Unstructured Region Research Articles

Nonphototrophic hypocotyl 3 domain proteins: traffic directors, hitchhikers, or both?

The nonphototrophic hypocotyl 3 (NPH3) domain is plant specific and of unknown function. It is nearly always attached to an N-terminal BTB domain and a largely unstructured C-terminal region. Recent reports revealed NPH3-domain GTPase activity and connection to intracellular trafficking, condensate formation, membrane attachment of the C-terminal region for some NPH3-domain proteins and, at the physiological level, drought-related function for at least one NPH3-domain protein. We integrate these new ideas of NPH3-domain protein function into two, nonexclusive, working models: the 'traffic director' model, whereby NPH3-domain proteins regulate intracellular trafficking and, the 'hitchhiker' model whereby NPH3-domain proteins ride the trafficking system to find ubiquitination targets. Determining which model best applies to uncharacterized NPH3-domain proteins will contribute to understanding intracellular trafficking and environmental responses.

Understanding amorphous gorge scenes based on the projection of spatial textures

Geographic information and topographical understanding are crucial for effective field environmental monitoring and autonomous navigation. Unfortunately, implementing this knowledge is often difficult due to unsolved problems such as understanding disorganized and unstructured mountainous regions. Traditional three-dimensional (3D) scene understanding methods from 3D point clouds or fused data are energy-consuming and unsuitable for a resource-constrained unmanned aerial vehicle (UAV) with limited computation, memory, and energy. This study proposes a methodology to understand unstructured gorges and reconstruct them in 3D environments from a low-cost monocular camera. The new approach assigns mountain texture projections to clusters and reshapes surfaces of unstructured mountains to follow geometric constraints regarding position and orientation. The relative relationships between surfaces and vanishing points are then analyzed to understand and reconstruct amorphous gorge scenes. This method is practical and efficient for navigating amorphous gorges based on understanding unstructured mountains. Fortunately, this method requires no prior training and is robust to color and illumination. We compared the percentage of incorrectly classified pixels with the ground truth. Experimental results have demonstrated that our approach can successfully understand amorphous gorges, meeting the requirements for autonomous flying in mountainous environments.

Dichotomous intronic polyadenylation profiles reveal multifaceted gene functions in the pan-cancer transcriptome

Alternative cleavage and polyadenylation within introns (intronic APA) generate shorter mRNA isoforms; however, their physiological significance remains elusive. In this study, we developed a comprehensive workflow to analyze intronic APA profiles using the mammalian target of rapamycin (mTOR)-regulated transcriptome as a model system. Our investigation revealed two contrasting effects within the transcriptome in response to fluctuations in cellular mTOR activity: an increase in intronic APA for a subset of genes and a decrease for another subset of genes. The application of this workflow to RNA-seq data from The Cancer Genome Atlas demonstrated that this dichotomous intronic APA pattern is a consistent feature in transcriptomes across both normal tissues and various cancer types. Notably, our analyses of protein length changes resulting from intronic APA events revealed two distinct phenomena in proteome programming: a loss of functional domains due to significant changes in protein length or minimal alterations in C-terminal protein sequences within unstructured regions. Focusing on conserved intronic APA events across 10 different cancer types highlighted the prevalence of the latter cases in cancer transcriptomes, whereas the former cases were relatively enriched in normal tissue transcriptomes. These observations suggest potential, yet distinct, roles for intronic APA events during pathogenic processes and emphasize the abundance of protein isoforms with similar lengths in the cancer proteome. Furthermore, our investigation into the isoform-specific functions of JMJD6 intronic APA events supported the hypothesis that alterations in unstructured C-terminal protein regions lead to functional differences. Collectively, our findings underscore intronic APA events as a discrete molecular signature present in both normal tissues and cancer transcriptomes, highlighting the contribution of APA to the multifaceted functionality of the cancer proteome.

MeCP2 is a naturally supercharged protein with cell membrane transduction capabilities.

The intrinsically disordered protein MeCP2 is a global transcriptional regulator encoded by the MECP2 gene. Although the structured domains of MeCP2 have been the subject of multiple studies, its unstructured regions have not been that extensively characterized. In this work, we show that MeCP2 possesses properties akin to those of supercharged proteins. By utilizing its unstructured portions, MeCP2 can successfully transduce across cell membranes and localize to heterochromatic foci in the nuclei, displaying uptake levels a third lower than a MeCP2 construct fused to the cell-penetrating peptide TAT. MeCP2 uptake can further be enhanced by the addition of compounds that promote endosomal escape following cellular trafficking by means of macropinocytosis. Using a combination of in silico prediction algorithms and live-cell imaging experiments, we mapped the sequence in MeCP2 responsible for its cellular incorporation, which bears a striking resemblance to TAT itself. Transduced MeCP2 was shown to interact with HDAC3. These findings provide valuable insight into the properties of MeCP2 and may be beneficial for devising future protein-based treatment strategies.

Untangling Zebrafish Genetic Annotation: Addressing Complexities and Nomenclature Issues in Orthologous Evaluation of TCOF1 and NOLC1.

Treacher Collins syndrome (TCS) is a genetic disorder affecting facial development, primarily caused by mutations in the TCOF1 gene. TCOF1, along with NOLC1, play important roles in ribosomal RNA transcription and processing. Previously, a zebrafish model of TCS successfully recapitulated the main characteristics of the syndrome by knocking down the expression of a gene on chromosome 13 (coding for Uniprot ID B8JIY2), which was identified as the TCOF1 orthologue. However, database updates renamed this gene as nolc1 and the zebrafish database (ZFIN) identified a different gene on chromosome 14 as the TCOF1 orthologue (coding for Uniprot ID E7F9D9). NOLC1 and TCOF1 are large proteins with unstructured regions and repetitive sequences that complicate alignments and comparisons. Also, the additional whole genome duplication of teleosts sets further difficulty. In this study, we present evidence that endorses that NOLC1 and TCOF1 are paralogs, and that the zebrafish gene on chromosome 14 is a low-complexity LisH domain-containing factor that displays homology to NOLC1 but lacks essential sequence features to accomplish TCOF1 nucleolar functions. Our analysis also supports the idea that zebrafish, as has been suggested for other non-tetrapod vertebrates, lack the TCOF1 gene that is associated with tripartite nucleolus. Using BLAST searches in a group of teleost genomes, we identified fish-specific sequences similar to E7F9D9 zebrafish protein. We propose naming them "LisH-containing Low Complexity Proteins" (LLCP). Interestingly, the gene on chromosome 13 (nolc1) displays the sequence features, developmental expression patterns, and phenotypic impact of depletion that are characteristic of TCOF1 functions. These findings suggest that in teleost fish, the nucleolar functions described for both NOLC1 and TCOF1 mediated by their repeated motifs, are carried out by a single gene, nolc1. Our study, which is mainly based on computational tools available as free web-based algorithms, could help to solve similar conflicts regarding gene orthology in zebrafish.

-catenin phosphorylation is elevated during mitosis to resist apical rounding and epithelial barrier leak.

Epithelial cell cohesion and barrier function critically depend on -catenin, an actin-binding protein and essential constituent of cadherin-catenin-based adherens junctions. -catenin undergoes actomyosin force-dependent unfolding of both actin-binding and middle domains to strongly engage actin filaments and its various effectors, where this mechanosensitivity is critical for adherens junction function. We previously showed that -catenin is highly phosphorylated in an unstructured region that links mechanosensitive middle- and actin-binding domains (known as the P-linker region), but the cellular processes that promote -catenin phosphorylation have remained elusive. Here, we leverage a previously published phosphor-proteomic data set to show that the -catenin P-linker region is maximally phosphorylated during mitosis. By reconstituting -catenin Crispr KO MDCK with wild-type, phospho- mutant and mimic forms of -catenin, we show that full phosphorylation restrains mitotic cell rounding in the apical direction, strengthening interactions between dividing and non-dividing neighbors to limit epithelial barrier leak. Since major scaffold components of adherens junctions, tight junctions and desmosomes are also differentially phosphorylated during mitosis, we reason that epithelial cell division may be a tractable system to understand how junction complexes are coordinately regulated to sustain barrier function under tension-generating morphogenetic processes.

Structural and biochemical characterization of the C-terminal region of the human RTEL1 helicase.

RTEL1 is an essential DNA helicase which plays an important role in various aspects of genome stability, from telomere metabolism to DNA replication, repair and recombination. RTEL1 has been implicated in a number of genetic diseases and cancer development, including glioma, breast, lung and gastrointestinal tumors. RTEL1 is a FeS helicase but, in addition to the helicase core, it comprises a long C-terminal region which includes a number of folded domains connected by intrinsically disordered loops and mediates RTEL1 interaction with factors involved in pivotal cellular pathways. However, information on the architecture and the function of this region is still limited. We expressed and purified a variety of fragments encompassing the folded domains and the unstructured regions. We determined the crystal structure of the second repeat, confirming that it has a fold similar to the harmonin homology domains. SAXS data provide low-resolution information on all the fragments and suggest that the presence of the RING domain affects the overall architecture of the C-terminal region, making the structure significantly more compact. NMR data provide experimental information on the interaction between PCNA and the RTEL1 C-terminal region, revealing a putative low-affinity additional site of interaction. A biochemical analysis shows that the C-terminal region, in addition to a preference for telomeric RNA and DNA G-quadruplexes, has a high affinity for R-loops and D-loops, consistent with the role played by the RTEL1 helicase in homologous recombination, telomere maintenance and preventing replication-transcription conflicts. We further dissected the contribution of each domain in binding different substrates.

Conserved cysteine-switches for redox sensing operate in the cyclin-dependent kinase inhibitor p21(CIP/KIP) protein family

The cell cycle is a tightly regulated, dynamic process controlled by multiple checkpoints. When the prevention of cell cycle progression is needed, key effectors such as members of the p21 (CIP/KIP) inhibit cyclin-dependent kinases (CDKs). It is accepted that p21 does not sense DNA damage and that stress signals affect p21 indirectly. A plethora of DNA damaging events activate the tumor suppressor p53, which in turn transcriptionally activates p21, steeply changing its levels to reach CDK inhibition. The levels of p21 are also controlled by phosphorylation and ubiquitination events, which are relevant as they modulate p21 activity, localization, and stability. Intriguingly, here we report the first evidence of the direct control of p21 cell proliferation inhibition by DNA damaging signals. Specifically, we have identified a redox regulating mechanism that controls p21 capacity to reduce cell proliferation. Using the human p21 protein, we identified two cysteine-switches that independently regulate its cyclin-binding and linker (LH) modules respectively. Additionally, we provide a mechanistic explanation of how reactive cysteines embedded in unstructured regions of intrinsically disordered proteins respond to ROS without the guidance of protein structure, contributing to a vastly unexplored area of research. Cellular experiments utilizing p21KID mutants that disrupt disulfide-based switches demonstrate their impact on the capacity of p21 to inhibit cell cycle progression, thus highlighting the functional relevance of our findings. Furthermore, our investigation reveals that reactive cysteine residues are highly conserved across the Kinase Inhibitory Domain (KID) sequences of p21 proteins from higher eukaryotes, and the p27 and p57 human paralogs. We propose that the presence of conserved regulatory cysteines within the KIDs of p21 family members from multiple taxa provides those proteins with the capability for directly sensing ROS, enabling the direct regulation of cyclin kinase activity by ROS levels.

Both the transcriptional activator, Bcd, and repressor, Cic, form small mobile oligomeric clusters

Transcription factors play an essential role in pattern formation during early embryo development, generating a strikingly fast and precise transcriptional response that results in sharp gene expression boundaries. To characterize the steps leading up to transcription, we performed a side-by-side comparison of the nuclear dynamics of two morphogens, a transcriptional activator, Bicoid (Bcd), and a transcriptional repressor, Capicua (Cic), both involved in body patterning along the anterior-posterior axis of the early Drosophila embryo. We used a combination of fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and single-particle tracking to access a wide range of dynamical timescales. Despite their opposite effects on gene transcription, we find that Bcd and Cic have very similar nuclear dynamics, characterized by the coexistence of a freely diffusing monomer population with a number of oligomeric clusters, which range from low stoichiometry and high mobility clusters to larger, DNA-bound hubs. Our observations are consistent with the inclusion of both Bcd and Cic into transcriptional hubs or condensates, while putting constraints on the mechanism by which these form. These results fit in with the recent proposal that many transcription factors might share a common search strategy for target gene regulatory regions that makes use of their large unstructured regions, and may eventually help explain how the transcriptional response they elicit can be at the same time so fast and so precise.

Quantitative Effects of the Loop Region on Topology, Thermodynamics, and Cation Binding of DNA G-quadruplexes.

The thermal stability of G-quadruplexes is important for their biological roles. G-quadruplexes are stable in the presence of cations such as K+ and Na+ because these cations coordinate in the G-quartet of four guanine bases. It is well known that the number of G-quartets and the configuration of the guanine bases affect the binding affinity of the cation. Recently, structures formed in the loop regions connecting the guanine stretches have attracted significant attention, because the loop region affects G-quadruplex properties, such as topology, thermal stability, and interactions with proteins and small molecules. Considering these effects, the loop region can also affect the binding affinity of the cations. Here, we designed a series of G-quadruplex-forming DNA sequences that contain a hairpin in a loop region and investigated the effects of the sequence and structure of the loop region on the cation binding affinity as well as the thermal stability of the G-quadruplex as a whole. First, structural analysis of the DNA sequences showed that the hairpin at the loop plays a key role in determining G4 topology (strand orientation). Second, in the case of the G-quadruplexes with the hairpin-forming loop region, it was found that a longer loop length led to a higher thermodynamic stability of the G-quadruplex as well as higher cation binding affinity. In contrast, an unstructured loop region did not lead to such effects. Interestingly, the cation binding affinity was correlated to the thermodynamic stability of the hairpin structure at the loop region. It was quantitatively demonstrated that the stable loop region stabilized the whole G-quadruplex structure, which induced higher cation binding affinity. These systematic and quantitative results showed that the loop region is one of the determinants of cation binding and expanded the possibilities of drug development targeting G4s by stabilizing the loop region.

3-Benzylaminomethyl Lithocholic Acid Derivatives Exhibited Potent and Selective Uncompetitive Inhibitory Activity Against Protein Tyrosine Phosphatase 1B (PTP1B).

Protein tyrosine phosphatase 1B (PTP1B) is a promising drug target for treating type 2 diabetes (T2DM) and obesity. As a result, developing new therapies that target PTP1B is an attractive strategy for treating these diseases. Herein, we detail the synthesis of 15 lithocholic acid (LA) derivatives, each containing different benzylaminomethyl groups attached to the C3 position of the steroid skeleton. The derivatives were assessed against two forms of PTP1B enzyme (hPTP1B1-400 and hPTP1B1-285), and the most potent compounds were then tested against T-cell protein tyrosine phosphatase (TCPTP) to determine their selectivity. The results showed that compounds 6m and 6n were more potent than the reference compounds (ursolic acid, chlorogenic acid, suramin, and TCS401). Additionally, both compounds exhibited greater potency over hPTP1B1-400. Furthermore, enzyme kinetic studies on hPTP1B1-400 revealed that these two lithocholic acid derivatives have an uncompetitive inhibition against hPTP1B1-400 with K i values of 2.5 and 3.4 μM, respectively. Interestingly, these compounds were around 75-fold more selective for PTP1B over TCPTP. Finally, docking studies and molecular dynamics simulations (MDS) were conducted to determine how these compounds interact with PTP1B. The docking studies revealed hydrophobic and H-bond interactions with amino acid residues in the unstructured region. MDS showed that these interactions persisted throughout the 200 ns simulation, indicating the crucial role of the unstructured zone in the biological activity and inhibition of PTP1B.

Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation.

The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

APCCdh1-mediated degradation of Cdh1 is necessary for faithful meiotic chromosome segregation in S. cerevisiae.

The Anaphase-Promoting Complex/Cyclosome (APC/C) is a ubiquitin ligase that promotes the ubiquitination and subsequent degradation of numerous cell cycle regulators during mitosis and in G1. Proteins are recruited to the APC/C by activator proteins such as Cdh1. During the cell cycle, Cdh1 is subject to precise regulation so that substrates are not degraded prematurely. We have explored the regulation of Cdh1 during the developmental transition into meiosis and sporulation in the budding yeast S. cerevisiae. Transition to sporulation medium triggers the degradation of Cdh1. Cdh1 degradation is mediated by the APC/C itself in a "trans" mechanism in which one molecule of Cdh1 recruits a second molecule of Cdh1 to the APC/C for ubiquitination. Degradation requires an intact glucose-sensing SNF1 protein kinase complex (orthologous to the mammalian AMPK nutritional sensor), which directly phosphorylates Cdh1 on Ser-200 within an unstructured N-terminal region. In the absence of phosphorylation, expression of a Cdh1-S200A mutant is fully stabilized, leading to chromosome instability and loss of viability. We hypothesize that Cdh1 degradation is necessary for the preservation of cell cycle regulators and chromosome cohesion proteins between the reductional and equational meiotic divisions, which occur without the intervening Gap or S phases found in mitotic cell cycles.

LiF-MS+, a revised technique for mapping peptide-protein interactions.

Short linear motifs are sequences of amino acids present in unstructured polypeptide regions that function as ligands for specific sites on folded protein domains. These interactions, which often occur with low to modest affinity, modulate dynamic biological processes such as signal transduction and membrane trafficking. We recently described Ligand Footprinting-Mass Spectrometry (LiF-MS), a technique that rapidly and precisely maps sites at which short peptide ligands bind their biologically relevant recognition sites on folded protein domains. This approach marks the binding location of a peptide ligand on a structured protein using a cleavable crosslinker appended to the ligand that leaves behind a stable chemical modification following cleavage. This modification serves as a mass tag detectable by mass spectrometry, pinpointing sites of peptide ligand binding. Here we present LiF-MS+, an improved version of the footprinting technique that replaces the butanol mass tag with 1-butylpyrrolidine, which is positively charged at neutral pH and thus aids in ionization of the crosslinked peptide for analysis by mass spectrometry. We show ligand-mediated butylpyrrolidine footprinting effectively maps the well characterized binding interaction of the p38α mitogen-activated protein kinase (MAPK) with a MKK6 D-motif short linear motif peptide ligand, uncovering additional binding site information not observed in our original experiment. LiF-MS+ is thus a straightforward improvement of our previously published methodology for mapping the binding of short linear motifs to folded protein domains.

Are the Head and Tail Domains of Intermediate Filaments Really Unstructured Regions?

Intermediate filaments (IFs) are integral components of the cytoskeleton which provide cells with tissue-specific mechanical properties and are involved in a plethora of cellular processes. Unfortunately, due to their intricate architecture, the 3D structure of the complete molecule of IFs has remained unresolved. Even though most of the rod domain structure has been revealed by means of crystallographic analyses, the flanked head and tail domains are still mostly unknown. Only recently have studies shed light on head or tail domains of IFs, revealing certainsecondary structures and conformational changes during IF assembly. Thus, a deeper understanding of their structure could provide insights into their function.

Structural anisotropy results in mechano-directional transport of proteins across nuclear pores

The nuclear pore complex regulates nucleocytoplasmic transport by means of a tightly synchronized suite of biochemical reactions. The physicochemical properties of the translocating cargos are emerging as master regulators of their shuttling dynamics. As well as being affected by molecular weight and surface-exposed amino acids, the kinetics of the nuclear translocation of protein cargos also depend on their nanomechanical properties, yet the mechanisms underpinning the mechanoselectivity of the nuclear pore complex are unclear. Here we show that proteins with locally soft regions in the vicinity of the nuclear-localization sequence exhibit higher nuclear-import rates, and that such mechanoselectivity is specifically impaired upon knocking down nucleoporin 153, a key protein in the nuclear pore complex. This allows us to design a short, easy-to-express and chemically inert unstructured peptide tag that accelerates the nuclear-import rate of stiff protein cargos. We also show that U2OS osteosarcoma cells expressing the peptide-tagged myocardin-related transcription factor import this mechanosensitive protein to the nucleus at higher rates and display faster motility. Locally unstructured regions lower the free-energy barrier of protein translocation and might offer a control mechanism for nuclear mechanotransduction.

Abstract 3104: A NanoBRET target engagement assay for querying domain selectivity at full-length polymerase theta in live cells

Abstract In HR-deficient cells, Polymerase Theta (Pol-theta) serves to repair double strand breaks and has emerged as a therapeutic target in HR-deficient cancers. Encoded by the POLQ gene, Pol-theta is a large (290 Kda) protein with two distinct functional domains (polymerase, helicase), separated by an unstructured region. Each domain of Pol-theta has proven vulnerable to small molecule inhibitors, with a number of molecules undergoing clinical evaluation. Unlike the helicase domain, target engagement at the polymerase domain requires cooperativity with DNA fragments. Here we describe a novel NanoBRET Target Engagement assay that enables domain-specific analysis of small molecule occupancy in live cells. To observe the high affinity-state of small molecule inhibitors to the polymerase domain, we have developed a cell-permeable pol domain NanoBRET probe, along with a novel work flow to introduce small DNA fragments into live cells. Together these components form a nucleoprotein complex with full length polymerase theta. This assay method can be used to quantify and rank-order engagement to the polymerase domain in live cells. To enable domain-selectivity analysis, we have developed a second NanoBRET probe directed to the helicase domain. Together these two NanoBRET probes allow for the mechanism of action studies at Pol-theta in living cells. Citation Format: Steven Edenson, Michael Slater, Cesear Corona, Michael Beck, James Vasta, Kelly Teske, Ani Michaud, Matthew Robers. A NanoBRET target engagement assay for querying domain selectivity at full-length polymerase theta in live cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3104.

Interaction of MLE with CLAMP zinc finger is involved in proper MSL proteins binding to chromosomes in Drosophila.

The Drosophila male-specific lethal (MSL) complex binds to the male X chromosome to activate transcription. It comprises five proteins (MSL1, MSL2, MSL3, male absent on the first (MOF), and maleless (MLE)) and two long noncoding RNAs (lncRNAs; roX1 and roX2). The MLE helicase remodels the roX lncRNAs, enabling the lncRNA-mediated assembly of the Drosophila dosage compensation complex. MSL2 is expressed only in males and interacts with the N-terminal zinc finger of the transcription factor chromatin-linked adapter for MSL proteins (CLAMP), which is important for the specific recruitment of the MSL complex to the male X chromosome. Here, we found that MLE's unstructured C-terminal region interacts with thesixth zinc-finger domain of CLAMP. In vitro, 4-5 zinc fingers are critical for the specific DNA-binding of CLAMP with GArepeats, which constitute the core motif at the high affinity binding sites for MSL proteins. Deleting the CLAMP binding region in MLE decreases the association of MSL proteins with the male X chromosome and increases male lethality. These results suggest that interactions of unstructured regions in MSL2 and MLE with CLAMP zinc finger domains are important for the specific recruitment of the MSL complex to the male X chromosome.

Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.

Unstructured linker regions play a role in the differential splicing activities of paralogous RNA binding proteins PTBP1 and PTBP2

RNA Binding Proteins regulate, in part, alternative pre-mRNA splicing and, in turn, gene expression patterns. Polypyrimidine tract binding proteins PTBP1 and PTBP2 are paralogous RNA binding proteins sharing 74% amino acid sequence identity. Both proteins contain four structured RNA-recognition motifs (RRMs) connected by linker regions and an N-terminal region. Despite their similarities, the paralogs have distinct tissue-specific expression patterns and can regulate discrete sets of target exons. How two highly structurally similar proteins can exert different splicing outcomes is not well understood. Previous studies revealed that PTBP2 is post-translationally phosphorylated in the unstructured N-terminal, Linker 1, and Linker 2 regions that share less sequence identity with PTBP1 signifying a role for these regions in dictating the paralogs distinct splicing activities. To this end, we conducted bioinformatics analysis to determine evolutionary conservation of RRMs vs linker regions in PTBP1 and PTBP2 across species. To determine the role of PTBP2 unstructured regions in splicing activity, we created hybrid PTBP1-PTBP2 constructs that had counterpart PTBP1 regions swapped to an otherwise PTBP2 protein and assayed on differentially regulated exons. We also conducted molecular dynamics studies to investigate how negative charges introduced by phosphorylation in PTBP2 unstructured regions can alter their physical properties. Collectively, results from our studies reveal an important role for PTBP2 unstructured regions and suggest a role for phosphorylation in the differential splicing activities of the paralogs on certain regulated exons.