scholarly journals RNA Gelation in Repeat Expansion Disorders

2017 ◽  
Author(s):  
Ankur Jain ◽  
Ronald D. Vale
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Neuromuscular Disorders ◽  
Cag Repeats ◽  
Live Cells ◽  
Critical Threshold ◽  
Rna Foci ◽  
Repeat Expansions ◽  
Nucleotide Repeats

Expansions of short nucleotide repeats in the protein coding and non-coding regions of >30 genes produce a variety of neurological and neuromuscular disorders including Huntington’s disease (CAG repeats), muscular dystrophy (CTG repeats) and amyotrophic lateral sclerosis (GGGGCC repeats) [1-3]. Expression of expanded repeats alone is sufficient to recapitulate disease pathology in animal models [4-6]. Repeat-containing transcripts accumulate in the nucleus as aberrant “RNA foci” [7-10] and sequester numerous RNA binding proteins [11,12], leading to a disruption of cellular homeostasis [13,14]. Interestingly, RNA foci, as well as the disease symptoms, only manifest at a critical threshold of nucleotide repeats: >30 for CAG/CTG expansions [1] and >7 for the GGGGCC expansion [15]. However, the reason for this characteristic threshold, as well as the molecular mechanism of foci formation, remain unresolved [16]. Here, we show that nucleotide repeat expansions in RNA create templates for multivalent Watson-Crick (CAG/CUG expansions) or Hoogsteen (GGGGCC expansion) base-pairing. These multivalent interactions cause purified RNAs containing repeat expansions to undergo a sol-gel transition and form micron-sized clusters. Reflecting an increase in the valency for intermolecular hybridization, the gelation of purified RNA only occurs above a critical number of trinucleotide or hexanucleotide repeats. These thresholds for in vitro RNA gelation are similar to those associated with manifestation of disease. By visualizing RNA in live cells, we show that nuclear foci form as a result of phase separation of the repeat-containing RNA and that these foci can be dissolved by agents that disrupt RNA gelation in vitro. Analogous to protein aggregation disorders, our results suggest that the sequence-specific gelation of RNAs could be a contributing factor to neurological disease.

scholarly journals Short Tandem Repeat Expansions and RNA-Mediated Pathogenesis in Myotonic Dystrophy

2019 ◽  
Vol 20 (13) ◽  
pp. 3365 ◽  
Author(s):  
Łukasz J. Sznajder ◽  
Maurice S. Swanson
Keyword(s):  
Myotonic Dystrophy ◽  
Tandem Repeat ◽  
Short Tandem Repeat ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Foci ◽  
Repeat Expansions ◽  
Multivalent Interactions ◽  
Short Tandem

Short tandem repeat (STR) or microsatellite, expansions underlie more than 50 hereditary neurological, neuromuscular and other diseases, including myotonic dystrophy types 1 (DM1) and 2 (DM2). Current disease models for DM1 and DM2 propose a common pathomechanism, whereby the transcription of mutant DMPK (DM1) and CNBP (DM2) genes results in the synthesis of CUG and CCUG repeat expansion (CUGexp, CCUGexp) RNAs, respectively. These CUGexp and CCUGexp RNAs are toxic since they promote the assembly of ribonucleoprotein (RNP) complexes or RNA foci, leading to sequestration of Muscleblind-like (MBNL) proteins in the nucleus and global dysregulation of the processing, localization and stability of MBNL target RNAs. STR expansion RNAs also form phase-separated gel-like droplets both in vitro and in transiently transfected cells, implicating RNA-RNA multivalent interactions as drivers of RNA foci formation. Importantly, the nucleation and growth of these nuclear foci and transcript misprocessing are reversible processes and thus amenable to therapeutic intervention. In this review, we provide an overview of potential DM1 and DM2 pathomechanisms, followed by a discussion of MBNL functions in RNA processing and how multivalent interactions between expanded STR RNAs and RNA-binding proteins (RBPs) promote RNA foci assembly.

scholarly journals An integrative study on ribonucleoprotein condensates identifies scaffolding RNAs and reveals a new player in Fragile X-associated Tremor/Ataxia Syndrome

2018 ◽  
Author(s):  
Fernando Cid-Samper ◽  
Mariona Gelabert-Baldrich ◽  
Benjamin Lang ◽  
Nieves Lorenzo-Gotor ◽  
Riccardo Delli Ponti ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Untranslated Regions ◽  
Binding Partners ◽  
Integrative Study ◽  
Splicing Regulator ◽  
Repeat Expansions ◽  
Ataxia Syndrome

SummaryRecent evidence indicates that specific RNAs promote formation of ribonucleoprotein condensates by acting as scaffolds for RNA-binding proteins (RBPs).We systematically investigated RNA-RBP interaction networks to understand ribonucleoprotein assembly. We found that highly-contacted RNAs are highly structured, have long untranslated regions (UTRs) and contain nucleotide repeat expansions. Among the RNAs with such properties, we identified the FMR1 3’ UTR that harbors CGG expansions implicated in Fragile X-associated Tremor/Ataxia Syndrome (FXTAS).We studied FMR1 binding partners in silico and in vitro and prioritized the splicing regulator TRA2A for further characterization. In a FXTAS cellular model we validated TRA2A-FRM1 interaction and investigated implications of its sequestration at both transcriptomic and post-transcriptomic levels. We found that TRA2A co-aggregates with FMR1 in a FXTAS mouse model and in post mortem human samples.Our integrative study identifies key components of ribonucleoprotein aggregates, providing links to neurodegenerative disease and allowing the discovery of new therapeutic targets.

scholarly journals Targeting Toxic Nuclear RNA Foci with CRISPR-Cas13 to Treat Myotonic Dystrophy

2019 ◽  
Author(s):  
Pornthida Poosala ◽  
Sean R. Lindley ◽  
Kelly M. Anderson ◽  
Douglas M. Anderson
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Rna Binding Proteins ◽  
Live Cells ◽  
Nuclear Retention ◽  
Rna Foci ◽  
Nuclear Rna ◽  
Nuclear Foci ◽  
Dystrophia Myotonica Protein Kinase

Human monogenetic diseases can arise from the aberrant expansion of tandem nucleotide repeat sequences, which when transcribed into RNA, can misfold and aggregate into toxic nuclear foci1. Nuclear retention of repeat-containing RNAs can disrupt their normal expression and induce widespread splicing defects by sequestering essential RNA binding proteins. Among the most prevalent of these disorders is myotonic dystrophy type 1 (DM1), a disease occurring from the expression of a noncoding CTG repeat expansion in the 3’UTR of the human dystrophia myotonica protein kinase (DMPK) gene2,3. Here we show that RNA-binding CRISPR-Cas13, with a robust non-classical nuclear localization signal, can be efficiently targeted to toxic nuclear RNA foci for either visualization or cleavage, tools we named hilightR and eraseR, respectively. HilightR combines catalytically dead Cas13b (dCas13b) with a fluorescent protein to directly visualize CUG repeat RNA foci in the nucleus of live cells, allowing for quantification of foci number and observation of foci dynamics. EraseR utilizes the intrinsic endoribonuclease activity of Cas13b, targeted to nuclear CUG repeat RNA, to disrupt nuclear foci. These studies demonstrate the potential for targeting toxic nuclear RNA foci directly with CRISPR-Cas13 for either the identification or treatment of DM1. The efficient and sequence programmable nature of CRISPR-Cas13 systems will allow for rapid targeting and manipulation of other human nuclear RNA disorders, without the associated risks of genome editing.

The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins

1991 ◽  
Vol 11 (2) ◽  
pp. 894-905
Author(s):  
R A Voelker ◽  
W Gibson ◽  
J P Graves ◽  
J F Sterling ◽  
M T Eisenberg
Keyword(s):  
Rna Processing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Rna Recognition Motif ◽  
Reading Frame ◽  
Cdna Sequences ◽  
Suppressor Of Sable

The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses.

scholarly journals Identification of mRNAs Associated with αCP2-Containing RNP Complexes

2003 ◽  
Vol 23 (19) ◽  
pp. 7055-7067 ◽  
Author(s):  
Shelly A. Waggoner ◽  
Stephen A. Liebhaber
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Cell Function ◽  
Rna Binding Proteins ◽  
Translation Control ◽  
Viral Mrnas ◽  
Posttranscriptional Gene Regulation ◽  
Direct Binding

ABSTRACT Posttranscriptional controls in higher eukaryotes are central to cell differentiation and developmental programs. These controls reflect sequence-specific interactions of mRNAs with one or more RNA binding proteins. The α-globin poly(C) binding proteins (αCPs) comprise a highly abundant subset of K homology (KH) domain RNA binding proteins and have a characteristic preference for binding single-stranded C-rich motifs. αCPs have been implicated in translation control and stabilization of multiple cellular and viral mRNAs. To explore the full contribution of αCPs to cell function, we have identified a set of mRNAs that associate in vivo with the major αCP2 isoforms. One hundred sixty mRNA species were consistently identified in three independent analyses of αCP2-RNP complexes immunopurified from a human hematopoietic cell line (K562). These mRNAs could be grouped into subsets encoding cytoskeletal components, transcription factors, proto-oncogenes, and cell signaling factors. Two mRNAs were linked to ceroid lipofuscinosis, indicating a potential role for αCP2 in this infantile neurodegenerative disease. Surprisingly, αCP2 mRNA itself was represented in αCP2-RNP complexes, suggesting autoregulatory control of αCP2 expression. In vitro analyses of representative target mRNAs confirmed direct binding of αCP2 within their 3′ untranslated regions. These data expand the list of mRNAs that associate with αCP2 in vivo and establish a foundation for modeling its role in coordinating pathways of posttranscriptional gene regulation.

scholarly journals Function of Pumilio Genes in Human Embryonic Stem Cells and Their Effect in Stemness and Cardiomyogenesis

2019 ◽  
Author(s):  
Isabelle Leticia Zaboroski Silva ◽  
Anny Waloski Robert ◽  
Guillermo Cabrera Cabo ◽  
Lucia Spangenberg ◽  
Marco Augusto Stimamiglio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Embryonic Stem ◽  
Mrna Levels ◽  
Human Escs ◽  
Mrna Targets ◽  
Cardiomyogenic Differentiation

AbstractPosttranscriptional regulation plays a fundamental role in the biology of embryonic stem cells (ESCs). Many studies have demonstrated that multiple mRNAs are coregulated by one or more RNA binding proteins (RBPs) that orchestrate the expression of these molecules. A family of RBPs, known as PUF (Pumilio-FBF), is highly conserved among species and has been associated with the undifferentiated and differentiated states of different cell lines. In humans, two homologs of the PUF family have been found: Pumilio 1 (PUM1) and Pumilio 2 (PUM2). To understand the role of these proteins in human ESCs (hESCs), we first demonstrated the influence of the silencing of PUM1 and PUM2 on pluripotency genes. OCT4 and NANOG mRNA levels decreased significantly with the knockdown of Pumilio, suggesting that PUMILIO proteins play a role in the maintenance of pluripotency in hESCs. Furthermore, we observed that the hESCs silenced for PUM1 and 2 exhibited an improvement in efficiency of in vitro cardiomyogenic differentiation. Using in silico analysis, we identified mRNA targets of PUM1 and PUM2 expressed during cardiomyogenesis. With the reduction of PUM1 and 2, these target mRNAs would be active and could be involved in the progression of cardiomyogenesis.

scholarly journals SSMART: Sequence-structure motif identification for RNA-binding proteins

2018 ◽  
Author(s):  
Alina Munteanu ◽  
Neelanjan Mukherjee ◽  
Uwe Ohler
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Sequence Motif ◽  
Primary Sequence ◽  
Sequence Structure ◽  
Rna Targets

AbstractMotivationRNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. There are hundreds of RBPs encoded in the eukaryotic genomes, and each recognize its RNA targets through a specific mixture of RNA sequence and structure properties. For most RBPs, however, only a primary sequence motif has been determined, while the structure of the binding sites is uncharacterized.ResultsWe developed SSMART, an RNA motif finder that simultaneously models the primary sequence and the structural properties of the RNA targets sites. The sequence-structure motifs are represented as consensus strings over a degenerate alphabet, extending the IUPAC codes for nucleotides to account for secondary structure preferences. Evaluation on synthetic data showed that SSMART is able to recover both sequence and structure motifs implanted into 3‘UTR-like sequences, for various degrees of structured/unstructured binding sites. In addition, we successfully used SSMART on high-throughput in vivo and in vitro data, showing that we not only recover the known sequence motif, but also gain insight into the structural preferences of the RBP.AvailabilitySSMART is freely available at https://ohlerlab.mdc-berlin.de/software/SSMART_137/[email protected]

scholarly journals Fluorescence Correlation and Cross-Correlation Spectroscopy Unveil Cytoplasmic mRNP Composition and Dynamics

2020 ◽  
Author(s):  
Àngels Mateu-Regué ◽  
Jan Christiansen ◽  
Christian Hellriegel ◽  
Finn Cilius Nielsen
Keyword(s):  
Life Cycle ◽  
Dynamic Analysis ◽  
Cross Correlation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Macromolecular Complexes ◽  
Fluorescence Correlation ◽  
Macromolecular Composition

ABSTRACTUnderstanding the mRNA life cycle requires analysis of the dynamic macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study mRNP complexes because they readily provide information about the molecular composition, stoichiometry, heterogeneity and dynamics of the particles. We developed FCS protocols for analysis of live cells and cellular lysates, and demonstrate the feasibility of analysing common cytoplasmic mRNPs composed of core factor YBX1, IMPs (or IGF2BPs) and their interactions with other RNA binding proteins such as PABPC1, ELAVL2 (HuB), STAU1 and FMRP. FCCS corroborated previously reported RNA dependent interactions between the factors and provided an estimate of the relative overlap between the factors in the mRNPs. In this way FCS and FCCS provide a new and useful approach for the quantitative and dynamic analysis of mRNP macromolecular complexes that may complement current biochemical approaches.

scholarly journals Distinct expression of select and transcriptome-wide isolated 3’UTRs suggests critical roles in development and transition states

2020 ◽  
Author(s):  
Shaoyi Ji ◽  
Ze Yang ◽  
Leonardi Gozali ◽  
Thomas Kenney ◽  
Arif Kocabas ◽  
...  
Keyword(s):  
Gene Ontology ◽  
Gene Regulation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Gene ◽  
Coding Region ◽  
Proliferating Cells ◽  
Coding Regions ◽  
Micro Rnas

AbstractMature mRNA molecules are typically considered to be comprised of a 5’UTR, a 3’UTR and a coding region (CDS), all attached until degradation. Unexpectedly, however, there have been multiple recent reports of widespread differential expression of mRNA 3’UTRs and their cognate coding regions, resulting in the expression of isolated 3’UTRs (i3’UTRs); these i3’UTRs can be highly expressed, often in reciprocal patterns to their cognate CDS. Similar to the role of other lncRNAs, isolated 3’UTRs are likely to play an important role in gene regulation but little is known about the contexts in which they are deployed. To begin to parse the functions of i3’UTRs, here we carry out in vitro, in vivo and in silico analyses of differential 3’UTR/CDS mRNA ratio usage across tissues, development and cell state changes both for a select list of developmentally important genes as well as through unbiased transcriptome-wide analyses. Across two developmental paradigms we find a distinct switch from high i3’UTR expression of stem cell related genes in proliferating cells compared to newly differentiated cells. Our unbiased transcriptome analysis across multiple gene sets shows that regardless of tissue, genes with high 3’UTR to CDS ratios belong predominantly to gene ontology categories related to cell-type specific functions while in contrast, the gene ontology categories of genes with low 3’UTR to CDS ratios are similar and relate to common cellular functions. In addition to these specific findings our data provide critical information from which detailed hypotheses for individual i3’UTRs can be tested-with a common theme that i3’UTRs appear poised to regulate cell-specific gene expression and state.Significance StatementThe widespread existence and expression of mRNA 3’ untranslated sequences in the absence of their cognate coding regions (called isolated 3’UTRs or i3’UTRs) opens up considerable avenues for gene regulation not previously envisioned. Each isolated 3’UTR may still bind and interact with micro RNAs, RNA binding proteins as well as other nucleic acid sequences, all in the absence or low levels of cognate protein production. Here we document the expression, localization and regulation of i3’UTRs both within particular biological systems as well as across the transcriptome. As this is an entirely new area of experimental investigation these early studies are seminal to this burgeoning field.

A synthetic RNA editing factor edits its target site in chloroplasts and bacteria

2020 ◽  
Author(s):  
Santana Royan ◽  
Bernard Gutmann ◽  
Catherine Colas des Francs-Small ◽  
Suvi Honkanen ◽  
Jason Schmidberger ◽  
...  
Keyword(s):  
Rna Editing ◽  
Protein Interactions ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Rna Binding Proteins ◽  
Cytidine Deaminase ◽  
Land Plant ◽  
Target Sequence ◽  
Pentatricopeptide Repeat

Abstract Targeted cytidine to uridine RNA editing is a widespread phenomenon throughout the land plant lineage. Members of the pentatricopeptide repeat (PPR) protein family act as the specificity factors in this process. These proteins consist of helix-turn-helix domains, each of which recognises a single RNA nucleotide following a well-elucidated code. A cytidine deaminase-like domain (present at the C-terminus of some PPR editing factors or provided in trans via protein-protein interactions) is the catalytic domain in the process. The huge expansion of the PPR superfamily in land plants provides the sequence variation required for design of novel consensus-based RNA-binding proteins. We used this approach to construct a synthetic RNA editing factor designed to target one of the two sites in the Arabidopsis chloroplast transcriptome naturally recognised by the RNA editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that this designed editing factor specifically recognises the target sequence in in vitro binding assays and can partially complement a clb19 mutant. The designed factor is specific for the target rpoA site and does not recognise or edit the other site recognised by CLB19 in the clpP1 transcript. We show that the designed editing factor can function equally specifically in the bacterium E. coli, and shows some activity even in the absence of the editing cofactors that are often required for natural editing factor activity in plants. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.

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