Stress-Triggered Phase Separation Is an Adaptive, Evolutionarily Tuned Response.

In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation, whereas Pab1's LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we create LCR mutations, which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations that impede phase separation reduce organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely mark stress onset, a broadly generalizable mechanism.

Regulation of Co-transcriptional Pre-mRNA Splicing by m6A through the Low-Complexity Protein hnRNPG.

N6-methyladenosine (m6A) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional m6A-dependent alternative splicing regulation is still poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an m6A reader protein that binds RNA through RRM and Arg-Gly-Gly (RGG) motifs. Here, we show that hnRNPG directly binds to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) using RGG motifs in its low-complexity region. Through interactions with the phosphorylated CTD and nascent RNA, hnRNPG associates co-transcriptionally with RNAPII and regulates alternative splicing transcriptome-wide. m6A near splice sites in nascent pre-mRNA modulates hnRNPG binding, which influences RNAPII occupancy patterns and promotes exon inclusion. Our results reveal an integrated mechanism of co-transcriptional m6A-mediated splicing regulation, in which an m6A reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing.

Evolution of Transcript Abundance is Influenced by Indels in Protein Low Complexity Regions.

Protein Protein low complexity regions (LCRs) are compositionally biased amino acid sequences, many of which have significant evolutionary impacts on the proteins which contain them. They are mutationally unstable experiencing higher rates of indels and substitutions than higher complexity regions. LCRs also impact the expression of their proteins, likely through multiple effects along the path from gene transcription, through translation, and eventual protein degradation. It has been observed that proteins which contain LCRs are associated with elevated transcript abundance (TAb), despite having lower protein abundance. We have gathered and integrated human data to investigate the co-evolution of TAb and LCRs through ancestral reconstructions and model inference using an approximate Bayesian calculation based method. We observe that on short evolutionary timescales TAb evolution is significantly impacted by changes in LCR length, with insertions driving TAb down. But in contrast, the observed data is best explained by indel rates in LCRs which are unaffected by shifts in TAb. Our work demonstrates a coupling between LCR and TAb evolution, and the utility of incorporating multiple responses into evolutionary analyses.

Common low complexity regions for SARS-CoV-2 and human proteomes as potential multidirectional risk factor in vaccine development.

BACKGROUND: The rapid spread of the COVID-19 demands immediate response from the scientific communities. Appropriate countermeasures mean thoughtful and educated choice of viral targets (epitopes). There are several articles that discuss such choices in the SARS-CoV-2 proteome, other focus on phylogenetic traits and history of the Coronaviridae genome/proteome. However none consider viral protein low complexity regions (LCRs). Recently we created the first methods that are able to compare such fragments. RESULTS: We show that five low complexity regions (LCRs) in three proteins (nsp3, S and N) encoded by the SARS-CoV-2 genome are highly similar to regions from human proteome. As many as 21 predicted T-cell epitopes and 27 predicted B-cell epitopes overlap with the five SARS-CoV-2 LCRs similar to human proteins. Interestingly, replication proteins encoded in the central part of viral RNA are devoid of LCRs. CONCLUSIONS: Similarity of SARS-CoV-2 LCRs to human proteins may have implications on the ability of the virus to counteract immune defenses. The vaccine targeted LCRs may potentially be ineffective or alternatively lead to autoimmune diseases development. These findings are crucial to the process of selection of new epitopes for drugs or vaccines which should omit such regions.

Cloning, expression and purification of the low-complexity region of RanBP9 protein.

Recombinant expression and purification of proteins is key for biochemical and biophysical investigations. Although this has become a routine and standard procedure for many proteins, intrinsically disordered ones and those with low complexity sequences pose difficulties. Proteins containing low complexity regions (LCRs) are increasingly becoming significant for their roles in both normal and pathological processes. Here, we report cloning, expression and purification of N-terminal LCR of RanBP9 protein (Nt-RanBP9). RanBP9 is a scaffolding protein present in both cytoplasm and nucleus that is implicated in many cellular processes. Nt-RanBP9 is a poorly understood region of the protein perhaps due to difficulties posed by the LCR. Indeed, conventional methods presented difficulties in Nt-RanBP9 cloning due to its high GC content resulting in insignificant protein expression. These led us to use a different approach of cloning by expressing the protein as a fusion construct containing mCherry or mEGFP using in vivo DNA recombination methods. Our results indicate that expression of mEGFP-tagged Nt-RanBP9 followed by thrombin cleavage of the tag was the most effective method to obtain the protein with >90% purity and good yields. We report and discuss the challenges in obtaining the N-terminal region of RanBP9, a protein with functional implications in multiple biological processes and neurodegenerative diseases.

Key Role of Amino Acid Repeat Expansions in the Functional Diversification of Duplicated Transcription Factors.

The high regulatory complexity of vertebrates has been related to two rounds of whole genome duplication (2R-WGD) that occurred before the divergence of the major vertebrate groups. Following these events, many developmental transcription factors (TFs) were retained in multiple copies and subsequently specialized in diverse functions, whereas others reverted to their singleton state. TFs are known to be generally rich in amino acid repeats or low-complexity regions (LCRs), such as polyalanine or polyglutamine runs, which can evolve rapidly and potentially influence the transcriptional activity of the protein. Here we test the hypothesis that LCRs have played a major role in the diversification of TF gene duplicates. We find that nearly half of the TF gene families originated during the 2R-WGD contains LCRs. The number of gene duplicates with LCRs is 155 out of 550 analyzed (28%), about twice as many as the number of single copy genes with LCRs (15 out of 115, 13%). In addition, duplicated TFs preferentially accumulate certain LCR types, the most prominent of which are alanine repeats. We experimentally test the role of alanine-rich LCRs in two different TF gene families, PHOX2A/PHOX2B and LHX2/LHX9. In both cases, the presence of the alanine-rich LCR in one of the copies (PHOX2B and LHX2) significantly increases the capacity of the TF to activate transcription. Taken together, the results provide strong evidence that LCRs are important driving forces of evolutionary change in duplicated genes.

RNA Granules and Diseases: A Case Study of Stress Granules in ALS and FTLD.

RNA granules are microscopically visible cellular structures that aggregate by protein-protein and protein-RNA interactions. Using stress granules as an example, we discuss the principles of RNA granule formation, which rely on the multivalency of RNA and multi-domain proteins as well as low-affinity interactions between proteins with prion-like/low-complexity domains (e.g. FUS and TDP-43). We then explore how dysregulation of RNA granule formation is linked to neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), and discuss possible strategies for therapeutic intervention.

Global and local genomic features together modulate the spontaneous single nucleotide mutation rate.

Spontaneous mutations are evolutionary engines as they generate variants for the evolutionary downstream processes that give rise to speciation and adaptation. Single nucleotide mutations (SNM) are the most abundant type of mutations among them. Here, we perform a meta-analysis to quantify the influence of selected global genomic parameters (genome size, genomic GC content, genomic repeat fraction, number of coding genes, gene count, and strand bias in prokaryotes) and local genomic features (local GC content, repeat content, CpG content and the number of SNM at CpG islands) on spontaneous SNM rates across the tree of life (prokaryotes, unicellular eukaryotes, multicellular eukaryotes) using wild-type sequence data in two different taxon classification systems. We find that the spontaneous SNM rates in our data are correlated with many genomic features in prokaryotes and unicellular eukaryotes irrespective of their sample sizes. On the other hand, only the number of coding genes was correlated with the spontaneous SNM rates in multicellular eukaryotes primarily contributed by vertebrates data. Considering local features, we notice that local GC content and CpG content significantly were correlated with the spontaneous SNM rates in the unicellular eukaryotes, while local repeat fraction is an important feature in prokaryotes and certain specific uni- and multi-cellular eukaryotes. Such predictive features of the spontaneous SNM rates often support non-linear models as the best fit compared to the linear model. We also observe that the strand asymmetry in prokaryotes plays an important role in determining the spontaneous SNM rates but the SNM spectrum does not.

A Census of Human Methionine-Rich Prion-like Domain-Containing Proteins.

Methionine-rich prion-like proteins can regulate liquid-liquid phase separation processes in response to stresses. To date, however, very few proteins have been identified as methionine-rich prion-like. Herein, we have performed a computational survey of the human proteome to search for methionine-rich prion-like domains. We present a census of 51 manually curated methionine-rich prion-like proteins. Our results show that these proteins tend to be modular in nature, with molecular sizes significantly greater than those we would expect due to random sampling effects. These proteins also exhibit a remarkably high degree of spatial compaction when compared to average human proteins, even when protein size is accounted for. Computational evidence suggests that such a high degree of compactness might be due to the aggregation of methionine residues, pointing to a potential redox regulation of compactness. Gene ontology and network analyses, performed to shed light on the biological processes in which these proteins might participate, indicate that methionine-rich and non-methionine-rich prion-like proteins share gene ontology terms related to the regulation of transcription and translation but, more interestingly, these analyses also reveal that proteins from the methionine-rich group tend to share more gene ontology terms among them than they do with their non-methionine-rich prion-like counterparts.

A de Novo ZMIZ1 Pathogenic Variant for Neurodevelopmental Disorder With Dysmorphic Facies and Distal Skeletal Anomalies.

Background: Neurodevelopmental disorder with dysmorphic facies and distal skeletal anomalies (NEDDFSA) is a rare syndromic disorder characterized by global neurodevelopmental delay, early-onset hypotonia, poor overall growth, poor speech/language ability, and additional common phenotypes such as eye anomalies, joint hypermobility, and skeletal anomalies of the hands and feet. NEDDFSA is caused by heterozygous pathogenic variants in the ZMIZ1 gene on chromosome 10q22.3 with autosomal dominant (AD) mode of inheritance. All the 32 reported cases with variants in ZMIZ1 gene had a genetic background in Caucasian, Hispanic, North African, and Southeastern Asian. Until now, there are no reports of Chinese patients with ZMIZ1 pathogenic variants. Methods: A 5-year-old girl was found to have the characteristic phenotypes of NEDDFSA. Array-Comparative Genomic Hybridization (array-CGH) and whole exome sequencing (WES) were applied for the trio of this female patient. Sanger sequencing was used to verify the selected variants. A comprehensive molecular analysis was carried out by protein structure prediction, evolutionary conservation, motif scanning, tissue-specific expression, and protein interaction network to elucidate pathogenicity of the identified ZMIZ1 variants. Results: The karyotype was 46, XX with no micro-chromosomal abnormalities identified by array-CGH. There were 20 variants detected in the female patient by WES. A de novo heterozygous missense variant (c.2330G > A, p.Gly777Glu, G777E) was identified in the exon 20 of ZMIZ1. No variants of ZMIZ1 were identified in the non-consanguineous parents and her healthy elder sister. It was predicted that G777E was pathogenic and detrimental to the spatial conformation of the MIZ/SP-RING zinc finger domain of ZMIZ1. Conclusion: Thus far, only four scientific articles reported deleterious variants in ZMIZ1 and most of the cases were from Western countries. This is the first report about a Chinese patient with ZMIZ1 variant. It will broaden the current knowledge of ZMIZ1 variants and variable clinical presentations for clinicians and genetic counselors.

Positional motif analysis reveals the extent of specificity of protein-RNA interactions observed by CLIP.

BACKGROUND: Crosslinking and immunoprecipitation (CLIP) is a method used to identify in vivo RNA-protein binding sites on a transcriptome-wide scale. With the increasing amounts of available data for RNA-binding proteins (RBPs), it is important to understand to what degree the enriched motifs specify the RNA-binding profiles of RBPs in cells. RESULTS: We develop positionally enriched k-mer analysis (PEKA), a computational tool for efficient analysis of enriched motifs from individual CLIP datasets, which minimizes the impact of technical and regional genomic biases by internal data normalization. We cross-validate PEKA with mCross and show that the use of input control for background correction is not required to yield high specificity of enriched motifs. We identify motif classes with common enrichment patterns across eCLIP datasets and across RNA regions, while also observing variations in the specificity and the extent of motif enrichment across eCLIP datasets, between variant CLIP protocols, and between CLIP and in vitro binding data. Thereby, we gain insights into the contributions of technical and regional genomic biases to the enriched motifs, and find how motif enrichment features relate to the domain composition and low-complexity regions of the studied proteins. CONCLUSIONS: Our study provides insights into the overall contributions of regional binding preferences, protein domains, and low-complexity regions to the specificity of protein-RNA interactions, and shows the value of cross-motif and cross-RBP comparison for data interpretation. Our results are presented for exploratory analysis via an online platform in an RBP-centric and motif-centric manner ( https://imaps.goodwright.com/apps/peka/ ).

A unified view of low complexity regions (LCRs) across species.

Low complexity regions (LCRs) play a role in a variety of important biological processes, yet we lack a unified view of their sequences, features, relationships, and functions. Here, we use dotplots and dimensionality reduction to systematically define LCR type/copy relationships and create a map of LCR sequence space capable of integrating LCR features and functions. By defining LCR relationships across the proteome, we provide insight into how LCR type and copy number contribute to higher order assemblies, such as the importance of K-rich LCR copy number for assembly of the nucleolar protein RPA43 in vivo and in vitro. With LCR maps, we reveal the underlying structure of LCR sequence space, and relate differential occupancy in this space to the conservation and emergence of higher order assemblies, including the metazoan extracellular matrix and plant cell wall. Together, LCR relationships and maps uncover and identify scaffold-client relationships among E-rich LCR-containing proteins in the nucleolus, and revealed previously undescribed regions of LCR sequence space with signatures of higher order assemblies, including a teleost-specific T/H-rich sequence space. Thus, this unified view of LCRs enables discovery of how LCRs encode higher order assemblies of organisms.

Flanking Regions Determine the Structure of the Poly-Glutamine in Huntingtin through Mechanisms Common among Glutamine-Rich Human Proteins.

The causative agent of Huntington's disease, the poly-Q homo-repeat in the N-terminal region of huntingtin (httex1), is flanked by a 17-residue-long fragment (N17) and a proline-rich region (PRR), which promote and inhibit the aggregation propensity of the protein, respectively, by poorly understood mechanisms. Based on experimental data obtained from site-specifically labeled NMR samples, we derived an ensemble model of httex1 that identified both flanking regions as opposing poly-Q secondary structure promoters. While N17 triggers helicity through a promiscuous hydrogen bond network involving the side chains of the first glutamines in the poly-Q tract, the PRR promotes extended conformations in neighboring glutamines. Furthermore, a bioinformatics analysis of the human proteome showed that these structural traits are present in many human glutamine-rich proteins and that they are more prevalent in proteins with longer poly-Q tracts. Taken together, these observations provide the structural bases to understand previous biophysical and functional data on httex1.

Aggregation of Respiratory Complex Subunits Marks the Onset of Proteotoxicity in Proteasome Inhibited Cells.

Proteostasis is maintained by optimal expression, folding, transport, and clearance of proteins. Deregulation of any of these processes triggers protein aggregation and is implicated in many age-related pathologies. In this study, using quantitative proteomics and microscopy, we show that aggregation of many nuclear-encoded mitochondrial proteins is an early protein destabilization event during short-term proteasome inhibition. Among these, respiratory chain complex (RCC) subunits represent a group of functionally related proteins consistently forming aggregates under multiple proteostasis stresses with varying aggregation propensities. Sequence analysis reveals that several RCC subunits, irrespective of the cleavable mitochondrial targeting sequence, contain low-complexity regions at the N-terminus. Using different chimeric and mutant constructs, we show that these low-complexity regions partially contribute to the intrinsic instability of multiple RCC subunits. Taken together, we propose that physicochemically driven aggregation of unassembled RCC subunits destabilizes their functional assembly inside mitochondria. This eventually deregulates the biogenesis of respiratory complexes and marks the onset of mitochondrial dysfunction.

Reversible, functional amyloids: towards an understanding of their regulation in yeast and humans.

Protein aggregates, and in particular amyloids, are generally considered to be inherently irreversible aberrant clumps, and are often associated with pathologies, such as Alzheimer's disease, Parkinson's disease, or systemic amyloidosis. However, recent evidence demonstrates that some aggregates are not only fully reversible, but also perform essential physiological functions. Despite these new findings, very little is known about how these functional protein aggregates are regulated in a physiological context. Here, we take the yeast pyruvate kinase Cdc19 as an example of a protein forming functional, reversible, solid, amyloid-like aggregates in response to stress conditions. Cdc19 aggregation is regulated via an aggregation-prone low complexity region (LCR). In favorable growth conditions, this LCR is prevented from aggregating by phosphorylation or oligomerization, while upon glucose starvation it becomes exposed and allows aggregation. We suggest that LCR phosphorylation, oligomerization or partner-binding may be general and widespread mechanisms regulating LCR-mediated reversible protein aggregation. Moreover, we show that, as predicted by computational tools, Cdc19 forms amyloid-like aggregates in vitro. Interestingly, we also observe striking similarities between Cdc19 and its mammalian counterpart, PKM2. Indeed, also PKM2 harbors a LCR and contains several peptides with high amyloidogenic propensity, which coincide with known phosphorylation sites. Thus, we speculate that the formation of reversible, amyloid-like aggregates may be a general physiological mechanism for cells to adapt to stress conditions, and that the underlying regulatory mechanisms may be conserved from yeast to humans.

Structural disorder and its role in proteasomal degradation.

The ubiquitin proteasome system is responsible for the controlled degradation of a vast number of intracellular proteins. It targets misfolded or otherwise aberrant proteins as well as proteins no longer needed at a given point in time. The 26S proteasome is a large macromolecular machine comprising 33 distinct subunits as well as a number of transiently associating cofactors. Being essentially a non-specific protease, specificity is conferred by the ubiquitin system, which selects and marks substrates for degradation. Here, we review our current understanding of the structure and function of the 26S proteasome; in doing so we highlight the role of disordered protein regions. Disordered segments in substrates promote their degradation, whereas low complexity regions prevent their proteolysis. In the 26S proteasome itself a main role of disordered segments seems to be rendering the ubiquitin receptors mobile, possibly supporting recruitment of polyubiquitylated substrates. Thus, these structural features of substrates as well as of the 26S proteasome itself likely play important roles at different stages of the protein degradation process.

Increased substitution rates surrounding low-complexity regions within primate proteins.

Previous studies have found that DNA-flanking low-complexity regions (LCRs) have an increased substitution rate. Here, the substitution rate was confirmed to increase in the vicinity of LCRs in several primate species, including humans. This effect was also found among human sequences from the 1000 Genomes Project. A strong correlation was found between average substitution rate per site and distance from the LCR, as well as the proportion of genes with gaps in the alignment at each site and distance from the LCR. Along with substitution rates, dN/dS ratios were also determined for each site, and the proportion of sites undergoing negative selection was found to have a negative relationship with distance from the LCR.

Hydrogel formation by multivalent IDPs: A reincarnation of the microtrabecular lattice?

Based on high-voltage electron microscopic (HVEM) data of fixed cultured cells, an elaborate three-dimensional network of filaments, including and interconnecting other elements of the cytoskeleton, was observed in cells some half a century ago. Despite many attempts and comparative studies, this "microtrabecular lattice" (MTL) of the cytoplasmic ground substance could not be established as a genuine component of the eukaryotic cell, and is mostly considered today as a sample-preparation artifact of protein adherence and cross-linking to the cytoskeleton. Here we elaborate on the provocative idea that recent observations of hydrogel-forming phase transitions of repetitive regions of intrinsically disordered proteins (IDPs) bear resemblance in creation, organization and physical appearance to the MTL. We review this phenomenon in detail, and suggest that phase transitions of actin regulatory proteins, neurofilament side-arms and other proteins could generate non-uniform spatial distribution of cytoplasmic material in the vicinity of the cytoskeleton that might even give rise to fixation phenomena resembling the MTL. Whether such hydrogel formation by IDPs is a general physical phenomenon, will remain to be seen, nevertheless, the underlying organizational principle provokes novel experimental studies to uncover the ensuing higher-level regulation of cell physiology, in which the despised and long-forgotten concept of MTL might give some interesting leads.