Advanced imaging techniques for studying protein phase separation in living cells and at single-molecule level.

Protein-protein and protein-RNA interactions are essential for cell function and survival. These interactions facilitate the formation of ribonucleoprotein complexes and biomolecular condensates via phase separation. Such assembly is involved in transcription, splicing, translation and stress response. When dysregulated, proteins and RNA can undergo irreversible aggregation which can be cytotoxic and pathogenic. Despite technical advances in investigating biomolecular condensates, achieving the necessary spatiotemporal resolution to deduce the parameters that govern their assembly and behavior has been challenging. Many laboratories have applied advanced microscopy methods for imaging condensates. For example, single molecule imaging methods have enabled the detection of RNA-protein interaction, protein-protein interaction, protein conformational dynamics, and diffusional motion of molecules that report on the intrinsic molecular interactions underlying liquid-liquid phase separation. This review will outline advances in both microscopy and spectroscopy techniques which allow single molecule detection and imaging, and how these techniques can be used to probe unique aspects of biomolecular condensates.

Reducing waste management scenario space for developing countries: A hierarchical clustering on principal components approach.

The complexity of waste management (WM) problems resulted in the explosion of scenarios that challenge focused discussion among stakeholders and hinder the integrity of policy responses in developing countries. Hence, drawing similarities is essential to reduce the number of scenarios to simplify the WM efforts. To extract similarities, measuring WM performance is not enough, but the background factors related to this performance should be incorporated. These factors form a unique system characteristic that facilitates or hinders WM functions. Thus, this study applied multivariate statistical analysis to clarify underlying characteristics that facilitate efficient WM scenario developments for developing countries. The study first analysed drivers associated with improved WM system performance using bivariate correlation analysis. As a result, twelve significant drivers associated with controlled solid waste were identified. Then, it mapped the countries based on their WM system characteristics using the combined principal component analysis and hierarchical clustering approach. Thirteen variables were examined to extract similarities between the countries. The results identified three homogenous clusters. The clusters were found considerably parallel to the global classifications based on income and human development index. Hence, the presented approach is efficient in explaining similarities that reduce WM scenarios and favours cooperation among countries.

Acculturation Style Is Associated with Stress and Tobacco Use among East African Immigrants.

While tobacco use rates are relatively high among East African immigrants in the U.S., factors contributing to this high rate are largely unknown. Acculturation is associated with stress and substance use. Whether acculturation styles are related to stress and current tobacco use has not been tested among this population. We conducted a cross-sectional study that included 376 East African adults who provided information on demographic background, acculturation style, acculturative stress, depressive symptoms, and tobacco use. Multivariate analysis indicated that individuals who were distant to both the culture of the host country and the culture of origin (marginalization style) had greater levels of acculturative stress than those who adopted both cultures (integration style; p < .001). Marginalized people were four to eight times and assimilated people were three to four times more likely than integration people to be a current tobacco user (p < .04). This relationship did not change after controlling for demographic information and stress. In this study, acculturation style was associated with perceived stress and current tobacco use among East African immigrants. Research focused on characterizing integrated individuals may guide efforts to develop culturally-relevant strategies to reduce tobacco-related disparities among East African individuals.

Variation in ubiquitin system genes creates substrate-specific effects on proteasomal protein degradation.

Precise control of protein degradation is critical for life, yet how natural genetic variation affects this essential process is largely unknown. Here, we developed a statistically powerful mapping approach to characterize how genetic variation affects protein degradation by the ubiquitin-proteasome system (UPS). Using the yeast Saccharomyces cerevisiae, we systematically mapped genetic influences on the N-end rule, a UPS pathway in which protein N-terminal amino acids function as degradation-promoting signals. Across all 20 possible N-terminal amino acids, we identified 149 genomic loci that influence UPS activity, many of which had pathway- or substrate-specific effects. Fine-mapping of four loci identified multiple causal variants in each of four ubiquitin system genes whose products process (NTA1), recognize (UBR1 and DOA10), and ubiquitinate (UBC6) cellular proteins. A cis-acting promoter variant that modulates UPS activity by altering UBR1 expression alters the abundance of 36 proteins without affecting levels of the corresponding mRNA transcripts. Our results reveal a complex genetic basis of variation in UPS activity.

TDP-43 represses cryptic exon inclusion in the FTD-ALS gene UNC13A.

A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord1. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing2-4. Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies5,6, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function.

Trans-acting genetic variation affects the expression of adjacent genes.

Gene expression differences among individuals are shaped by trans-acting expression quantitative trait loci (eQTLs). Most trans-eQTLs map to hotspot locations that influence many genes. The molecular mechanisms perturbed by hotspots are often assumed to involve "vertical" cascades of effects in pathways that can ultimately affect the expression of thousands of genes. Here, we report that trans-eQTLs can affect the expression of adjacent genes via "horizontal" mechanisms that extend along a chromosome. Genes affected by trans-eQTL hotspots in the yeast Saccharomyces cerevisiae were more likely to be located next to each other than expected by chance. These paired hotspot effects tended to occur at adjacent genes that also show coexpression in response to genetic and environmental perturbations, suggesting shared mechanisms. Physical proximity and shared chromatin state, in addition to regulation of adjacent genes by similar transcription factors, were independently associated with paired hotspot effects among adjacent genes. Paired effects of trans-eQTLs can occur at neighboring genes even when these genes do not share a common function. This phenomenon could result in unexpected connections between regulatory genetic variation and phenotypes.

Decreased mechanotransduction prevents nuclear collapse in a Caenorhabditis elegans laminopathy.

The function of the nucleus depends on the integrity of the nuclear lamina, an intermediate filament network associated with the linker of nucleoskeleton and cytoskeleton (LINC) complex. The LINC complex spans the nuclear envelope and mediates nuclear mechanotransduction, the process by which mechanical signals and forces are transmitted across the nuclear envelope. In turn, the AAA+ ATPase torsinA is thought to regulate force transmission from the cytoskeleton to the nucleus. In humans, mutations affecting nuclear envelope-associated proteins cause laminopathies, including progeria, myopathy, and dystonia, though the extent to which endogenous mechanical stresses contribute to these pathologies is unclear. Here, we use the Caenorhabditis elegans germline as a model to investigate mechanisms that maintain nuclear integrity as germ cell nuclei progress through meiotic development and migrate for gametogenesis-processes that require LINC complex function. We report that decreasing the function of the C. elegans torsinA homolog, OOC-5, rescues the sterility and premature aging caused by a null mutation in the single worm lamin homolog. We show that decreasing OOC-5/torsinA activity prevents nuclear collapse in lamin mutants by disrupting the function of the LINC complex. At a mechanistic level, OOC-5/torsinA promotes the assembly or maintenance of the lamin-associated LINC complex and this activity is also important for interphase nuclear pore complex insertion into growing germline nuclei. These results demonstrate that LINC complex-transmitted forces damage nuclei with a compromised nuclear lamina. Thus, the torsinA-LINC complex nexus might comprise a therapeutic target for certain laminopathies by preventing damage from endogenous cellular forces.

Insights into the Involvement of Spliceosomal Mutations in Myelodysplastic Disorders from Analysis of SACY-1/DDX41 in Caenorhabditis elegans.

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia (AML). DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase that is recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of AML. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here, we conduct a comprehensive analysis of the Caenorhabditis elegans DDX41 ortholog, SACY-1 Biochemical analyses defined SACY-1 as a component of the C. elegans spliceosome, and genetic analyses revealed synthetic lethal interactions with spliceosomal components. We used the auxin-inducible degradation system to analyze the consequence of SACY-1 depletion on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-dependent and splicing-independent mechanisms. Altered 3' splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 in the second step of splicing. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. The transcriptome changes observed after SACY-1 depletion suggest that disruption of the spliceosome induces a stress response, which could contribute to the cellular phenotypes conferred by sacy-1 mutant alleles. Multiple sacy-1/ddx41 missense mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that their incorporation into the spliceosome is detrimental. Antagonistic variants that perturb the function of the spliceosome may be relevant to the disease-causing mutations, including DDX41, affecting highly conserved components of the spliceosome in humans.