scholarly journals FMRP promotes RNA localization to neuronal projections through interactions between its RGG domain and G-quadruplex RNA sequences

2019 ◽  
Author(s):  
Raeann Goering ◽  
Laura I. Hudish ◽  
Bryan B. Guzman ◽  
Nisha Raj ◽  
Gary J. Bassell ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Rna Localization ◽  
Null Mouse ◽  
Rna Sequences ◽  
Local Protein Synthesis ◽  
Rna Molecules ◽  
G Quadruplex

ABSTRACTThe sorting of RNA molecules to distinct subcellular locations facilitates the activity of spatially restricted processes through local protein synthesis. This process affects thousands of transcripts yet precisely how these RNAs are trafficked to their destinations remains generally unclear. Here we have analyzed subcellular transcriptomes of FMRP-null mouse neuronal cells to identify transcripts that depend on FMRP for efficient transport to neurites. We found that these FMRP RNA localization targets contain a large enrichment of G-quadruplex sequences, particularly in their 3′ UTRs, suggesting that FMRP recognizes these sequences to promote the localization of transcripts that contain them. Fractionation of neurons derived from human Fragile X Syndrome patients revealed a high degree of conservation in the identity of FMRP localization targets between human and mouse as well as an enrichment of G-quadruplex sequences in human FMRP RNA localization targets. Using high-throughput RNA/protein interaction assays and single-molecule RNA FISH, we identified the RGG domain of FMRP as important for both interaction with G-quadruplex RNA sequences and the neuronal transport of G-quadruplex-containing transcripts. Finally, we used ribosome footprinting to identify translational regulatory targets of FMRP. The translational regulatory targets were not enriched for G-quadruplex sequences and were largely distinct from the RNA localization targets of FMRP, indicating that the two functions can be biochemically separated and are mediated through different target recognition mechanisms. These results establish a molecular mechanism underlying FMRP-mediated neuronal RNA localization and provide a framework for the elucidation of similar mechanisms governed by other RNA-binding proteins.

scholarly journals FMRP promotes RNA localization to neuronal projections through interactions between its RGG domain and G-quadruplex RNA sequences

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Raeann Goering ◽  
Laura I Hudish ◽  
Bryan B Guzman ◽  
Nisha Raj ◽  
Gary J Bassell ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Rna Localization ◽  
Null Mouse ◽  
Rna Sequences ◽  
Rna Molecules ◽  
G Quadruplex

The sorting of RNA molecules to subcellular locations facilitates the activity of spatially restricted processes. We have analyzed subcellular transcriptomes of FMRP-null mouse neuronal cells to identify transcripts that depend on FMRP for efficient transport to neurites. We found that these transcripts contain an enrichment of G-quadruplex sequences in their 3′ UTRs, suggesting that FMRP recognizes them to promote RNA localization. We observed similar results in neurons derived from Fragile X Syndrome patients. We identified the RGG domain of FMRP as important for binding G-quadruplexes and the transport of G-quadruplex-containing transcripts. Finally, we found that the translation and localization targets of FMRP were distinct and that an FMRP mutant that is unable to bind ribosomes still promoted localization of G-quadruplex-containing messages. This suggests that these two regulatory modes of FMRP may be functionally separated. These results provide a framework for the elucidation of similar mechanisms governed by other RNA-binding proteins.

scholarly journals Differences in Vaccine and SARS-CoV-2 Replication Derived mRNA: Implications for Cell Biology and Future Disease

2021 ◽  
Author(s):  
Kevin McKernan ◽  
Anthony M. Kyriakopoulos ◽  
Peter McCullough
Keyword(s):  
Binding Sites ◽  
Cell Biology ◽  
Protein Production ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Codon Optimization ◽  
Synonymous Codon ◽  
Gc Content ◽  
Rna Sequences ◽  
G Quadruplex

Codon optimization describes the process used to increase protein production by use of alternative but synonymous codon changes. In SARS-CoV-2 mRNA vaccines codon optimizations can result in differential secondary conformations that inevitably affect a protein’s function with significant consequences to the cell. Importantly, when codon optimization increases the GC content of synthetic mRNAs, there can be an inevitable enrichment of G-quartets which potentially form G-quadruplex structures. The emerging G-quadruplexes are favorable binding sites of RNA binding proteins like helicases that inevitably affect epigenetic reprogramming of the cell by altering transcription, translation and replication. In this study, we performed a RNAfold analysis to investigate alterations in secondary structures of mRNAs in SARS-CoV-2 vaccines due to codon optimization. We show a significant increase in the GC content of mRNAs in vaccines as compared to native SARS-CoV-2 RNA sequences encoding the spike protein. As the GC enrichment leads to more G-quadruplex structure formations, these may contribute to potential pathological processes initiated by SARS-CoV-2 molecular vaccination.

scholarly journals PTB/hnRNP I Is Required for RNP Remodeling during RNA Localization in Xenopus Oocytes

2007 ◽  
Vol 28 (2) ◽  
pp. 678-686 ◽  
Author(s):  
Raymond A. Lewis ◽  
James A. Gagnon ◽  
Kimberly L. Mowry
Keyword(s):  
Protein Interactions ◽  
Xenopus Oocytes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Localization ◽  
Rna Transport ◽  
Rna Sequences ◽  
Cytoplasmic Rna ◽  
Embryonic Polarity ◽  
Specific Mrnas

ABSTRACT Transport of specific mRNAs to defined regions within the cell cytoplasm is a fundamental mechanism for regulating cell and developmental polarity. In the Xenopus oocyte, Vg1 RNA is transported to the vegetal cytoplasm, where localized expression of the encoded protein is critical for embryonic polarity. The Vg1 localization pathway is directed by interactions between key motifs within Vg1 RNA and protein factors recognizing those RNA sequences. We have investigated how RNA-protein interactions could be modulated to trigger distinct steps in the localization pathway and found that the Vg1 RNP is remodeled during cytoplasmic RNA transport. Our results implicate two RNA-binding proteins with key roles in Vg1 RNA localization, PTB/hnRNP I and Vg1RBP/vera, in this process. We show that PTB/hnRNP I is required for remodeling of the interaction between Vg1 RNA and Vg1RBP/vera. Critically, mutations that block this remodeling event also eliminate vegetal localization of the RNA, suggesting that RNP remodeling is required for localization.

scholarly journals Nuclear RNP complex assembly initiates cytoplasmic RNA localization

2004 ◽  
Vol 165 (2) ◽  
pp. 203-211 ◽  
Author(s):  
Tracy L. Kress ◽  
Young J. Yoon ◽  
Kimberly L. Mowry
Keyword(s):  
Protein Interactions ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Localization ◽  
Rna Sequences ◽  
Cytoplasmic Rna ◽  
Rnp Complex ◽  
Maternal Mrnas ◽  
Protein Components

Cytoplasmic localization of mRNAs is a widespread mechanism for generating cell polarity and can provide the basis for patterning during embryonic development. A prominent example of this is localization of maternal mRNAs in Xenopus oocytes, a process requiring recognition of essential RNA sequences by protein components of the localization machinery. However, it is not yet clear how and when such protein factors associate with localized RNAs to carry out RNA transport. To trace the RNA–protein interactions that mediate RNA localization, we analyzed RNP complexes from the nucleus and cytoplasm. We find that an early step in the localization pathway is recognition of localized RNAs by specific RNA-binding proteins in the nucleus. After transport into the cytoplasm, the RNP complex is remodeled and additional transport factors are recruited. These results suggest that cytoplasmic RNA localization initiates in the nucleus and that binding of specific RNA-binding proteins in the nucleus may act to target RNAs to their appropriate destinations in the cytoplasm.

scholarly journals Detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct, long-read sequencing

2021 ◽  
Author(s):  
Sepideh Tavakoli ◽  
Mohammad Nabizadehmashhadtoroghi ◽  
Amr Makhamreh ◽  
Howard Gamper ◽  
Neda Rezapour ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Parameters ◽  
Type I ◽  
High Confidence ◽  
Rna Molecules ◽  
Base Calling ◽  
Long Read

Enzyme-mediated chemical modifications to mRNAs have the potential to fine-tune gene expression in response to environmental stimuli. Notably, pseudouridine-modified mRNAs are more resistant to RNase-mediated degradation, more responsive to cellular stress, and have the potential to modulate immunogenicity and enhance translation in vivo. However, the precise biological functions of pseudouridine modification on mRNAs remain unclear due to the lack of sensitive and accurate tools for mapping. We developed a semi-quantitative method for mapping pseudouridylated sites with high confidence directly on mammalian mRNA transcripts via direct RNA, long-read nanopore sequencing. By analysis of a modification-free transcriptome, we demonstrate that the depth of coverage and intrinsic errors associated with specific k-mer sequences are critical parameters for accurate base-calling. We adjust these parameters for high-confidence U-to-C base-calling errors that occur at pseudouridylated sites, which are benchmarked against sites that were identified previously by biochemical methods. We also uncovered new pseudouridylated sites, many of which fall on genes that encode RNA binding proteins and on uridine-rich k-mers. Sites identified by U-to-C base calling error were verified using 1000mer synthetic RNA controls bearing a single pseudouridine in the center position, demonstrating that 1. the U-to-C base-calling error occurs at the site of pseudouridylation, and 2. the basecalling error is systematically under-calling the pseudouridylated sites. High-occupancy sites with >40% U-to-C basecalling error are classified as sites of hyper modification type I, whereas genes with more than one site of pseudouridylation are classified as having type II hyper modification which is confirmed by single-molecule analysis. We report the discovery of mRNAs with up to 7 unique sites of pseudouridine modification. Here we establish an innovative pipeline for direct identification, quantification, and detection of pseudouridine modifications and type I/II hypermodifications on native RNA molecules using long-read sequencing without resorting to RNA amplification, chemical reactions on RNA, enzyme-based replication, or DNA sequencing steps.

RNA-Binding Proteins and Translation in Neurodegenerative Disease

2020 ◽  
Author(s):  
Kent E. Duncan
Keyword(s):  
Neurodegenerative Diseases ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Potential Contribution ◽  
Fused In Sarcoma ◽  
Specific Mrnas ◽  
Ataxia Syndrome ◽  
Research Frontiers

Both RNA-binding proteins (RBPs) and translation are increasingly implicated in several neurodegenerative diseases, but their specific roles in promoting disease are not yet fully defined. This chapter critically evaluates the evidence that altered translation of specific mRNAs mediated by RNA-binding proteins plays an important role in driving specific neurodegenerative diseases. First, diseases are discussed where a causal role for RNA-binding proteins in disease appears solid, but whether this involves altered translation is less clear. The main foci here are TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS) in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Subsequently, diseases are presented where altered translation is believed to contribute, but involvement of RNA-binding proteins is less clear. These include Huntington’s and other repeat expansion disorders such as fragile X tremor/ataxia syndrome (FXTAS), where repeat-induced non-AUG-initiated (RAN) translation is a focus. The potential contribution of both canonical and non-canonical RBPs to altered translation in Parkinson’s disease is discussed. The chapter closes by proposing key research frontiers for the field to explore and outlining methodological advances that could help to address them.

scholarly journals Secondary structural choice of DNA and RNA associated with CGG/CCG trinucleotide repeat expansion rationalizes the RNA misprocessing in FXTAS

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yogeeshwar Ajjugal ◽  
Narendar Kolimi ◽  
Thenmalarchelvi Rathinavelan
Keyword(s):  
Rna Binding ◽  
Trinucleotide Repeat ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Repeat Expansion ◽  
Mobility Shift ◽  
Cgg Repeat ◽  
Dna And Rna ◽  
Structural Choice ◽  
Sense Strand

AbstractCGG tandem repeat expansion in the 5′-untranslated region of the fragile X mental retardation-1 (FMR1) gene leads to unusual nucleic acid conformations, hence causing genetic instabilities. We show that the number of G…G (in CGG repeat) or C…C (in CCG repeat) mismatches (other than A…T, T…A, C…G and G…C canonical base pairs) dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C…C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.

scholarly journals Coupled Transcription-Translation in Prokaryotes: An Old Couple With New Surprises

2021 ◽  
Vol 11 ◽  
Author(s):  
Mikel Irastortza-Olaziregi ◽  
Orna Amster-Choder
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Small Rnas ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Localization ◽  
Molecular Architecture ◽  
Independent Manner ◽  
New Information ◽  
Shed Light

Coupled transcription-translation (CTT) is a hallmark of prokaryotic gene expression. CTT occurs when ribosomes associate with and initiate translation of mRNAs whose transcription has not yet concluded, therefore forming “RNAP.mRNA.ribosome” complexes. CTT is a well-documented phenomenon that is involved in important gene regulation processes, such as attenuation and operon polarity. Despite the progress in our understanding of the cellular signals that coordinate CTT, certain aspects of its molecular architecture remain controversial. Additionally, new information on the spatial segregation between the transcriptional and the translational machineries in certain species, and on the capability of certain mRNAs to localize translation-independently, questions the unanimous occurrence of CTT. Furthermore, studies where transcription and translation were artificially uncoupled showed that transcription elongation can proceed in a translation-independent manner. Here, we review studies supporting the occurrence of CTT and findings questioning its extent, as well as discuss mechanisms that may explain both coupling and uncoupling, e.g., chromosome relocation and the involvement of cis- or trans-acting elements, such as small RNAs and RNA-binding proteins. These mechanisms impact RNA localization, stability, and translation. Understanding the two options by which genes can be expressed and their consequences should shed light on a new layer of control of bacterial transcripts fate.

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.

scholarly journals Visualizing the translation and packaging of HIV-1 full-length RNA

2020 ◽  
Vol 117 (11) ◽  
pp. 6145-6155 ◽  
Author(s):  
Jianbo Chen ◽  
Yang Liu ◽  
Bin Wu ◽  
Olga A. Nikolaitchik ◽  
Preeti R. Mohan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Rna Binding ◽  
Virus Assembly ◽  
Full Length ◽  
Rna Molecules ◽  
Rna Translation ◽  
Translation Template ◽  
A Genome ◽  
Hiv 1

HIV-1 full-length RNA (HIV-1 RNA) plays a central role in viral replication, serving as a template for Gag/Gag-Pol translation and as a genome for the progeny virion. To gain a better understanding of the regulatory mechanisms of HIV-1 replication, we adapted a recently described system to visualize and track translation from individual HIV-1 RNA molecules in living cells. We found that, on average, half of the cytoplasmic HIV-1 RNAs are being actively translated at a given time. Furthermore, translating and nontranslating RNAs are well mixed in the cytoplasm; thus, Gag biogenesis occurs throughout the cytoplasm without being constrained to particular subcellular locations. Gag is an RNA binding protein that selects and packages HIV-1 RNA during virus assembly. A long-standing question in HIV-1 gene expression is whether Gag modulates HIV-1 RNA translation. We observed that despite its RNA-binding ability, Gag expression does not alter the proportion of translating HIV-1 RNA. Using single-molecule tracking, we found that both translating and nontranslating RNAs exhibit dynamic cytoplasmic movement and can reach the plasma membrane, the major HIV-1 assembly site. However, Gag selectively packages nontranslating RNA into the assembly complex. These studies illustrate that although HIV-1 RNA serves two functions, as a translation template and as a viral genome, individual RNA molecules carry out only one function at a time. These studies shed light on previously unknown aspects of HIV-1 gene expression and regulation.

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