association, ER nucleic acid complex isomerization, and product "feedback" on isomerization rate recapitulated the general kinetic trends for both ER DNA and RNA binding. Collectively, our findings reveal that Sox2 and ER bind RNA and DNA with previously unappreciated multiphasic binding kinetics, and that their reaction mechanisms differ with ER binding nucleic acids via a novel reaction mechanism.

biophysics↗

bioRxiv2023

The RNA-binding domain of hnRNP U extends beyond the RGG/RG motifs

Heterogeneous nuclear ribonucleoprotein U (hnRNP U) is a ubiquitously expressed protein that regulates chromatin architecture through its interactions with numerous DNA, protein, and RNA partners. The RNA-binding domain (RBD) of hnRNP U was previously mapped to an RGG/RG element within its disordered C-terminal region, but little is understood about its binding mode and potential for selective RNA recognition. Analysis of publicly available hnRNP U enhanced UV crosslinking and immunoprecipitation (eCLIP) data identified high-confidence binding sites within human RNAs. We synthesized a set of diverse RNAs encompassing eleven of these identified crosslink sites for biochemical characterization using a combination of fluorescence anisotropy and electrophoretic mobility shift assays. These in vitro binding experiments with a rationally designed set of RNAs and hnRNP U domains revealed that the RGG/RG element is a small part of a more expansive RBD that encompasses most of the disordered C-terminal region. This RBD contains a second, previously experimentally uncharacterized RGG/RG element with RNA-binding properties comparable to the canonical RGG/RG element. These RGG/RG elements serve redundant functions, with neither serving as the primary RBD. While in isolation each RGG/RG element has modest affinity for RNA, together they significantly enhance the association of hnRNP U with RNA, enabling binding of most of the designed RNA set with low to mid-nanomolar binding affinities. Identification and characterization of the complete hnRNP U RBD highlights the perils of a reductionist approach to defining biochemical activities in this system and paves the way for a detailed investigation of its RNA-binding specificity.

biochemistry↗

bioRxiv2021

CST does not evict elongating telomerase but prevents initiation by ssDNA binding

The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol -primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol -primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

biochemistry↗

bioRxiv2020

Cyp33 Binds AU-Rich RNA Motifs via an Extended Interface that Competitively Disrupts the Gene Repressive Cyp33-MLL1 Interaction in vitro

Cyp33 is an essential human cyclophilin prolyl isomerase that plays myriad roles in splicing and chromatin remodeling. In addition to a canonical cyclophilin (Cyp) domain, Cyp33 contains an RNA-recognition motif (RRM) domain, and RNA-binding triggers proline isomerase activity. One prominent role for Cyp33 is through a direct interaction with the mixed lineage leukemia protein 1 (MLL1, also known as KMT2A) complex, which is a histone methyltransferase that serves as a global regulator of human transcription. MLL activity is regulated by Cyp33, which isomerizes a key proline in the linker between the PHD3 and Bromo domains of MLL1, acting as a switch between gene activation and repression. The direct interaction between MLL1 and Cyp33 is critical, as deletion of the MLL1-PHD3 domain responsible for this interaction results in oncogenesis. The Cyp33 RRM is central to these activities, as it binds both the PHD3 domain and RNA. To better understand how RNA binding drives the action of Cyp33, we performed RNA-SELEX against full-length Cyp33 accompanied by deep sequencing. We have identified an enriched Cyp33 binding motif (AAUAAUAA) broadly represented in the cellular RNA pool as well as tightly binding RNA aptamers with affinities comparable and competitive with the Cyp33 MLL1-PHD3 interaction. RNA binding extends beyond the canonical RRM domain, but not to the Cyp domain, suggesting an indirect mechanism of interaction. NMR chemical shift mapping confirms an overlapping, but not identical, interface on Cyp33 for RNA and PHD3 binding. This finding suggests RNA can disrupt the gene repressive Cyp33-MLL1 complex providing another layer of regulation for chromatin remodeling by MLL1.

biochemistry↗

bioRxiv2020

Spontaneous replication fork collapse regulates telomere length homeostasis in wild type cells

In most eukaryotes, the enzyme telomerase maintains the termini of linear chromosomes through the addition of repetitive telomeric sequences. It is widely assumed that the primary site of action for telomerase is the single-stranded G-rich overhang at the ends of linear chromosomes. We show here that a second substrate, created by spontaneous replication fork collapse during duplex telomeric DNA replication in wild type budding yeast, is elongated by telomerase at a much higher frequency ([~]50%) than fully replicated chromosome termini. Furthermore, as much as [~]200 nucleotides can be added in a single cell division to these newly collapsed forks, indicating that spontaneous replication fork collapse and the subsequent response by telomerase is a major determinant of telomere length homeostasis. This challenges a long-standing model for telomere length regulation which posits a length-sensing mechanism that assesses individual telomeres to determine whether chromosome ends are in "telomerase-extendible" or "telomerase-non-extendible" states. We propose that these two states are instead structurally and temporally distinct substrates for telomerase, generated by two different processes (fork collapse vs. completion of DNA replication). We also show that replication fork collapse at telomeres is kept in check by a telomere-dedicated Cdc13/Stn1/Ten1 complex in collaboration with the canonical RPA complex, indicating that these two complexes bind single-stranded DNA exposed at the replication fork to facilitate replisome progression through duplex telomeric DNA. Although failures during DNA replication are often genotoxic events, this represents an opposing example in which fork collapse has been co-opted to promote genome stability. Significance StatementIn most eukaryotes, the termini of linear chromosomes are composed of arrays of short repeats that are continually replenished by the enzyme telomerase. If telomerase is unable to act, gradual loss of these terminal repeats results in an eventual block to cell division. Therefore, in cells that depend on continuous cell division, the mechanism(s) by which telomerase is directed to chromosome ends is tightly regulated. This study shows that in addition to the ends of fully replicated chromosomes, a second site of action for telomerase is generated when replication through duplex telomeric DNA is disrupted. These results suggest that the disparate response of telomerase to two temporally and structurally distinct substrates is a major determinant of telomere length homeostasis.

genetics↗

bioRxiv2019

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation

Dosage compensation between the sexes has emerged independently multiple times during evolution, often harnessing long noncoding RNAs (lncRNAs) to alter gene expression on the sex chromosomes. In eutherian mammals, X chromosome inactivation (XCI) in females proceeds via the lncRNA Xist, which coats one of the two X chromosomes and recruits repressive proteins to epigenetically silence gene expression in cis1,2. How Xist evolved new functional RNA domains to recruit ancient, pleiotropic protein partners is of great interest. Here we show that Spen, an Xist-binding repressor protein essential for XCI3-7, binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen inactivation leads to de-repression of a subset of endogenous retroviral (ERV) elements in embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing8-9. ERV RNA and Xist A-repeat bind the RRM3 domain of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in local gene silencing in cis. These results suggest that insertion of an ERV element into proto-Xist may have been a critical evolutionary event, which allowed Xist to coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.

genomics↗

bioRxiv2019

The cryo-EM structure of human CST reveals a two-megadalton decameric assembly bound to telomeric DNA

The single-stranded DNA-binding CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and genome-wide replication recovery, processes that are critical for genome stability. Here, we report the 2.95 [A] cryo-EM structure of human CST bound to telomeric single-stranded DNA, which unexpectedly assembles as a decameric supercomplex. The atomic model of the 134 kDa CTC1, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. In situ arrangements of STN1 and TEN1 are revealed, with STN1 interacting with CTC1 at two separated sites, allowing allosteric mediation of CST decameric assembly. Surprisingly, CTC1 lacks the anticipated structural homology to yeast Cdc13 but instead shares similarity with a form of Replication Protein A. The atomic-resolution model of human CST provides crucial mechanistic understanding of CST mutations associated with human diseases. Moreover, the decameric form of CST suggests the intriguing possibility of ssDNA architectural organization similar to what the nucleosome provides for dsDNA.\n\nOne Sentence SummaryHuman telomeric single-stranded DNA triggers the assembly of a decameric protein supercomplex solved by cryo-EM.

biophysics↗