Abstract P351: The Post-transcriptional Regulations Of Centrosomal Plp Mrna In Drosophila

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Junnan Fang
Keyword(s):  
Translational Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Cell Types ◽  
Mrna Localization ◽  
Rna Localization ◽  
Embryonic Lethality ◽  
Cellular Processes ◽  
Pericentriolar Material ◽  
And Function

Centrosomes, functioning as microtubule organizing centers, are composed of a proteinaceous matrix of pericentriolar material (PCM) that surrounds a pair of centrioles. Drosophila Pericentrin (Pcnt)-like protein (PLP) is a key component of the centrosome that serves as a scaffold for PCM assembly. The disruption of plp in Drosophila results in embryonic lethality, while the deregulation of Pcnt in humans is associated with MOPD II and Trisomy 21.We recently found plp mRNA localizes to Drosophila embryonic centrosomes. While RNA is known to associate with centrosomes in diverse cell types, the elements required for plp mRNA localization to centrosomes remains completely unknown. Additionally, how plp translation is regulated to accommodate rapid cell divisions during early embryogenesis is unclear. RNA localization coupled with translational control is a conserved mechanism that functions in diverse cellular processes. Control of mRNA localization and translation is mediated by RNA-binding proteins (RBPs). We find PLP protein expression is specifically promoted by an RNA-binding protein, Orb, during embryogenesis; moreover, plp mRNA interacts with Orb. Importantly, we find overexpression of full-length PLP can rescue cell division defects and embryonic lethality caused by orb depletion. We aim to uncover the mechanisms underlying embryonic plp mRNA localization and function and how Orb regulates plp translation.

Messages on the move: the role of the cytoskeleton in mRNA localization and translation in plant cellsThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 572-580 ◽  
Author(s):  
Douglas G. Muench ◽  
Nam-Il Park
Keyword(s):  
Translational Control ◽  
Cell Biology ◽  
Protein Targeting ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Plant Cells ◽  
Mrna Localization ◽  
Cell Trafficking ◽  
Cellular Processes

The cytoskeleton plays an important role in numerous cellular processes, including subcellular mRNA localization and translation. Several examples of mRNA localization have emerged in plant cells, and these appear to function in protein targeting, the establishment of polarity, and cell-to-cell trafficking. The identification of several cytoskeleton-associated RNA-binding proteins in plant cells has made available candidate proteins that mediate the interaction between mRNA and the cytoskeleton, and possibly play a role in mRNA localization and translational control. We propose a model that links mRNA–microtubule interactions to translational autoregulation, a process that may assist in the efficient and regulated binding of proteins to microtubules.

scholarly journals Expanding Role of Ubiquitin in Translational Control

2020 ◽  
Vol 21 (3) ◽  
pp. 1151 ◽  
Author(s):  
Shannon E. Dougherty ◽  
Austin O. Maduka ◽  
Toshifumi Inada ◽  
Gustavo M. Silva
Keyword(s):  
Translational Control ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Protein Quality ◽  
Rna Binding Proteins ◽  
Ubiquitin Chain ◽  
Molecular Aspects ◽  
And Function

The eukaryotic proteome has to be precisely regulated at multiple levels of gene expression, from transcription, translation, and degradation of RNA and protein to adjust to several cellular conditions. Particularly at the translational level, regulation is controlled by a variety of RNA binding proteins, translation and associated factors, numerous enzymes, and by post-translational modifications (PTM). Ubiquitination, a prominent PTM discovered as the signal for protein degradation, has newly emerged as a modulator of protein synthesis by controlling several processes in translation. Advances in proteomics and cryo-electron microscopy have identified ubiquitin modifications of several ribosomal proteins and provided numerous insights on how this modification affects ribosome structure and function. The variety of pathways and functions of translation controlled by ubiquitin are determined by the various enzymes involved in ubiquitin conjugation and removal, by the ubiquitin chain type used, by the target sites of ubiquitination, and by the physiologic signals triggering its accumulation. Current research is now elucidating multiple ubiquitin-mediated mechanisms of translational control, including ribosome biogenesis, ribosome degradation, ribosome-associated protein quality control (RQC), and redox control of translation by ubiquitin (RTU). This review discusses the central role of ubiquitin in modulating the dynamism of the cellular proteome and explores the molecular aspects responsible for the expanding puzzle of ubiquitin signals and functions in translation.

scholarly journals The Nuclear RNA-binding Protein Sam68 Translocates to the Cytoplasm and Associates with the Polysomes in Mouse Spermatocytes

2006 ◽  
Vol 17 (1) ◽  
pp. 14-24 ◽  
Author(s):  
Maria Paola Paronetto ◽  
Francesca Zalfa ◽  
Flavia Botti ◽  
Raffaele Geremia ◽  
Claudia Bagni ◽  
...  
Keyword(s):  
Translational Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Male Meiosis ◽  
Binding Motif ◽  
Mitogen Activated Protein ◽  
Pachytene Spermatocytes ◽  
And Function

Translational control plays a crucial role during gametogenesis in organisms as different as worms and mammals. Mouse knockout models have highlighted the essential function of many RNA-binding proteins during spermatogenesis. Herein we have investigated the expression and function during mammalian male meiosis of Sam68, an RNA-binding protein implicated in several aspects of RNA metabolism. Sam68 expression and localization within the cells is stage specific: it is expressed in the nucleus of spermatogonia, it disappears at the onset of meiosis (leptotene/zygotene stages), and it accumulates again in the nucleus of pachytene spermatocytes and round spermatids. During the meiotic divisions, Sam68 translocates to the cytoplasm where it is found associated with the polysomes. Translocation correlates with serine/threonine phosphorylation and it is blocked by inhibitors of the mitogen activated protein kinases ERK1/2 and of the maturation promoting factor cyclinB-cdc2 complex. Both kinases associate with Sam68 in pachytene spermatocytes and phosphorylate the regulatory regions upstream and downstream of the Sam68 RNA-binding motif. Molecular cloning of the mRNAs associated with Sam68 in mouse spermatocytes reveals a subset of genes that might be posttranscriptionally regulated by this RNA-binding protein during spermatogenesis. We also demonstrate that Sam68 shuttles between the nucleus and the cytoplasm in secondary spermatocytes, suggesting that it may promote translation of specific RNA targets during the meiotic divisions.

scholarly journals Imaging-based pooled CRISPR screening reveals regulators of lncRNA localization

2019 ◽  
Vol 116 (22) ◽  
pp. 10842-10851 ◽  
Author(s):  
Chong Wang ◽  
Tian Lu ◽  
George Emanuel ◽  
Hazen P. Babcock ◽  
Xiaowei Zhuang
Keyword(s):  
High Throughput ◽  
Noncoding Rna ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Localization ◽  
Molecular Organization ◽  
Dynamic Regulation ◽  
Guide Rna ◽  
Cellular Processes

Pooled-library CRISPR screening provides a powerful means to discover genetic factors involved in cellular processes in a high-throughput manner. However, the phenotypes accessible to pooled-library screening are limited. Complex phenotypes, such as cellular morphology and subcellular molecular organization, as well as their dynamics, require imaging-based readout and are currently beyond the reach of pooled-library CRISPR screening. Here we report an all imaging-based pooled-library CRISPR screening approach that combines high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification in individual cells. In this approach, sgRNAs are codelivered to cells with corresponding barcodes placed at the 3′ untranslated region of a reporter gene using a lentiviral delivery system with reduced recombination-induced sgRNA-barcode mispairing. Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is used to read out the barcodes and hence identify the sgRNAs with high accuracy. We used this approach to screen 162 sgRNAs targeting 54 RNA-binding proteins for their effects on RNA localization to nuclear compartments and uncovered previously unknown regulatory factors for nuclear RNA localization. Notably, our screen revealed both positive and negative regulators for the nuclear speckle localization of a long noncoding RNA, MALAT1, suggesting a dynamic regulation of lncRNA localization in subcellular compartments.

scholarly journals Complexity and graded regulation of neuronal cell type-specific alternative splicing revealed by single-cell RNA sequencing

2021 ◽  
Author(s):  
Huijuan Feng ◽  
Daniel F. Moakley ◽  
Shuonan Chen ◽  
Melissa G. McKenzie ◽  
Vilas Menon ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Neuronal Cell ◽  
Cell Types ◽  
Mammalian Brain ◽  
Differential Splicing ◽  
Hierarchical Levels ◽  
Neuronal Types ◽  
And Function

AbstractThe enormous neuronal cellular diversity in the mammalian brain, which is highly prototypical and organized in a hierarchical manner, is dictated by cell type-specific gene regulatory programs at the molecular level. Although prevalent in the brain, contribution of alternative splicing (AS) to the molecular diversity across neuronal cell types is just starting to emerge. Here we systematically investigated AS regulation across over 100 transcriptomically defined neuronal types of the adult mouse cortex using deep single cell RNA-sequencing (scRNA-seq) data. We found distinct splicing programs between glutamatergic and GABAergic neurons and between subclasses within each neuronal class, consisting of overlapping sets of alternative exons showing differential splicing at multiple hierarchical levels. Using an integrative approach, our analysis suggests that RNA-binding proteins (RBPs) Celf1/2, Mbnl2 and Khdrbs3 are preferentially expressed and more active in glutamatergic neurons, while Elavl2 and Qk are preferentially expressed and more active in GABAergic neurons. Importantly, these and additional RBPs also contribute to differential splicing between neuronal subclasses at multiple hierarchical levels, and some RBPs drive splicing dynamics that do not conform to the hierarchical structure defined by the transcriptional profiles. Thus, our results suggest graded regulation of AS across neuronal cell types, which provides a molecular mechanism orthogonal to, rather than downstream of, transcriptional regulation in specifying neuronal identity and function.SignificanceAlternative splicing (AS) is extensively used in the mammalian brain, but its contribution to the molecular and cellular diversity across neuronal cell types remains poorly understood. Through systematic and integrative analysis of AS regulation across over 100 transcriptomically defined cortical neuronal types, we found neuronal subclass-specific splicing regulatory programs consists of overlapping alternative exons showing differential splicing at multiple hierarchical levels. This graded AS regulation is controlled by unique combinations of RNA-binding proteins (RBPs). Importantly, these RBPs also drive splicing dynamics across neuronal cell types that do not conform to the hierarchical taxonomy established based on transcriptional profiles, suggesting that the graded AS regulation provides a molecular mechanism orthogonal to transcriptional regulation in specifying neuronal identity and function.

scholarly journals Emerging Roles for 3′ UTRs in Neurons

2020 ◽  
Vol 21 (10) ◽  
pp. 3413
Author(s):  
Bongmin Bae ◽  
Pedro Miura
Keyword(s):  
Translational Control ◽  
Transcriptional Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Alternative Polyadenylation ◽  
Mrna Localization ◽  
Structural Features ◽  
Mrna Isoforms ◽  
Rna Sequences ◽  
Neuronal Functions

The 3′ untranslated regions (3′ UTRs) of mRNAs serve as hubs for post-transcriptional control as the targets of microRNAs (miRNAs) and RNA-binding proteins (RBPs). Sequences in 3′ UTRs confer alterations in mRNA stability, direct mRNA localization to subcellular regions, and impart translational control. Thousands of mRNAs are localized to subcellular compartments in neurons—including axons, dendrites, and synapses—where they are thought to undergo local translation. Despite an established role for 3′ UTR sequences in imparting mRNA localization in neurons, the specific RNA sequences and structural features at play remain poorly understood. The nervous system selectively expresses longer 3′ UTR isoforms via alternative polyadenylation (APA). The regulation of APA in neurons and the neuronal functions of longer 3′ UTR mRNA isoforms are starting to be uncovered. Surprising roles for 3′ UTRs are emerging beyond the regulation of protein synthesis and include roles as RBP delivery scaffolds and regulators of alternative splicing. Evidence is also emerging that 3′ UTRs can be cleaved, leading to stable, isolated 3′ UTR fragments which are of unknown function. Mutations in 3′ UTRs are implicated in several neurological disorders—more studies are needed to uncover how these mutations impact gene regulation and what is their relationship to disease severity.

scholarly journals Transcription Is Just the Beginning of Gene Expression Regulation: The Functional Significance of RNA-Binding Proteins to Post-transcriptional Processes in Plants

2019 ◽  
Vol 60 (9) ◽  
pp. 1939-1952 ◽  
Author(s):  
Wil Prall ◽  
Bishwas Sharma ◽  
Brian D Gregory
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Gene Expression Regulation ◽  
Rna Binding Proteins ◽  
Global Climate ◽  
Genome Mining ◽  
Valuable Insight ◽  
Cellular Processes ◽  
High Level ◽  
And Function

Abstract Plants have developed sophisticated mechanisms to compensate and respond to ever-changing environmental conditions. Research focus in this area has recently shifted towards understanding the post-transcriptional mechanisms that contribute to RNA transcript maturation, abundance and function as key regulatory steps in allowing plants to properly react and adapt to these never-ending shifts in their environments. At the center of these regulatory mechanisms are RNA-binding proteins (RBPs), the functional mediators of all post-transcriptional processes. In plants, RBPs are becoming increasingly appreciated as the critical modulators of core cellular processes during development and in response to environmental stimuli. With the majority of research on RBPs and their functions historically in prokaryotic and mammalian systems, it has more recently been unveiled that plants have expanded families of conserved and novel RBPs compared with their eukaryotic counterparts. To better understand the scope of RBPs in plants, we present past and current literature detailing specific roles of RBPs during stress response, development and other fundamental transition periods. In this review, we highlight examples of complex regulation coordinated by RBPs with a focus on the diverse mechanisms of plant RBPs and the unique processes they regulate. Additionally, we discuss the importance for additional research into understanding global interactions of RBPs on a systems and network-scale, with genome mining and annotation providing valuable insight for potential uses in improving crop plants in order to maintain high-level production in this era of global climate change.

scholarly journals A massively parallel reporter assay reveals focused and broadly encoded RNA localization signals in neurons

2021 ◽  
Author(s):  
Martin Mikl ◽  
Davide Eletto ◽  
Minkyoung Lee ◽  
Atefeh Lafzi ◽  
Farah Mhamedi ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Neuronal Cell ◽  
Mrna Localization ◽  
Rna Localization ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Massively Parallel Reporter Assay ◽  
Localization Behavior

AbstractAsymmetric subcellular localization of mRNA is a common cellular phenomenon that is thought to contribute to spatial gene regulation. In highly polar neurons, subcellular transcript localization and translation are thought to enhance cellular efficiency and timely responses to external cues. Although mRNA localization has been observed in many tissues and numerous examples of the functional importance of this process exist, we still lack a systematic understanding of how the transcript sorting machinery works in a sequence-specific manner.Here, we addressed these gaps by combining subcellular transcriptomics and rationally designed sequence libraries. We developed a massively parallel reporter assay (MPRA) for mRNA localization and tested ~50,000 sequences for their ability to drive RNA localization to neurites of neuronal cell lines. By scanning the 3’UTR of >300 genes we identified many previously unknown localization regions and mapped the localization potential of endogenous sequences. Our data suggest two ways the localization potential can be encoded in the 3’UTR: focused localization motifs and broadly encoded localization potential based on small contributions.We identified sequence motifs enriched in dendritically localized transcripts and tested the potential of these motifs to affect the localization behavior of an mRNA. This assay revealed sequence elements with the ability to bias localization towards neurite as well as soma. Depletion of RNA binding proteins predicted or experimentally shown to bind these motifs abolished the effect on localization, suggesting that these motifs act by recruiting specific RNA-binding proteins.Based on our dataset we developed machine learning models that accurately predict the localization behavior of novel sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native transcripts.Applying similar systematic high-throughput approaches to other cell types will open the door for a comparative perspective on RNA localization across tissues and reveal the commonalities and differences of this crucial regulatory mechanism.

scholarly journals C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons

2021 ◽  
Author(s):  
Eun Seon Kim ◽  
Chang Geon Chung ◽  
Jeong Hyang Park ◽  
Byung Su Ko ◽  
Sung Soon Park ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Accumulation ◽  
Heterozygous Mutation ◽  
Pathological Feature ◽  
Dependent Manner ◽  
Cellular Processes ◽  
Drosophila Model ◽  
Post Transcriptional Regulation

Abstract RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP, Staufen, may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS—but not with mutated endogenous NLS—potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/−), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/− is protective. Stau+/− also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.

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