scholarly journals A massively parallel reporter assay reveals focused and broadly encoded RNA localization signals in neurons

2021 ◽  
Author(s):  
Martin Mikl ◽  
Davide Eletto ◽  
Minkyoung Lee ◽  
Atefeh Lafzi ◽  
Farah Mhamedi ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Neuronal Cell ◽  
Mrna Localization ◽  
Rna Localization ◽  
Massively Parallel ◽  
Reporter Assay ◽  
Massively Parallel Reporter Assay ◽  
Localization Behavior

AbstractAsymmetric subcellular localization of mRNA is a common cellular phenomenon that is thought to contribute to spatial gene regulation. In highly polar neurons, subcellular transcript localization and translation are thought to enhance cellular efficiency and timely responses to external cues. Although mRNA localization has been observed in many tissues and numerous examples of the functional importance of this process exist, we still lack a systematic understanding of how the transcript sorting machinery works in a sequence-specific manner.Here, we addressed these gaps by combining subcellular transcriptomics and rationally designed sequence libraries. We developed a massively parallel reporter assay (MPRA) for mRNA localization and tested ~50,000 sequences for their ability to drive RNA localization to neurites of neuronal cell lines. By scanning the 3’UTR of >300 genes we identified many previously unknown localization regions and mapped the localization potential of endogenous sequences. Our data suggest two ways the localization potential can be encoded in the 3’UTR: focused localization motifs and broadly encoded localization potential based on small contributions.We identified sequence motifs enriched in dendritically localized transcripts and tested the potential of these motifs to affect the localization behavior of an mRNA. This assay revealed sequence elements with the ability to bias localization towards neurite as well as soma. Depletion of RNA binding proteins predicted or experimentally shown to bind these motifs abolished the effect on localization, suggesting that these motifs act by recruiting specific RNA-binding proteins.Based on our dataset we developed machine learning models that accurately predict the localization behavior of novel sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native transcripts.Applying similar systematic high-throughput approaches to other cell types will open the door for a comparative perspective on RNA localization across tissues and reveal the commonalities and differences of this crucial regulatory mechanism.

scholarly journals Massively parallel identification of zipcodes in primary cortical neurons

2021 ◽  
Author(s):  
Nicolai von Kuegelgen ◽  
Samantha Mendonsa ◽  
Sayaka Dantsuji ◽  
Maya Ron ◽  
Marieluise Kirchner ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Primary Cortical Neurons ◽  
Subcellular Compartments ◽  
Local Functions ◽  
Massively Parallel Reporter Assay

Cells adopt highly polarized shapes and form distinct subcellular compartments largely due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called "zipcodes." Their recognition by RNA-binding proteins (RBPs) leads to the integration of the mRNAs into macromolecular complexes and their localization. While there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here, we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3'UTRs. This approach combines a method of separating the principal subcellular compartments of neurons - cell bodies and neurites - with a massively parallel reporter assay. Our analysis identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons and suggests a strategy for detecting many more.

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 The crystal structure of Staufen1 in complex with a physiological RNA sheds light on substrate selectivity

2018 ◽  
Vol 1 (5) ◽  
pp. e201800187 ◽  
Author(s):  
Daniela Lazzaretti ◽  
Lina Bandholz-Cajamarca ◽  
Christiane Emmerich ◽  
Kristina Schaaf ◽  
Claire Basquin ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Information ◽  
Target Selection ◽  
Mrna Localization ◽  
In Vitro Binding ◽  
Vitro Binding

During mRNA localization, RNA-binding proteins interact with specific structured mRNA localization motifs. Although several such motifs have been identified, we have limited structural information on how these interact with RNA-binding proteins. Staufen proteins bind structured mRNA motifs through dsRNA-binding domains (dsRBD) and are involved in mRNA localization in Drosophila and mammals. We solved the structure of two dsRBDs of human Staufen1 in complex with a physiological dsRNA sequence. We identified interactions between the dsRBDs and the RNA sugar–phosphate backbone and direct contacts of conserved Staufen residues to RNA bases. Mutating residues mediating nonspecific backbone interactions only affected Staufen function in Drosophila when in vitro binding was severely reduced. Conversely, residues involved in base-directed interactions were required in vivo even when they minimally affected in vitro binding. Our work revealed that Staufen can read sequence features in the minor groove of dsRNA and suggests that these influence target selection in vivo.

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 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 Rab1b and ARF5 are novel RNA-binding proteins involved in FMDV IRES–driven RNA localization

2019 ◽  
Vol 2 (1) ◽  
pp. e201800131 ◽  
Author(s):  
Javier Fernandez-Chamorro ◽  
Rosario Francisco-Velilla ◽  
Jorge Ramajo ◽  
Encarnación Martinez-Salas
Keyword(s):  
Binding Proteins ◽  
Protein Transport ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Localization ◽  
Dominant Negative ◽  
Entry Site ◽  
Initiation Of Translation ◽  
Mouth Disease Virus ◽  
Domain 3

Internal ribosome entry site (IRES) elements are organized in domains that guide internal initiation of translation. Here, we have combined proteomic and imaging analysis to study novel foot-and-mouth disease virus IRES interactors recognizing specific RNA structural subdomains. Besides known picornavirus IRES–binding proteins, we identified novel factors belonging to networks involved in RNA and protein transport. Among those, Rab1b and ARF5, two components of the ER-Golgi, revealed direct binding to IRES transcripts. However, whereas Rab1b stimulated IRES function, ARF5 diminished IRES activity. RNA-FISH studies revealed novel features of the IRES element. First, IRES-RNA formed clusters within the cell cytoplasm, whereas cap-RNA displayed disperse punctate distribution. Second, the IRES-driven RNA localized in close proximity with ARF5 and Rab1b, but not with the dominant-negative of Rab1b that disorganizes the Golgi. Thus, our data suggest a role for domain 3 of the IRES in RNA localization around ER-Golgi, a ribosome-rich cellular compartment.

scholarly journals Massively parallel identification of zipcodes in primary cortical neurons

2021 ◽  
Author(s):  
Marina Chekulaeva ◽  
Nicolai von Kügelgen ◽  
Samantha Mendonsa ◽  
Sayaka Dantsuji ◽  
Maya Ron ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Regulatory Elements ◽  
Massively Parallel ◽  
Primary Cortical Neurons ◽  
Subcellular Compartments ◽  
Local Functions ◽  
Massively Parallel Reporter Assay

Abstract Cells adopt highly polarized shapes and form distinct subcellular compartments largely due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called "zipcodes." Their recognition by RNA-binding proteins (RBPs) leads to the integration of the mRNAs into macromolecular complexes and their localization. While there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here, we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3'UTRs. This approach combines a method of separating the principal subcellular compartments of neurons – cell bodies and neurites - with a massively parallel reporter assay. Our analysis identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons and suggests a strategy for detecting many more.

scholarly journals β-actin mRNA interactome mapping by proximity biotinylation

2018 ◽  
Author(s):  
Joyita Mukherjee ◽  
Orit Hermesh ◽  
Carolina Eliscovich ◽  
Nicolas Nalpas ◽  
Mirita Franz-Wachtel ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Molecular Motor ◽  
Mrna Localization ◽  
Biotin Ligase ◽  
Kh Domain ◽  
Actin Mrna ◽  
Actin Protein

AbstractThe molecular function and fate of mRNAs are controlled by RNA-binding proteins (RBPs). However, identification of the interacting proteome of a specific mRNA in vivo is still very challenging. Based on the widely-used RNA tagging with MS2 aptamers for RNA visualization, we developed a novel RNA proximity biotinylation (RNA-BioID) method by tethering biotin ligase (BirA*) via MS2 coat protein (MCP) at the 3’-UTR of endogenous MS2 tagged β-actin mRNA (MBS) in mouse embryonic fibroblasts (MEFs). We demonstrate the dynamics of the β-actin mRNA interactome by characterizing its changes upon serum-induced localization of the mRNA. Apart from the previously known interactors, we identified over 60 additional β-actin associated RBPs by RNA-BioID. Among them the KH-domain containing protein FUBP3/MARTA2 has shown to be required for β-actin mRNA localization. We found that FUBP3 binds to the 3’-UTR of β-actin mRNA, is essential for β-actin mRNA localization but does not interact with the characterized β-actin zipcode element. RNA-BioID provides a tool to identify new mRNA interactors and to study the dynamic view of the interacting proteome of endogenous mRNAs in space and time.Significance statementTransport of specific mRNAs to defined sites in the cytoplasm allows local protein production and contributes to cell polarity, embryogenesis, and neuronal function. These localized mRNAs contain signals (zipcodes) that help directing them to their destination site. Zipcodes are recognized by RNA-binding proteins that, with the help of molecular motor proteins and supplementary factors, mediate mRNA trafficking. To identify all proteins assembling with a localized mRNA we advanced a proximity labeling method (BioID) by tethering a biotin ligase to the 3’ untranslated region of mRNA encoding the conserved beta-actin protein. We demonstrate that this method allows the identification of novel, functionally important proteins that are required for mRNA localization.

scholarly journals Rab1b and ARF5 are novel RNA-binding proteins involved in IRES-driven RNA localization

2018 ◽  
Author(s):  
Javier Fernandez-Chamorro ◽  
Rosario Francisco-Velilla ◽  
Jorge Ramajo ◽  
Encarnación Martinez-Salas
Keyword(s):  
Binding Proteins ◽  
Protein Transport ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Localization ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Entry Site ◽  
Initiation Of Translation ◽  
Domain 3

ABSTRACTInternal ribosome entry site (IRES) elements are organized in domains that guide internal initiation of translation. Here we have combined proteomic and imaging analysis to study novel IRES interactors recognizing specific RNA structural subdomains. Besides known IRES-binding proteins, we identified novel factors belonging to networks involved in RNA and protein transport. Among those, Rab1b and ARF5, two components of the ER-Golgi, revealed direct binding to IRES transcripts. However, these proteins exert different effects on translation. While a dominant-negative mutant of Rab1b decreased IRES function, ARF5 silencing stimulated IRES activity. RNA FISH studies revealed novel features of the IRES element. First, IRES-RNA formed clusters within the cell cytoplasm, whereas cap-RNA displayed disperse punctuated distribution. Second, the IRES-driven RNA colocalized with ARF5 and Rab1b, but not with the dominant-negative of Rab1b. Thus, our data suggest a role for domain 3 of the IRES in RNA localization around ER-Golgi, a ribosome-rich cellular compartment.

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