The RNA-binding Selectivity of the RGG/RG Motifs of hnRNP U is Abolished by Elements Within the C-terminal 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 U's 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.

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 U's 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.

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 k on, k off, and K d for DNA and RNA binding of two model human TFs, ERα and Sox2. Unexpectedly, we found that both proteins exhibit multiphasic nucleic acid-binding kinetics. We propose that Sox2 RNA and DNA multiphasic binding kinetics can be explained by a conventional model for sequential Sox2 monomer association and dissociation. In contrast, ERα nucleic acid binding exhibited biphasic dissociation paired with novel triphasic association behavior, in which 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 ERα nucleic acid-binding data. Instead, simulations with a model incorporating sequential ERα monomer association, ERα nucleic acid complex isomerization, and product "feedback" on isomerization rate recapitulated the general kinetic trends for both ERα DNA and RNA binding. Collectively, our findings reveal that Sox2 and ERα bind RNA and DNA with previously unappreciated multiphasic binding kinetics, and that their reaction mechanisms differ with ERα binding nucleic acids via a novel reaction mechanism.

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, ERα 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, ERα 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 ERα nucleic acid binding data. Instead, simulations with a model incorporating sequential ERα monomer association, ERα nucleic acid complex isomerization, and product "feedback" on isomerization rate recapitulated the general kinetic trends for both ERα DNA and RNA binding. Collectively, our findings reveal that Sox2 and ERα bind RNA and DNA with previously unappreciated multiphasic binding kinetics, and that their reaction mechanisms differ with ERα binding nucleic acids via a novel reaction mechanism.

Guardians of the Genome: How the Single-Stranded DNA-Binding Proteins RPA and CST Facilitate Telomere Replication.

Telomeres act as the protective caps of eukaryotic linear chromosomes; thus, proper telomere maintenance is crucial for genome stability. Successful telomere replication is a cornerstone of telomere length regulation, but this process can be fraught due to the many intrinsic challenges telomeres pose to the replication machinery. In addition to the famous "end replication" problem due to the discontinuous nature of lagging strand synthesis, telomeres require various telomere-specific steps for maintaining the proper 3' overhang length. Bulk telomere replication also encounters its own difficulties as telomeres are prone to various forms of replication roadblocks. These roadblocks can result in an increase in replication stress that can cause replication forks to slow, stall, or become reversed. Ultimately, this leads to excess single-stranded DNA (ssDNA) that needs to be managed and protected for replication to continue and to prevent DNA damage and genome instability. RPA and CST are single-stranded DNA-binding protein complexes that play key roles in performing this task and help stabilize stalled forks for continued replication. The interplay between RPA and CST, their functions at telomeres during replication, and their specialized features for helping overcome replication stress at telomeres are the focus of this review.

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 motif 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 cross-linking and immunoprecipitation (eCLIP) data identified high-confidence binding sites within human RNAs. We synthesized a set of diverse RNAs encompassing 11 of these identified cross-link 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 motif 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 motif with RNA-binding properties comparable to those of the canonical RGG/RG motif. These RGG/RG motifs serve redundant functions, with neither serving as the primary RBD. While in isolation, each RGG/RG motif has modest affinity for RNA, together they significantly enhance the association of hnRNP U with RNA, enabling the binding of most of the designed RNA set with low to midnanomolar binding affinities. Identification and characterization of the complete hnRNP U RBD highlight the perils of a reductionist approach to defining biochemical activities in this system and pave the way for a detailed investigation of its RNA-binding specificity.

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.

Quantification of transcriptome changes to investigate the role of glucocorticoid receptor-RNA binding during dexamethasone treatment.

OBJECTIVES: The glucocorticoid receptor (GR) is a well-studied, ligand-activated transcription factor and a common target of anti-inflammatory treatments. Recently, several studies have drawn attention the effects of binding of GR to RNA rather than DNA and the potential implications of this activity for GR function. The objective of our study was to further characterize the relationship between GR function and RNA binding by measuring changes in the glucocorticoid-driven transcriptome in the presence of a GR mutant that exhibited reduced RNA affinity. DATA DESCRIPTION: GR was activated in three cell lines containing GR constructs (GR-HaloTag). One of the cell lines contained a wild-type GR-HaloTag. Another contained GR-HaloTag with a mutation that reduced RNA affinity and slightly reduced DNA affinity. The third cell line contained GR-HaloTag with a mutation that only slightly reduced DNA affinity. All three cell lines were treated with dexamethasone, a GR agonist. RNA-seq samples were collected every hour for 3 h. Moreover, transcriptome quantification was accomplished via labeling of RNAs transcribed in the final hour of dexamethasone treatment using 4-thiouridine. These labeled RNAs were then purified and sequenced. This data set is the first of its kind for GR and contains valuable insights into the function of RNA binding by GR.

RNA binding by the glucocorticoid receptor attenuates dexamethasone-induced gene activation.

The glucocorticoid receptor (GR) is a ligand-activated transcription factor that regulates a suite of genes through direct binding of GR to specific DNA promoter elements. GR also interacts with RNA, but the function of this RNA-binding activity remains elusive. Current models speculate that RNA could repress the transcriptional activity of GR. To investigate the function of the GR-RNA interaction on GR's transcriptional activity, we generated cells that stably express a mutant of GR with reduced RNA binding affinity and treated the cells with the GR agonist dexamethasone. Changes in the dexamethasone-driven transcriptome were quantified using 4-thiouridine labeling of RNAs followed by high-throughput sequencing. We find that while many genes are unaffected, GR-RNA binding is repressive for specific subsets of genes in both dexamethasone-dependent and independent contexts. Genes that are dexamethasone-dependent are activated directly by chromatin-bound GR, suggesting a competition-based repression mechanism in which increasing local concentrations of RNA may compete with DNA for binding to GR at sites of transcription. Unexpectedly, genes that are dexamethasone-independent instead display a localization to specific chromosomal regions, which points to changes in chromatin accessibility or architecture. These results show that RNA binding plays a fundamental role in regulating GR function and highlights potential functions for transcription factor-RNA interactions.

RPA-like single-stranded DNA-binding protein complexes including CST serve as specialized processivity factors for polymerases.

Telomeres and other single-stranded regions of the genome require specialized management to maintain stability and for proper progression of DNA metabolism pathways. Human Replication Protein A and CTC1-STN1-TEN1 are structurally similar heterotrimeric protein complexes that have essential ssDNA-binding roles in DNA replication, repair, and telomeres. Yeast and ciliates have related ssDNA-binding proteins with strikingly conserved structural features to these human heterotrimeric protein complexes. Recent breakthrough structures have extended our understanding of these commonalities by illuminating a common mechanism used by these proteins to act as processivity factors for their partner polymerases through their ability to manage ssDNA.

RPA engages telomeric G-quadruplexes more effectively than CST.

G-quadruplexes (G4s) are a set of stable secondary structures that form within guanine-rich regions of single-stranded nucleic acids that pose challenges for DNA maintenance. The G-rich DNA sequence at telomeres has a propensity to form G4s of various topologies. The human protein complexes Replication Protein A (RPA) and CTC1-STN1-TEN1 (CST) are implicated in managing G4s at telomeres, leading to DNA unfolding and allowing telomere replication to proceed. Here, we use fluorescence anisotropy equilibrium binding measurements to determine the ability of these proteins to bind various telomeric G4s. We find that the ability of CST to specifically bind G-rich ssDNA is substantially inhibited by the presence of G4s. In contrast, RPA tightly binds telomeric G4s, showing negligible changes in affinity for G4 structure compared to linear ssDNAs. Using a mutagenesis strategy, we found that RPA DNA-binding domains work together for G4 binding, and simultaneous disruption of these domains reduces the affinity of RPA for G4 ssDNA. The relative inability of CST to disrupt G4s, combined with the greater cellular abundance of RPA, suggests that RPA could act as a primary protein complex responsible for resolving G4s at telomeres.

The DNA binding high mobility group box protein family functionally binds RNA.

Nucleic acid binding proteins regulate transcription, splicing, RNA stability, RNA localization, and translation, together tailoring gene expression in response to stimuli. Upon discovery, these proteins are typically classified as either DNA or RNA binding as defined by their in vivo functions; however, recent evidence suggests dual DNA and RNA binding by many of these proteins. High mobility group box (HMGB) proteins have a DNA binding HMGB domain, act as transcription factors and chromatin remodeling proteins, and are increasingly understood to interact with RNA as means to regulate gene expression. Herein, multiple layers of evidence that the HMGB family are dual DNA and RNA binding proteins is comprehensively reviewed. For example, HMGB proteins directly interact with RNA in vitro and in vivo, are localized to RNP granules involved in RNA processing, and their protein interactors are enriched in RNA binding proteins involved in RNA metabolism. Importantly, in cell-based systems, HMGB-RNA interactions facilitate protein-protein interactions, impact splicing outcomes, and modify HMGB protein genomic or cellular localization. Misregulation of these HMGB-RNA interactions are also likely involved in human disease. This review brings to light that as a family, HMGB proteins are likely to bind RNA which is essential to HMGB protein biology. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Replication Protein A Utilizes Differential Engagement of Its DNA-Binding Domains to Bind Biologically Relevant ssDNAs in Diverse Binding Modes.

Replication protein A (RPA) is a ubiquitous ssDNA-binding protein that functions in many DNA processing pathways to maintain genome integrity. Recent studies suggest that RPA forms a highly dynamic complex with ssDNA that can engage with DNA in many modes that are orchestrated by the differential engagement of the four DNA-binding domains (DBDs) in RPA. To understand how these modes influence RPA interaction with biologically relevant ligands, we performed a comprehensive and systematic evaluation of RPA's binding to a diverse set of ssDNA ligands that varied in sequence, length, and structure. These equilibrium binding data show that WT RPA binds structured ssDNA ligands differently from its engagement with minimal ssDNAs. Next, we investigated each DBD's contributions to RPA's binding modes through mutation of conserved, functionally important aromatic residues. Mutations in DBD-A and -B have a much larger effect on binding when ssDNA is embedded into DNA secondary structures compared to their association with unstructured minimal ssDNA. As our data support a complex interplay of binding modes, it is critical to define the trimer core DBDs' role in binding these biologically relevant ligands. We found that DBD-C is important for engaging DNA with diverse binding modes, including, unexpectedly, at short ssDNAs. Thus, RPA uses its constituent DBDs to bind biologically diverse ligands in unanticipated ways. These findings lead to a better understanding of how RPA carries out its functions at diverse locations of the genome and suggest a mechanism through which dynamic recognition can impact differential downstream outcomes.

An Extended DNA Binding Domain of the Estrogen Receptor Alpha Directly Interacts with RNAs in Vitro.

Estrogen receptor alpha (ERα) is a ligand-responsive transcription factor critical for sex determination and development. Recent reports challenge the canonical view of ERα function by suggesting an activity beyond binding dsDNA at estrogen-responsive promotor elements: association with RNAs in vivo. Whether these interactions are direct or indirect remains unknown, which limits the ability to understand the extent, specificity, and biological role of ERα-RNA binding. Here we demonstrate that an extended DNA-binding domain of ERα directly binds a wide range of RNAs in vitro with structural specificity. ERα binds RNAs that adopt a range of hairpin-derived structures independent of sequence, while interacting poorly with single- and double-stranded RNA. RNA affinities are only 4-fold weaker than consensus dsDNA and significantly tighter than nonconsensus dsDNA sequences. Moreover, RNA binding is competitive with DNA binding. Together, these data show that ERα utilizes an extended DNA-binding domain to achieve a high-affinity/low-specificity mode for interacting with RNA.

The DNA-Binding High-Mobility Group Box Domain of Sox Family Proteins Directly Interacts with RNA In Vitro.

There is a growing body of evidence that a substantial number of protein domains identified as DNA-binding also interact with RNA to regulate biological processes. Several recent studies have revealed that the Sox2 transcription factor binds RNA through its high-mobility group box (HMGB) domain in vitro and in vivo. A high degree of conservation of this domain among members of the Sox family of transcription factors suggests that RNA-binding activity may be a general feature of these proteins. To address this hypothesis, we examined a subset of HMGB domains from human Sox family of proteins for their ability to bind both DNA and RNA in vitro. We observed selective, high-affinity interactions between Sox family HMGB domains and various model RNA elements, including a four-way junction RNA, a hairpin RNA with an internal bulge, G-quadruplex RNA, and a fragment of long noncoding RNA ES2, which is known to directly interact with Sox2. Importantly, the HMGB domains bind these RNA ligands significantly tighter than nonconsensus dsDNA and in some cases with affinities rivaling those of their consensus dsDNA sequences. These data suggest that RNA binding is a conserved feature of the Sox family of transcription factors with the potential to modulate unappreciated biological functions.

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 α-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 α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

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.

hnRNPK recognition of the B motif of Xist and other biological RNAs.

Heterogeneous nuclear ribonuclear protein K (hnRNPK) is an abundant RNA-binding protein crucial for a wide variety of biological processes. While its binding preference for multi-cytosine-patch (C-patch) containing RNA is well documented, examination of binding to known cellular targets that contain C-patches reveals an unexpected breadth of binding affinities. Analysis of in-cell crosslinking data reinforces the notion that simple C-patch preference is not fully predictive of hnRNPK localization within transcripts. The individual RNA-binding domains of hnRNPK work together to interact with RNA tightly, with the KH3 domain being neither necessary nor sufficient for binding. Rather, the RG/RGG domain is implicated in providing essential contributions to RNA-binding, but not DNA-binding, affinity. hnRNPK is essential for X chromosome inactivation, where it interacts with Xist RNA specifically through the Xist B-repeat region. We use this interaction with an RNA motif derived from this B-repeat region to determine the RNA-structure dependence of C-patch recognition. While the location preferences of hnRNPK for C-patches are conformationally restricted within the hairpin, these structural constraints are relieved in the absence of RNA secondary structure. Together, these results illustrate how this multi-domain protein's ability to accommodate and yet discriminate between diverse cellular RNAs allows for its broad cellular functions.

The structure of human CST reveals a decameric assembly bound to telomeric DNA.

The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex. The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly. CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome's organization of double-stranded DNA.