DDX49 is an RNA helicase that affects translation by regulating mRNA export and the levels of pre-ribosomal RNA.

Among the proteins predicted to be a part of the DExD box RNA helicase family, the functions of DDX49 are unknown. Here, we characterize the enzymatic activities and functions of DDX49 by comparing its properties with the well-studied RNA helicase, DDX39B. We find that DDX49 exhibits a robust ATPase and RNA helicase activity, significantly higher than that of DDX39B. DDX49 is required for the efficient export of poly (A)+ RNA from nucleus in a splicing-independent manner. Furthermore, DDX49 is a resident protein of nucleolus and regulates the steady state levels of pre-ribosomal RNA by regulating its transcription and stability. These dual functions of regulating mRNA export and pre-ribosomal RNA levels enable DDX49 to modulate global translation. Phenotypically, DDX49 promotes proliferation and colony forming potential of cells. Strikingly, DDX49 is significantly elevated in diverse cancer types suggesting that the increased abundance of DDX49 has a role in oncogenic transformation of cells. Taken together, this study shows the physiological role of DDX49 in regulating distinct steps of mRNA and pre-ribosomal RNA metabolism and hence translation and potential pathological role of its dysregulation, especially in cancers.

Function and Regulation of Phase-Separated Biological Condensates.

Achieving functional specificity while minimizing cost to fitness is a key constraint during evolution. Formation of biological condensates by liquid-liquid phase separation (LLPS) appears to serve as an important regulatory mechanism to generate moderate specificity in molecular recognition while maintaining a reasonable cost for fitness in terms of design complexity. Formation of biological condensates serves as a unique mechanism of molecular recognition achieving some level of specificity without a huge cost to fitness. Rapid formation of biological condensates in vivo induced by specific cellular or environmental triggers has been shown to be an important mechanism for increasing cellular fitness. Here we discuss the functions and regulation of biological condensates, especially those formed by LLPS, involving interactions between proteins and nucleic acids. These condensates are spatially isolated within the cytosol or nucleus and can facilitate specific biochemical functions under conditions such as stress. The misregulation of biological condensates resulting in nondynamic aggregates has been implicated in a number of diseases. Understanding the functional importance of biological condensates and their regulation opens doors for development of therapies targeting dysfunctional biological condensates, as well as spatiotemporal engineering of functions in cells.

DDX39B promotes translation through regulation of pre-ribosomal RNA levels.

DDX39B, a DExD RNA helicase, is known to be involved in various cellular processes such as mRNA export, splicing and translation. Previous studies showed that the overexpression of DDX39B promotes the global translation but inhibits the mRNA export in a dominant negative manner. This presents a conundrum as to how DDX39B overexpression would increase the global translation if it inhibits the nuclear export of mRNAs. We resolve this by showing that DDX39B affects the levels of pre-ribosomal RNA by regulating its stability as well as synthesis. Furthermore, DDX39B promotes proliferation and colony forming potential of cells and its levels are significantly elevated in diverse cancer types. Thus, increase in DDX39B enhances global translation and cell proliferation through upregulation of pre-ribosomal RNA. This highlights a possible mechanism by which dysregulation of DDX39B expression could lead to oncogenesis.

Centrosomes and mitotic spindle poles: a recent liaison?

Centrosomes comprise two cylindrical centrioles embedded in the pericentriolar material (PCM). The PCM is an ordered assembly of large scaffolding molecules, providing an interaction platform for proteins involved in signalling, trafficking and most importantly microtubule nucleation and organization. In mitotic cells, centrosomes are located at the spindle poles, sites where spindle microtubules converge. However, certain cell types and organisms lack centrosomes, yet contain focused spindle poles, highlighting that despite their juxtaposition in cells, centrosomes and mitotic spindle poles are distinct physical entities. In the present paper, we discuss the origin of centrosomes and summarize their contribution to mitotic spindle assembly and cell division. We then describe the key molecular players that mediate centrosome attachment to mitotic spindle poles and explore why co-segregation of centrosomes and spindle poles into daughter cells is of potential benefit to organisms.

Intrauterine Viral Infections: Impact of Inflammation on Fetal Neurodevelopment.

Intrauterine viral infections during pregnancy by pathogens such as Zika virus, Cytomegalovirus, Rubella and Herpes Simplex virus can lead to prenatal as well as postnatal neurodevelopmental disorders. Although maternal viral infections are common during pregnancy, viruses rarely penetrate the trophoblast. When they do cross, viruses can cause adverse congenital health conditions for the fetus. In this context, maternal inflammatory responses to these neurotropic pathogens play a significant role in negatively affecting neurodevelopment. For instance, intrauterine inflammation poses an increased risk of neurodevelopmental disorders such as microcephaly, schizophrenia, autism spectrum disorder, cerebral palsy and epilepsy. Severe inflammatory responses have been linked to stillbirths, preterm births, abortions and microcephaly. In this review, we discuss the mechanistic basis of how immune system shapes the landscape of the brain and how different neurotropic viral pathogens evoke inflammatory responses. Finally, we list the consequences of neuroinflammation on fetal brain development and discuss directions for future research and intervention strategies.

Corrigendum: Intrauterine Viral Infections: Impact of Inflammation on Fetal Neurodevelopment.

[This corrects the article DOI: 10.3389/fnins.2021.771557.].

PRMT3 interacts with ALDH1A1 and regulates gene-expression by inhibiting retinoic acid signaling.

Protein arginine methyltransferase 3 (PRMT3) regulates protein functions by introducing asymmetric dimethylation marks at the arginine residues in proteins. However, very little is known about the interaction partners of PRMT3 and their functional outcomes. Using yeast-two hybrid screening, we identified Retinal dehydrogenase 1 (ALDH1A1) as a potential interaction partner of PRMT3 and confirmed this interaction using different methods. ALDH1A1 regulates variety of cellular processes by catalyzing the conversion of retinaldehyde to retinoic acid. By molecular docking and site-directed mutagenesis, we identified the specific residues in the catalytic domain of PRMT3 that facilitate interaction with the C-terminal region of ALDH1A1. PRMT3 inhibits the enzymatic activity of ALDH1A1 and negatively regulates the expression of retinoic acid responsive genes in a methyltransferase activity independent manner. Our findings show that in addition to regulating protein functions by introducing methylation modifications, PRMT3 could also regulate global gene expression through protein-protein interactions.

SET7/9 interacts and methylates the ribosomal protein, eL42 and regulates protein synthesis.

Methylation of proteins is emerging to be an important regulator of protein function. SET7/9, a protein lysine methyltransferase, catalyses methylation of several proteins involved in diverse biological processes. SET7/9-mediated methylation often regulates the stability, sub-cellular localization and protein-protein interactions of its substrate proteins. Here, we aimed to identify novel biological processes regulated by SET7/9 by identifying new interaction partners. For this we used yeast two-hybrid screening and identified the large subunit ribosomal protein, eL42 as a potential interactor of SET7/9. We confirmed the SET7/9-eL42 interaction by co-immunoprecipitation and GST pulldown studies. The N-terminal MORN domain of SET7/9 is essential for its interaction with eL42. Importantly, we identified that SET7/9 methylates eL42 at three different lysines - Lys53, Lys80 and Lys100 through site-directed mutagenesis. By puromycin incorporation assay, we find that SET7/9-mediated methylation of eL42 affects global translation. This study identifies a new role of the functionally versatile SET7/9 lysine methyltransferase in the regulation of global protein synthesis.

Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins.

Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype-phenotype landscape of a population, thereby contributing to adaptation and fitness.

Neurodevelopmental protein Musashi-1 interacts with the Zika genome and promotes viral replication.

A recent outbreak of Zika virus in Brazil has led to a simultaneous increase in reports of neonatal microcephaly. Zika targets cerebral neural precursors, a cell population essential for cortical development, but the cause of this neurotropism remains obscure. Here we report that the neural RNA-binding protein Musashi-1 (MSI1) interacts with the Zika genome and enables viral replication. Zika infection disrupts the binding of MSI1 to its endogenous targets, thereby deregulating expression of factors implicated in neural stem cell function. We further show that MSI1 is highly expressed in neural progenitors of the human embryonic brain and is mutated in individuals with autosomal recessive primary microcephaly. Selective MSI1 expression in neural precursors could therefore explain the exceptional vulnerability of these cells to Zika infection.

A CEP215-HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer.

Numerical centrosome aberrations underlie certain developmental abnormalities and may promote cancer. A cell maintains normal centrosome numbers by coupling centrosome duplication with segregation, which is achieved through sustained association of each centrosome with a mitotic spindle pole. Although the microcephaly- and primordial dwarfism-linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic profiling, we identify the minus end-directed microtubule motor protein HSET as a direct binding partner of CEP215. Targeted deletion of the HSET-binding domain of CEP215 in vertebrate cells causes centrosome detachment and results in HSET depletion at centrosomes, a phenotype also observed in CEP215-deficient patient-derived cells. Moreover, in cancer cells with centrosome amplification, the CEP215-HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosome-spindle pole interface by the CEP215-HSET complex could promote survival of cancer cells containing supernumerary centrosomes.

Tackling centrosome biology through gene targeting in chicken B cells.

The centrosome proteome comprises hundreds of proteins whose function at the organelle and in the cellular context is unknown. Loss-of-function studies present a powerful tool to probe the roles of these individual constituents and hence improve our insight into key questions of centrosome biology such as how centrosomes are built, how they duplicate, and which cellular processes they partake in. In cultured cells ribonucleic acid (RNA) interference remains the most widely used method to achieve protein depletion, but due to the remarkable stability of many centrosome components depletion is often incomplete. In such instances genome editing provides a viable alternative. The exceptionally high homologous recombination rate of chicken DT40 cells makes this lymphocytic cell line ideal for genetic manipulation. Here we describe methods for the design and generation of knockouts and in situ tagging of genes in these cells. Furthermore, we report an optimized technique that allows isolation of centrosomes from DT40 cells for use in in vitro functional assays and proteomic analysis. Gene editing by CRISPR-Cas9 technology is fast replacing RNA interference as a method of choice for loss-of-function studies, but the combination of the fast cell cycle, the robustness in culture and ease of gene targeting, will continue to make DT40 cells a useful model system for studies of vertebrate protein function.

Small organelle, big responsibility: the role of centrosomes in development and disease.

The centrosome, a key microtubule organizing centre, is composed of centrioles, embedded in a protein-rich matrix. Centrosomes control the internal spatial organization of somatic cells, and as such contribute to cell division, cell polarity and migration. Upon exiting the cell cycle, most cell types in the human body convert their centrioles into basal bodies, which drive the assembly of primary cilia, involved in sensing and signal transduction at the cell surface. Centrosomal genes are targeted by mutations in numerous human developmental disorders, ranging from diseases exclusively affecting brain development, through global growth failure syndromes to diverse pathologies associated with ciliary malfunction. Despite our much-improved understanding of centrosome function in cellular processes, we know remarkably little of its role in the organismal context, especially in mammals. In this review, we examine how centrosome dysfunction impacts on complex physiological processes and speculate on the challenges we face when applying knowledge generated from in vitro and in vivo model systems to human development.

TLX controls angiogenesis through interaction with the von Hippel-Lindau protein.

TLX is known as the orphan nuclear receptor indispensable for maintaining neural stem cells in adult neurogenesis. We report here that neuroblastoma cell lines express high levels of TLX, which further increase in hypoxia to enhance the angiogenic capacity of these cells. The proangiogenetic activity of TLX appears to be induced by its direct binding to the von Hippel-Lindau protein (pVHL), which stabilizes TLX. In turn, TLX competes with hydroxylated hypoxia-inducible factor (HIF-α) for binding to pVHL, which contributes to the stabilization of HIF-2α in neuroblastoma during normoxia. Upon hypoxia, TLX increases in the nucleus where it binds in close proximity of the HIF-response element on the VEGF-promoter chromatin, and, together with HIF-2α, recruits RNA polymerase II to induce VEGF expression. Conversely, depletion of TLX by shRNA decreases the expression of HIF-2α and VEGF as well as the growth-promoting and colony-forming capacity of the neuroblastoma cell lines IMR-32 and SH-SY5Y. On the contrary, silencing HIF-2α will slightly increase TLX, suggesting that TLX acts to maintain a hypoxic environment when HIF-2α is decreasing. Our results demonstrate TLX to play a key role in controlling angiogenesis by regulating HIF-2α. TLX and pVHL might counterbalance each other in important fate decisions such as self-renewal and differentiation, as well as angiogenesis and anti-angiogenesis.