Structural Diversity of Sense and Antisense RNA Hexanucleotide Repeats Associated with ALS and FTLD

The hexanucleotide expansion GGGGCC located in C9orf72 gene represents the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). Since the discovery one of the non-exclusive mechanisms of expanded hexanucleotide G4C2 repeats involved in ALS and FTLD is RNA toxicity, which involves accumulation of pathological sense and antisense RNA transcripts. Formed RNA foci sequester RNA-binding proteins, causing their mislocalization and, thus, diminishing their biological function. Therefore, structures adopted by pathological RNA transcripts could have a key role in pathogenesis of ALS and FTLD. Utilizing NMR spectroscopy and complementary methods, we examined structures adopted by both guanine-rich sense and cytosine-rich antisense RNA oligonucleotides with four hexanucleotide repeats. While both oligonucleotides tend to form dimers and hairpins, the equilibrium of these structures differs with antisense oligonucleotide being more sensitive to changes in pH and sense oligonucleotide to temperature. In the presence of K+ ions, guanine-rich sense RNA oligonucleotide also adopts secondary structures called G-quadruplexes. Here, we also observed, for the first time, that antisense RNA oligonucleotide forms i-motifs under specific conditions. Moreover, simultaneous presence of sense and antisense RNA oligonucleotides promotes formation of heterodimer. Studied structural diversity of sense and antisense RNA transcripts not only further depicts the complex nature of neurodegenerative diseases but also reveals potential targets for drug design in treatment of ALS and FTLD.

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Architecture of RNA influences plant biology

Journal of Experimental Botany ◽

10.1093/jxb/erab030 ◽

2021 ◽

Author(s):

Jiaying Zhu ◽

Changhao Li ◽

Xu Peng ◽

Xiuren Zhang

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Mirna Biogenesis ◽

Plant Responses ◽

Tertiary Structures ◽

Rna Transcripts ◽

Environmental Variations ◽

Living Organisms ◽

Processing Stability

Abstract The majority of the genome is transcribed to RNA in living organisms. RNA transcripts can form astonishing arrays of secondary and tertiary structures via Watson-Crick, Hoogsteen or wobble base pairing. In vivo, RNA folding is not a simple thermodynamics event of minimizing free energy. Instead, the process is constrained by transcription, RNA binding proteins (RBPs), steric factors and micro-environment. RNA secondary structure (RSS) plays myriad roles in numerous biological processes, such as RNA processing, stability, transportation and translation in prokaryotes and eukaryotes. Emerging evidence has also implicated RSS in RNA trafficking, liquid-liquid phase separation and plant responses to environmental variations such as temperature and salinity. At the molecular level, RSS is correlated with regulating splicing, polyadenylation, protein systhsis, and miRNA biogenesis and functions. In this review, we summarized newly reported methods for probing RSS in vivo and functions and mechanisms of RSS in plant physiology.

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ALBA proteins are stage regulated during trypanosome development in the tsetse fly and participate in differentiation

Molecular Biology of the Cell ◽

10.1091/mbc.e11-06-0511 ◽

2011 ◽

Vol 22 (22) ◽

pp. 4205-4219 ◽

Cited By ~ 70

Author(s):

Ines Subota ◽

Brice Rotureau ◽

Thierry Blisnick ◽

Sandra Ngwabyt ◽

Mickaël Durand-Dubief ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Fluorescent Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Protozoan Parasite ◽

Tsetse Fly ◽

First Time

The protozoan parasite Trypanosoma brucei is responsible for sleeping sickness and alternates between mammal and tsetse fly hosts, where it has to adapt to different environments. We investigated the role of two members of the ALBA family, which encodes hypothetical RNA-binding proteins conserved in most eukaryotes. We show that ALBA3/4 proteins colocalize with the DHH1 RNA-binding protein and with a subset of poly(A+) RNA in stress granules upon starvation. Depletion of ALBA3/4 proteins by RNA interference in the cultured procyclic stage produces cell modifications mimicking several morphogenetic aspects of trypanosome differentiation that usually take place in the fly midgut. A combination of immunofluorescence data and videomicroscopy analysis of live trypanosomes expressing endogenously ALBA fused with fluorescent proteins revealed that ALBA3/4 are present throughout the development of the parasite in the tsetse fly, with the striking exception of the transition stages found in the proventriculus region. This involves migration of the nucleus toward the posterior end of the cell, a phenomenon that is perturbed upon forced expression of ALBA3 during the differentiation process, showing for the first time the involvement of an RNA-binding protein in trypanosome development in vivo.

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Post-Transcriptional Regulation of Gnrhr: A Checkpoint for Metabolic Control of Female Reproduction

International Journal of Molecular Sciences ◽

10.3390/ijms22073312 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3312

Author(s):

Angela K. Odle ◽

Melanie C. MacNicol ◽

Gwen V. Childs ◽

Angus M. MacNicol

Keyword(s):

Hormone Receptors ◽

Rna Binding ◽

Rna Binding Proteins ◽

Transcriptional Regulators ◽

Receptor Expression ◽

Reproductive Capacity ◽

Complex Nature ◽

Female Reproduction ◽

Normal Pattern ◽

Post Transcriptional Regulation

The proper expression of gonadotropin-releasing hormone receptors (GnRHRs) by pituitary gonadotropes is critical for maintaining maximum reproductive capacity. GnRH receptor expression must be tightly regulated in order to maintain the normal pattern of expression through the estrous cycle in rodents, which is believed to be important for interpreting the finely tuned pulses of GnRH from the hypothalamus. Much work has shown that Gnrhr expression is heavily regulated at the level of transcription. However, researchers have also discovered that Gnrhr is regulated post-transcriptionally. This review will discuss how RNA-binding proteins and microRNAs may play critical roles in the regulation of GnRHR expression. We will also discuss how these post-transcriptional regulators may themselves be affected by metabolic cues, specifically with regards to the adipokine leptin. All together, we present evidence that Gnrhr is regulated post-transcriptionally, and that this concept must be further explored in order to fully understand the complex nature of this receptor.

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CLUH interactome reveals an association to SPAG5 and a proximity to the translation of mitochondrial protein

10.1101/2021.07.08.451585 ◽

2021 ◽

Author(s):

Mickaële Hemono ◽

Alexandre Haller ◽

Johana Chicher ◽

Anne-Marie Duchêne ◽

Richard Patryk Ngondo

Keyword(s):

Mammalian Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mitochondrial Protein ◽

Embryonic Stem ◽

Nuclear Genome ◽

Mrna Translation ◽

Mitochondrial Proteins ◽

Molecular Function ◽

First Time

Mitochondria require thousands of proteins to fulfil their essential function in energy production and other fundamental biological processes. These proteins are mostly encoded by the nuclear genome, translated in the cytoplasm before being imported into the organelle. RNA binding proteins (RBPs) are central players in the regulation of this process by affecting mRNA translation, stability or localization. CLUH is an RBP recognizing specifically mRNAs coding for mitochondrial proteins, but its precise molecular function and interacting partners remain undiscovered in mammals. Here we reveal for the first time CLUH interactome in mammalian cells. Using both co-IP and BioID proximity-labeling approaches, we identify novel molecular partners interacting stably or transiently with CLUH in HCT116 cells and mouse embryonic stem cells. We reveal a stable RNA-independent interaction of CLUH with itself and with SPAG5 in cytosolic granular structures. More importantly, we uncover an unexpected proximity of CLUH to mitochondrial proteins and their cognate mRNAs in the cytosol. Additionally, our data highlight the importance of CLUH TPR domain for its interactions with both proteins and mRNAs. Overall, through the analysis of CLUH interactome, our study sheds a new light on CLUH molecular function by highlighting its association to the translation and subcellular localization of some mRNAs coding for mitochondrial proteins.

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SFPQ regulates the accumulation of RNA foci and dipeptide repeat proteins from the expanded repeat mutation in C9orf72

Journal of Cell Science ◽

10.1242/jcs.256602 ◽

2021 ◽

Vol 134 (4) ◽

pp. jcs256602 ◽

Cited By ~ 1

Author(s):

Mirjana Malnar ◽

Boris Rogelj

Keyword(s):

Antisense Rna ◽

Rna Binding ◽

Dipeptide Repeat Proteins ◽

Rna Transcripts ◽

Repeat Proteins ◽

Rna Foci ◽

Hek Cells ◽

Lateral Sclerosis ◽

Dipeptide Repeat

ABSTRACTThe expanded GGGGCC repeat mutation in the C9orf72 gene is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion is transcribed to sense and antisense RNA, which form RNA foci and bind cellular proteins. This mechanism of action is considered cytotoxic. Translation of the expanded RNA transcripts also leads to the accumulation of toxic dipeptide repeat proteins (DPRs). The RNA-binding protein splicing factor proline and glutamine rich (SFPQ), which is being increasingly associated with ALS and FTD pathology, binds to sense RNA foci. Here, we show that SFPQ plays an important role in the C9orf72 mutation. Overexpression of SFPQ resulted in higher numbers of both sense and antisense RNA foci and DPRs in transfected human embryonic kidney (HEK) cells. Conversely, reduced SPFQ levels resulted in lower numbers of RNA foci and DPRs in both transfected HEK cells and C9orf72 mutation-positive patient-derived fibroblasts and lymphoblasts. Therefore, we have revealed a role of SFPQ in regulating the C9orf72 mutation that has implications for understanding and developing novel therapeutic targets for ALS and FTD.This article has an associated First Person interview with the first author of the paper.

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Individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to determine protein-RNA interactions

10.1101/108118 ◽

2017 ◽

Cited By ~ 1

Author(s):

Christopher R. Sibley

Keyword(s):

High Throughput ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cdna Libraries ◽

Cross Linking ◽

Rna Transcripts ◽

Rna Targets ◽

Disease Aetiology ◽

Nucleotide Resolution ◽

Rna Complexes

AbstractRNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. In doing so they help direct many essential roles in cellular physiology, whilst their perturbed activity can contribute to disease aetiology. In this chapter we detail a functional genomics approach, termed individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), that can determine the interactions of RBPs with their RNA targets in high throughput and at nucleotide resolution. iCLIP achieves this by exploiting UV-induced covalent crosslinks formed between RBPs and their target RNAs to both purify the RBP-RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct crosslink sites in the high throughput sequenced cDNA libraries.

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The interactome of CLUH reveals its association to SPAG5 and its co-translational proximity to mitochondrial proteins

BMC Biology ◽

10.1186/s12915-021-01213-y ◽

2022 ◽

Vol 20 (1) ◽

Author(s):

Mickaële Hémono ◽

Alexandre Haller ◽

Johana Chicher ◽

Anne-Marie Duchêne ◽

Richard Patryk Ngondo

Keyword(s):

Mammalian Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Embryonic Stem ◽

Nuclear Genome ◽

Mrna Translation ◽

Mitochondrial Proteins ◽

Molecular Function ◽

First Time ◽

Tpr Domain

Abstract Background Mitochondria require thousands of proteins to fulfill their essential function in energy production and other fundamental biological processes. These proteins are mostly encoded by the nuclear genome, translated in the cytoplasm before being imported into the organelle. RNA binding proteins (RBPs) are central players in the regulation of this process by affecting mRNA translation, stability, or localization. CLUH is an RBP recognizing specifically mRNAs coding for mitochondrial proteins, but its precise molecular function and interacting partners remain undiscovered in mammals. Results Here we reveal for the first time CLUH interactome in mammalian cells. Using both co-IP and BioID proximity-labeling approaches, we identify novel molecular partners interacting stably or transiently with CLUH in HCT116 cells and mouse embryonic stem cells. We reveal stable RNA-independent interactions of CLUH with itself and with SPAG5 in cytosolic granular structures. More importantly, we uncover an unexpected proximity of CLUH to mitochondrial proteins and their cognate mRNAs in the cytosol. We show that this interaction occurs during the process of active translation and is dependent on CLUH TPR domain. Conclusions Overall, through the analysis of CLUH interactome, our study sheds a new light on CLUH molecular function by revealing new partners and by highlighting its link to the translation and subcellular localization of some mRNAs coding for mitochondrial proteins.

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Emergence of order in condensates composed of multi-valent, multi-domain proteins

10.1101/2021.11.15.468745 ◽

2021 ◽

Author(s):

Srivastav Ranganathan ◽

Eugene Shakhnovich

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Orientational Order ◽

Structural Diversity ◽

Coarse Grained ◽

Post Translational Modifications ◽

First Order Transition ◽

Dynamics Simulations ◽

Common Architecture ◽

Folded Rna

Many RNA-binding proteins (RBPs) that assemble into membraneless organelles, have a common architecture including disordered prion-like domain (PLD) and folded RNA-binding domain (RBD). An enrichment of PLD within the condensed phase gives rise to formation, on longer time scales, amyloid-like fibrils (aging). In this study, we employ coarse-grained Langevin dynamics simulations to explore the physical basis for the structural diversity in condensed phases of multi-domain RBPs. We discovered a highly cooperative first order transition between disordered (liquid-like) structures and an ordered (solid-like) phase whereby chains of PLD organize in fibrils with high nematic orientational order. Cooperativity of this liquid-solid transition makes fibril formation highly malleable to mutations or post-translational modifications. An interplay between homo-domain (PLD-PLD) and hetero-domain (PLD-RBD) interactions results in variety of structures with distinct spatial architectures. Our results provide a mechanistic understanding of how multi-domain RBPs could form assemblies with distinct structural and, potentially, material properties.

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