scholarly journals La Deletion from Mouse Brain Alters Pre-tRNA Metabolism and Accumulation of Pre-5.8S rRNA, with Neuron Death and Reactive Astrocytosis

2017 ◽  
Vol 37 (10) ◽  
Author(s):  
Nathan H. Blewett ◽  
James R. Iben ◽  
Sergei Gaidamakov ◽  
Richard J. Maraia
Keyword(s):  
Rna Binding ◽  
Rna Polymerase Iii ◽  
Neuronal Loss ◽  
Fold Increase ◽  
Nuclear Accumulation ◽  
Neuron Death ◽  
Brain Mass ◽  
5.8S Rrna ◽  
La Antigen ◽  
Progressive Neurodegeneration

ABSTRACT Human La antigen (Sjögren's syndrome antigen B [SSB]) is an abundant multifunctional RNA-binding protein. In the nucleoplasm, La binds to and protects from 3′ exonucleases, the ends of precursor tRNAs, and other transcripts synthesized by RNA polymerase III and facilitates their maturation, while a nucleolar isoform has been implicated in rRNA biogenesis by multiple independent lines of evidence. We showed previously that conditional La knockout (La cKO) from mouse cortex neurons results in defective tRNA processing, although the pathway(s) involved in neuronal loss thereafter was unknown. Here, we demonstrate that La is stably associated with a spliced pre-tRNA intermediate. Microscopic evidence of aberrant nuclear accumulation of 5.8S rRNA in La cKO is supported by a 10-fold increase in a pre-5.8S rRNA intermediate. To identify pathways involved in subsequent neurodegeneration and loss of brain mass in the cKO cortex, we employed mRNA sequencing (mRNA-Seq), immunohistochemistry, and other approaches. This revealed robust enrichment of immune and astrocyte reactivity in La cKO cortex. Immunohistochemistry, including temporal analyses, demonstrated neurodegeneration, followed by astrocyte invasion associated with immune response and decreasing cKO cortex size over time. Thus, deletion of La from postmitotic neurons results in defective pre-tRNA and pre-rRNA processing and progressive neurodegeneration with loss of cortical brain mass.

scholarly journals 5′ Processing of tRNA Precursors Can Be Modulated by the Human La Antigen Phosphoprotein

1998 ◽  
Vol 18 (6) ◽  
pp. 3201-3211 ◽  
Author(s):  
Hao Fan ◽  
John L. Goodier ◽  
Joel R. Chamberlain ◽  
David R. Engelke ◽  
Richard J. Maraia
Keyword(s):  
Rna Binding ◽  
Rna Polymerase Iii ◽  
Binding Domain ◽  
Sequence Motif ◽  
Polymerase Iii ◽  
La Protein ◽  
La Antigen ◽  
Rna Binding Domain ◽  
Nascent Transcripts ◽  
Basic Region

ABSTRACT Eukaryotic precursor (pre)-tRNAs are processed at both ends prior to maturation. Pre-tRNAs and other nascent transcripts synthesized by RNA polymerase III are bound at their 3′ ends at the sequence motif UUUOH [3′ oligo(U)] by the La antigen, a conserved phosphoprotein whose role in RNA processing has been associated previously with 3′-end maturation only. We show that in addition to its role in tRNA 3′-end maturation, human La protein can also modulate 5′ processing of pre-tRNAs. Both the La antigen’s N-terminal RNA-binding domain and its C-terminal basic region are required for attenuation of pre-tRNA 5′ processing. RNA binding and nuclease protection assays with a variety of pre-tRNA substrates and mutant La proteins indicate that 5′ protection is a highly selective activity of La. This activity is dependent on 3′ oligo(U) in the pre-tRNA for interaction with the N-terminal RNA binding domain of La and interaction of the C-terminal basic region of La with the 5′ triphosphate end of nascent pre-tRNA. Phosphorylation of La is known to occur on serine 366, adjacent to the C-terminal basic region. We show that this modification interferes with the La antigen’s ability to protect pre-tRNAi Met from 5′ processing either by HeLa extract or purified RNase P but that it does not affect interaction with the 3′ end of pre-tRNA. These findings provide the first evidence to indicate that tRNA 5′-end maturation may be regulated in eukaryotes. Implications of triphosphate recognition is discussed as is a role for La phosphoprotein in controlling transcriptional and posttranscriptional events in the biogenesis of polymerase III transcripts.

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.

Characterization and purification of lupus antigen La, and RNA-binding protein

1985 ◽  
Vol 5 (3) ◽  
pp. 586-590
Author(s):  
A M Francoeur ◽  
E K Chan ◽  
J I Garrels ◽  
M B Mathews
Keyword(s):  
Hela Cell ◽  
Gel Electrophoresis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
In Vitro Translation ◽  
Cell Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
La Antigen

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.

Eukaryotic transcription termination factor La mediates transcript release and facilitates reinitiation by RNA polymerase III

1994 ◽  
Vol 14 (3) ◽  
pp. 2147-2158
Author(s):  
R J Maraia ◽  
D J Kenan ◽  
J D Keene
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
Class Iii ◽  
Ample Evidence ◽  
Polymerase Iii ◽  
Transcription Complexes ◽  
Pol Iii

Ample evidence indicates that Alu family interspersed elements retrotranspose via primary transcripts synthesized by RNA polymerase III (pol III) and that this transposition sometimes results in genetic disorders in humans. However, Alu primary transcripts can be processed posttranscriptionally, diverting them away from the transposition pathway. The pol III termination signal of a well-characterized murine B1 (Alu-equivalent) element inhibits RNA 3' processing, thereby stabilizing the putative transposition intermediary. We used an immobilized template-based assay to examine transcription termination by VA1, 7SL, and Alu class III templates and the role of transcript release in the pol III terminator-dependent inhibition of processing of B1-Alu transcripts. We found that the RNA-binding protein La confers this terminator-dependent 3' processing inhibition on transcripts released from the B1-Alu template. Using pure recombinant La protein and affinity-purified transcription complexes, we also demonstrate that La facilitates multiple rounds of transcription reinitiation by pol III. These results illustrate an important role for La in RNA production by demonstrating its ability to clear the termination sites of class III templates, thereby promoting efficient use of transcription complexes by pol III. The role of La as a potential regulatory factor in transcript maturation and how this might apply to Alu interspersed elements is discussed.

C25, an essential RNA polymerase III subunit related to the RNA polymerase II subunit RPB7

1994 ◽  
Vol 14 (9) ◽  
pp. 6164-6170
Author(s):  
P P Sadhale ◽  
N A Woychik
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rna Polymerase Iii ◽  
Sodium Dodecyl ◽  
Polymerase Iii ◽  
Conditional Mutant ◽  
5.8S Rrna ◽  
Cell Growth And Viability

We identified a partially sequenced Saccharomyces cerevisiae gene which encodes a protein related to the S. cerevisiae RNA polymerase II subunit, RPB7. Several lines of evidence suggest that this related gene, YKL1, encodes the RNA polymerase III subunit C25. C25, like RPB7, is present in submolar ratios, easily dissociates from the enzyme, is essential for cell growth and viability, but is not required in certain transcription assays in vitro. YKL1 has ABF-1 and PAC upstream sequences often present in RNA polymerase subunit genes. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of the YKL1 gene product is equivalent to that of the RNA polymerase III subunit C25. Finally, a C25 conditional mutant grown at the nonpermissive temperature synthesizes tRNA at reduced rates relative to 5.8S rRNA, a hallmark of all characterized RNA polymerase III mutants.

scholarly journals An interdomain bridge influences RNA binding of the human La protein

2018 ◽  
Vol 294 (5) ◽  
pp. 1529-1540 ◽  
Author(s):  
Stefano A. Marrella ◽  
Kerene A. Brown ◽  
Farnaz Mansouri-Noori ◽  
Jennifer Porat ◽  
Derek J. Wilson ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Polymerase Iii ◽  
Conformational Dynamics ◽  
Direct Interaction ◽  
Rna Recognition Motif ◽  
Binding Modes ◽  
Nuclear Retention ◽  
Time Resolved ◽  
La Protein ◽  
C Terminus

La proteins are RNA chaperones that perform various functions depending on distinct RNA-binding modes and their subcellular localization. In the nucleus, they help process UUU-3′OH–tailed nascent RNA polymerase III transcripts, such as pre-tRNAs, whereas in the cytoplasm they contribute to translation of poly(A)-tailed mRNAs. La accumulation in the nucleus and cytoplasm is controlled by several trafficking elements, including a canonical nuclear localization signal in the extreme C terminus and a nuclear retention element (NRE) in the RNA recognition motif 2 (RRM2) domain. Previous findings indicate that cytoplasmic export of La due to mutation of the NRE can be suppressed by mutations in RRM1, but the mechanism by which the RRM1 and RRM2 domains functionally cooperate is poorly understood. In this work, we use electromobility shift assays (EMSA) to show that mutations in the NRE and RRM1 affect binding of human La to pre-tRNAs but not UUU-3′OH or poly(A) sequences, and we present compensatory mutagenesis data supporting a direct interaction between the RRM1 and RRM2 domains. Moreover, we use collision-induced unfolding and time-resolved hydrogen–deuterium exchange MS analyses to study the conformational dynamics that occur when this interaction is intact or disrupted. Our results suggest that the intracellular distribution of La may be linked to its RNA-binding modes and provide the first evidence for a direct protein–protein interdomain interaction in La proteins.

scholarly journals Transcriptomic analysis of frontotemporal lobar degeneration with TDP-43 pathology reveals cellular alterations across multiple brain regions

2021 ◽  
Author(s):  
Rahat Hasan ◽  
Jack Humphrey ◽  
Conceicao Bettencourt ◽  
Tammaryn Lashley ◽  
Pietro Fratta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Frontotemporal Lobar Degeneration ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Temporal Cortex ◽  
Neuronal Loss ◽  
Underlying Disease ◽  
Brain Regions ◽  
Neuronal Markers ◽  
The Brain

Frontotemporal lobar degeneration (FTLD) is a group of heterogeneous neurodegenerative disorders affecting the frontal and temporal lobes of the brain. Nuclear loss and cytoplasmic aggregation of the RNA-binding protein TDP-43 represents the major FTLD pathology, known as FTLD-TDP. To date, there is no effective treatment for FTLD-TDP due to an incomplete understanding of the molecular mechanisms underlying disease development. Here we compared post-mortem tissue RNA-seq transcriptomes from the frontal cortex, temporal cortex and cerebellum between 28 controls and 30 FTLD-TDP patients to profile changes in cell-type composition, gene expression and transcript usage. We observed downregulation of neuronal markers in all three regions of the brain, accompanied by upregulation of microglia, astrocytes, and oligodendrocytes, as well as endothelial cells and pericytes, suggesting shifts in both immune activation and within the vasculature. We validate our estimates of neuronal loss using neuropathological atrophy scores and show that neuronal loss in the cortex can be mainly attributed to excitatory neurons, and that increases in microglial and endothelial cell expression are highly correlated with neuronal loss. All our analyses identified a strong involvement of the cerebellum in the neurodegenerative process of FTLD-TDP. Altogether, our data provides a detailed landscape of gene expression alterations to help unravel relevant disease mechanisms in FTLD.

scholarly journals Cytoplasmic accumulation of FUS triggers early behavioral alterations linked to cortical neuronal hyperactivity and defects in inhibitory synapses

2020 ◽  
Author(s):  
Jelena Scekic-Zahirovic ◽  
Inmaculada Sanjuan-Ruiz ◽  
Vanessa Kan ◽  
Salim Megat ◽  
Pierre De Rossi ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Rna Binding ◽  
Gene Mutations ◽  
Neuronal Loss ◽  
Inhibitory Neurons ◽  
Inhibitory Synapses ◽  
Behavioral Alterations ◽  
Behavioral Abnormalities ◽  
Cytoplasmic Accumulation

AbstractGene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS, lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a profound increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo. Importantly, RNAseq analysis suggested involvement of defects in inhibitory neurons, that was confirmed by ultrastructural and morphological defects of inhibitory synapses and increased synaptosomal levels of mRNAs involved in inhibitory neurotransmission. Thus, cytoplasmic FUS triggers inhibitory synaptic deficits, leading to increased neuronal activity and behavioral phenotypes. FUS mislocalization may trigger deleterious phenotypes beyond motor neuron impairment in ALS, but also in other neurodegenerative diseases with FUS mislocalization.

scholarly journals Extracellular CIRP Activates the IL-6Rα/STAT3/Cdk5 Pathway in Neurons

2021 ◽  
Author(s):  
Archna Sharma ◽  
Max Brenner ◽  
Asha Jacob ◽  
Philippe Marambaud ◽  
Ping Wang
Keyword(s):  
Rna Binding ◽  
Neuronal Damage ◽  
Fold Increase ◽  
Time Dependent ◽  
Dependent Manner ◽  
N2a Cells ◽  
Stat3 Phosphorylation ◽  
Bv2 Cells ◽  
Equilibrium Dissociation ◽  
Bv2 Microglial Cells

Abstract Extracellular cold-inducible RNA-binding protein (eCIRP) stimulates microglial inflammation causing neuronal damage during ischemic stroke and is a critical mediator of alcohol-induced cognitive impairment. However, the precise role of eCIRP in mediating neuroinflammation remains unknown. In this study, we report that eCIRP activates neurotoxic cyclin-dependent kinase-5 (Cdk5)/p25 through the induction of IL-6Rα/STAT3 pathway in neurons. Amyloid b (Aβ)-mediated neuronal stress, which is associated with Alzheimer’s disease, increased levels of eCIRP released from BV2 microglial cells. The released eCIRP levels from BV2 cells increased 3.2-fold upon stimulation with conditioned medium from Neuro-2a (N2a) cells containing Aβ compared to control N2a supernatant in a time-dependent manner. Stimulation of N2a cells and primary neurons with eCIRP upregulated the neuronal Cdk5 activator p25 expression in a dose- and time-dependent manner. eCIRP directly induced neuronal STAT3 phosphorylation and p25 increase via its novel receptor IL-6Rα. Next, we showed using surface plasmon resonance that eCIRP-derived peptide C23 inhibited the binding of eCIRP to IL-6Rα at 25 mM, with a 40-fold increase in equilibrium dissociation constant (Kd) value (from 8.08 x 10-8 M to 3.43 x 10-6 M), and completely abrogated the binding at 50 mM. Finally, C23 reversed the eCIRP-induced increase in neuronal STAT3 phosphorylation and p25 levels. In conclusion, the current study demonstrates that upregulation of neuronal IL-6Rα/STAT3/Cdk5 pathway is a key mechanism of eCIRP’s role in neuroinflammation and that C23 as a potent inhibitor of this pathway, has translational potential in neurodegenerative pathologies controlled by eCIRP.

Analysis of behavior using genetical genomics in mice as a model: from alcohol preferences to gene expression differences

Genome ◽  
2007 ◽  
Vol 50 (10) ◽  
pp. 877-897 ◽  
Author(s):  
Shiva M. Singh ◽  
Julie Treadwell ◽  
Morgan L. Kleiber ◽  
Michelle Harrison ◽  
Raihan K. Uddin
Keyword(s):  
Gene Expression ◽  
Rna Binding ◽  
Human Subjects ◽  
Fold Increase ◽  
Global Gene Expression ◽  
Genetic Dissection ◽  
Behavioral Phenotypes ◽  
Neurotransmitter Transport ◽  
Genetic Strains ◽  
Different Strains

Most familial behavioral phenotypes result from the complex interaction of multiple genes. Studies of such phenotypes involving human subjects are often inconclusive owing to complexity of causation and experimental limitations. Studies of animal models argue for the use of established genetic strains as a powerful tool for genetic dissection of behavioral disorders and have led to the identification of rare genes and genetic mechanisms implicated in such phenotypes. We have used microarrays to study global gene expression in adult brains of four genetic strains of mice (C57BL/6J, DBA/2J, A/J, and BALB/c). Our results demonstrate that different strains show expression differences for a number of genes in the brain, and that closely related strains have similar patterns of gene expression as compared with distantly related strains. In addition, among the 24 000 genes and ESTs on the microarray, 77 showed at least a 1.5-fold increase in the brains of C57BL/6J mice as compared with those of DBA/2J mice. These genes fall into such functional categories as gene regulation, metabolism, cell signaling, neurotransmitter transport, and DNA/RNA binding. The importance of these findings as a novel genetic resource and their use and application in the genetic analysis of complex behavioral phenotypes, susceptibilities, and responses to drugs and chemicals are discussed.

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