Non-coding function for mRNAs in Focal Adhesion Architecture and Mechanotransduction

AbstractMessenger RNA (mRNA) compartmentalization within the cytosol is well-recognized as a key mechanism of local translation-mediated regulation of protein levels, but whether such localization could be a means of exercising non-coding mRNA function is unknown. Here, we explore non-coding functions for mRNAs associated with focal adhesions (FAs), cellular structures responsible for mediating cell adhesion and response to changes in the extracellular matrix (ECM). Using high-throughput single molecule imaging and genomic profiling approaches, we find that mRNAs with distinct sequence characteristics localize to FAs in different human cell types. Notably, ∼85% of FA-mRNAs are not translationally active at steady state or under conditions of FA dissolution or activation. Untranslated mRNA sequences are anchored to FA based on their functional states by the RNA binding protein, G3BP1, forming biomolecular granules. Removing RNA or G3BP1, but not blocking new polypeptide synthesis, dramatically changes FA protein composition and organization, resulting in loss of cell contractility and cellular ability to adapt to changing ECM. We have therefor uncovered a novel, non-coding role for mRNAs as scaffolds to maintain FA structure and function, broadening our understating of noncanonical mRNA functions.

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

Circulation Research ◽

10.1161/res.129.suppl_1.p351 ◽

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.

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Loss of Fbxw7 in Sertoli cells impairs testis development and causes infertility in mice†

Biology of Reproduction ◽

10.1093/biolre/ioz230 ◽

2019 ◽

Vol 102 (4) ◽

pp. 963-974

Author(s):

Hanbin Zhang ◽

Feilong Chen ◽

Heling Dong ◽

Minyu Xie ◽

Huan Zhang ◽

...

Keyword(s):

Messenger Rna ◽

Cell Loss ◽

Mutant Mice ◽

Tubular Atrophy ◽

Testis Development ◽

Protein Levels ◽

Conditional Mutant ◽

F Box Protein ◽

And Function ◽

Specific Deletion

Abstract F-box and WD-40 domain protein 7 (Fbxw7) is a component of the Skp1-Cdc53/Cullin-F-box-protein complex (SCF/β-TrCP), which is an E3 ubiquitin ligase that mediates protein degradation. This complex has recently been shown to negatively regulate spermatogonial stem cell self-renewal; however, its roles in Sertoli cell (SC) proliferation, differentiation, and function remain to be established. In this study, we generated conditional mutant mice with SC-specific deletion of Fbxw7 via the Cre-loxP system. Fbxw7 deficiency in SCs impaired testis development, which is characterized by age-dependent tubular atrophy, excessive germ cell loss, and spermatogenic arrest, and the mutant males were infertile at 7 months old. Fbxw7 ablation also compromised cytoskeletal organization and cell polarity of SCs, as well as integrity of the blood-testis barrier. In addition, the transcript levels of cell markers for germ cells, Leydig cells, and SCs were significantly decreased in Fbxw7 mutant mice. Importantly, protein levels of GATA-4, a transcription factor that plays a crucial role in SC maturation and testis development, were progressively decreased in control SCs after postnatal day 14, whereas levels were aberrantly elevated in Fbxw7-deleted SCs. Interestingly, the Gata-4 messenger RNA levels remained stable following Fbxw7 deletion. Fbxw7 silencing in SCs also induced progressive Leydig cell inefficiency and testosterone insufficiency. Collectively, these results demonstrate that Fbxw7 expression is required for SC maturation and function, potentially through degradation of GATA-4, to support pubertal testis development and spermatogenesis.

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Tanycyte-independent control of hypothalamic leptin signaling

10.1101/528794 ◽

2019 ◽

Author(s):

Sooyeon Yoo ◽

David Cha ◽

Dong Won Kim ◽

Thanh V. Hoang ◽

Seth Blackshaw

Keyword(s):

Gene Expression ◽

Single Molecule ◽

Leptin Receptor ◽

Cell Types ◽

Leptin Signaling ◽

And Function ◽

Induced Changes ◽

The Brain ◽

Specific Deletion

AbstractLeptin is secreted by adipocytes to regulate appetite and body weight. Recent studies have reported that tanycytes actively transport circulating leptin across the brain barrier into the hypothalamus, and are required for normal levels of hypothalamic leptin signaling. However, direct evidence for leptin receptor (LepR) expression is lacking, and the effect of tanycyte-specific deletion of LepR has not been investigated. In this study, we analyze the expression and function of the tanycytic LepR in mice. Using single-molecule fluorescent in situ hybridization (smfISH), RT-qPCR, single-cell RNA sequencing (scRNA-Seq), and selective deletion of the LepR in tanycytes, we are unable to detect expression of LepR in the tanycytes. Tanycyte-specific deletion of LepR likewise did not affect leptin-induced pSTAT3 expression in hypothalamic neurons, regardless of whether leptin was delivered by intraperitoneal or intracerebroventricular injection. Finally, we use activity-regulated scRNA-Seq (act-Seq) to comprehensively profile leptin-induced changes in gene expression in all cell types in mediobasal hypothalamus. Clear evidence for leptin signaling is only seen in endothelial cells and subsets of neurons, although virtually all cell types show leptin-induced changes in gene expression. We thus conclude that LepR expression in tanycytes is either absent or undetectably low, that tanycytes do not directly regulate hypothalamic leptin signaling through a LepR-dependent mechanism, and that leptin regulates gene expression in diverse hypothalamic cell types through both direct and indirect mechanisms.

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Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors

10.1101/2020.01.29.924654 ◽

2020 ◽

Author(s):

Shiri Kult ◽

Tsviya Olender ◽

Marco Osterwalder ◽

Sharon Krief ◽

Ronnie Blecher-Gonen ◽

...

Keyword(s):

Transcription Factors ◽

Cell Fate ◽

Single Molecule ◽

Cell Types ◽

Underlying Mechanism ◽

Regulatory Elements ◽

Transcriptional Enhancer ◽

Molecular Identity ◽

And Function

AbstractThe connection between different tissues is vital for the development and function of any organs and systems. In the musculoskeletal system, the attachment of elastic tendons to stiff bones poses a mechanical challenge that is solved by the formation of a transitional tissue, which allows the transfer of muscle forces to the skeleton without tearing. Here, we show that tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, which is regulated by sharing regulatory elements with these cells and by Krüppel-like factors transcription factors (KLF).To uncover the molecular identity of attachment cells, we first applied high-throughput RNA sequencing to murine humeral attachment cells. The results, which were validated by in situ hybridization and single-molecule in situ hybridization, reveal that attachment cells express hundreds of chondrogenic and tenogenic genes. In search for the underlying mechanism allowing these cells to express these genes, we performed ATAC sequencing and found that attachment cells share a significant fraction of accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis further revealed transcriptional enhancer signatures for the majority of these regions. We then examined a subset of these regions using transgenic mouse enhancer reporter. Results verified the shared activity of some of these enhancers, supporting the possibility that the transcriptome of attachment cells is regulated by enhancers with shared activities in tenocytes or chondrocytes. Finally, integrative chromatin and motif analyses, as well as the transcriptome data, indicated that KLFs are regulators of attachment cells. Indeed, blocking the expression of Klf2 and Klf4 in the developing limb mesenchyme led to abnormal differentiation of attachment cells, establishing these factors as key regulators of the fate of these cells.In summary, our findings show how the molecular identity of bi-fated attachment cells enables the formation of the unique transitional tissue that connect tendon to bone. More broadly, we show how mixing the transcriptomes of two cell types through shared enhancers and a dedicated set of transcription factors can lead to the formation of a new cell fate that connects them.

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Defective Ca2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy

The Journal of Cell Biology ◽

10.1083/jcb.200703187 ◽

2007 ◽

Vol 179 (1) ◽

pp. 139-149 ◽

Cited By ~ 100

Author(s):

Sibylle Jablonka ◽

Marcus Beck ◽

Barbara Dorothea Lechner ◽

Christine Mayer ◽

Michael Sendtner

Keyword(s):

Spinal Muscular Atrophy ◽

Growth Cone ◽

Messenger Rna ◽

Muscular Atrophy ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Axon Terminals ◽

Protein Levels ◽

Reduced Frequency ◽

And Function

Proximal spinal muscular atrophy (SMA) is a motoneuron disease for which there is currently no effective treatment. In animal models of SMA, spinal motoneurons exhibit reduced axon elongation and growth cone size. These defects correlate with reduced β-actin messenger RNA and protein levels in distal axons. We show that survival motoneuron gene (Smn)–deficient motoneurons exhibit severe defects in clustering Cav2.2 channels in axonal growth cones. These defects also correlate with a reduced frequency of local Ca2+ transients. In contrast, global spontaneous excitability measured in cell bodies and proximal axons is not reduced. Stimulation of Smn production from the transgenic SMN2 gene by cyclic adenosine monophosphate restores Cav2.2 accumulation and excitability. This may lead to the development of new therapies for SMA that are not focused on enhancing motoneuron survival but instead investigate restoration of growth cone excitability and function.

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Regulation and Function of RNA Pseudouridylation in Human Cells

Annual Review of Genetics ◽

10.1146/annurev-genet-112618-043830 ◽

2020 ◽

Vol 54 (1) ◽

pp. 309-336

Author(s):

Erin K. Borchardt ◽

Nicole M. Martinez ◽

Wendy V. Gilbert

Keyword(s):

Messenger Rna ◽

Noncoding Rna ◽

Protein Production ◽

Rna Binding ◽

Rna Binding Proteins ◽

Human Cells ◽

Rna Sequences ◽

Rna Targets ◽

Pseudouridine Synthases ◽

And Function

Recent advances in pseudouridine detection reveal a complex pseudouridine landscape that includes messenger RNA and diverse classes of noncoding RNA in human cells. The known molecular functions of pseudouridine, which include stabilizing RNA conformations and destabilizing interactions with varied RNA-binding proteins, suggest that RNA pseudouridylation could have widespread effects on RNA metabolism and gene expression. Here, we emphasize how much remains to be learned about the RNA targets of human pseudouridine synthases, their basis for recognizing distinct RNA sequences, and the mechanisms responsible for regulated RNA pseudouridylation. We also examine the roles of noncoding RNA pseudouridylation in splicing and translation and point out the potential effects of mRNA pseudouridylation on protein production, including in the context of therapeutic mRNAs.

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Predominance of spliceosomal complex formation over polyadenylation site selection in TDP-43 autoregulation

Nucleic Acids Research ◽

10.1093/nar/gkt1343 ◽

2013 ◽

Vol 42 (5) ◽

pp. 3362-3371 ◽

Cited By ~ 20

Author(s):

Sara Bembich ◽

Jeremias S. Herzog ◽

Laura De Conti ◽

Cristiana Stuani ◽

S. Eréndira Avendaño-Vázquez ◽

...

Keyword(s):

Complex Formation ◽

Messenger Rna ◽

Protein Production ◽

Rna Binding ◽

Rna Binding Proteins ◽

Alternative Polyadenylation ◽

Cellular Viability ◽

Protein Levels ◽

Polyadenylation Sites ◽

Nucleocytoplasmic Distribution

AbstractTDP-43 is a nuclear protein involved in many aspects of RNA metabolism. To ensure cellular viability, its expression levels within cells must be tightly regulated. We have previously demonstrated that TDP-43 autoregulation occurs through the activation of a normally silent intron in its 3′-UTR sequence that results in the use of alternative polyadenylation sites. In this work, we analyse which is the dominant event in autoregulation: the recognition of the splice sites of 3′-UTR intron 7 or the intrinsic quality of the alternative polyadenylation sites. A panel of minigene constructs was tested for autoregulation functionality, protein production and subcellular messenger RNA localization. Our data clearly indicate that constitutive spliceosome complex formation across intron 7 does not lead to high protein production but, on the contrary, to lower TDP-43 messenger RNA and protein levels. This is due to altered nucleocytoplasmic distribution of the RNA that is mostly retained in the nucleus and degraded. This study provides a novel in-depth characterization of how RNA binding proteins can autoregulate their own levels within cells, an essential regulatory process in maintaining cellular viability.

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Mitotic Centromere-Associated Kinesin (MCAK/KIF2C) Regulates Cell Migration and Invasion by Modulating Microtubule Dynamics and Focal Adhesion Turnover

Cancers ◽

10.3390/cancers13225673 ◽

2021 ◽

Vol 13 (22) ◽

pp. 5673

Author(s):

Ha Hyung Moon ◽

Nina-Naomi Kreis ◽

Alexandra Friemel ◽

Susanne Roth ◽

Dorothea Schulte ◽

...

Keyword(s):

Cell Motility ◽

Molecular Mechanisms ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Focal Adhesions ◽

Protein Composition ◽

Cell Types ◽

Migration And Invasion ◽

And Migration ◽

Cytoskeleton Dynamics

The microtubule (MT) cytoskeleton is crucial for cell motility and migration by regulating multiple cellular activities such as transport and endocytosis of key components of focal adhesions (FA). The kinesin-13 family is important in the regulation of MT dynamics and the best characterized member of this family is the mitotic centromere-associated kinesin (MCAK/KIF2C). Interestingly, its overexpression has been reported to be related to increased metastasis in various tumor entities. Moreover, MCAK is involved in the migration and invasion behavior of various cell types. However, the precise molecular mechanisms were not completely clarified. To address these issues, we generated CRISPR/dCas9 HeLa and retinal pigment epithelium (RPE) cell lines overexpressing or downregulating MCAK. Both up- or downregulation of MCAK led to reduced cell motility and poor migration in malignant as well as benign cells. Specifically, it’s up- or downregulation impaired FA protein composition and phosphorylation status, interfered with a proper spindle and chromosome segregation, disturbed the assembly and disassembly rate of FA, delayed cell adhesion, and compromised the plus-tip dynamics of MTs. In conclusion, our data suggest MCAK act as an important regulator for cell motility and migration by affecting the actin-MT cytoskeleton dynamics and the FA turnover, providing molecular mechanisms by which deregulated MCAK could promote malignant progression and metastasis of tumor cells.

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Smaug1 membrane-less organelles respond to AMPK/mTOR and affect mitochondrial function‡

Journal of Cell Science ◽

10.1242/jcs.253591 ◽

2021 ◽

Author(s):

Ana J. Fernández-Alvarez ◽

María Gabriela Thomas ◽

Malena L. Pascual ◽

Martín Habif ◽

Jerónimo Pimentel ◽

...

Keyword(s):

Single Molecule ◽

Mitochondrial Respiration ◽

Rna Binding ◽

Cell Types ◽

Mitochondrial Enzymes ◽

Mitochondrial Network ◽

Loss Of Function ◽

Mitochondrial Complex ◽

Mitochondrial Uncoupling ◽

Mitochondrial Defects

Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule FISH assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial Complex I inhibition by rotenone –but not strong mitochondrial uncoupling by CCCP– rapidly induced Smaug1 MLOs dissolution. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMPK. Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK/mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins.

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

10.1101/2021.01.27.428525 ◽

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.

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