scholarly journals Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Laura Arribas-Hernández ◽  
Sarah Rennie ◽  
Tino Köster ◽  
Carlotta Porcelli ◽  
Martin Lewinski ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Activity ◽  
Sequence Motifs ◽  
Intrinsically Disordered ◽  
Eukaryotic Gene ◽  
Yth Domain ◽  
Nucleotide Resolution ◽  
Mrna Targeting

Specific recognition of N6-methyladenosine (m6A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH-domain protein ECT2 is thought to bind to mRNA at URU(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A binding activity. Here, we apply iCLIP (individual-nucleotide resolution cross-linking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2, and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A-sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m6A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants, and reveals new insight into the mode of RNA recognition by YTH-domain-containing proteins.

scholarly journals Principles of mRNA targeting and regulation via the Arabidopsis m6A-binding proteins ECT2 and ECT3

2021 ◽  
Author(s):  
Laura Arribas-Hernández ◽  
Sarah Rennie ◽  
Tino Köster ◽  
Michael Schon ◽  
Carlotta Porcelli ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Target Identification ◽  
Super Resolution ◽  
Binding Activity ◽  
Intrinsically Disordered ◽  
Mrna Targets ◽  
Yth Domain

AbstractGene regulation dependent on N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A through a YTH domain. The Arabidopsis YTH-domain protein ECT2 is thought to influence mRNA 3’-end formation via binding to URU(m6A)Y sites, an unexpected conclusion given that ECT2 functions require its m6A binding activity, and that RR(m6A)CH is the m6A consensus site in all eukaryotes. Here, we apply the orthogonal techniques individual nucleotide-resolution UV-crosslinking and immunoprecipitation (iCLIP) and HyperTRIBE to define high-quality target sets of the YTH-domain proteins ECT2 and ECT3. The results show that in vivo, ECT2 does in fact bind to RR(m6A)CH. URUAY and other pyrimidine-rich motifs are enriched around, but not at m6A-sites, reflecting a preference for N6-adenosine methylation of RRACH islands in pyrimidine-rich regions. Such regions may also be implicated in ECT2-binding. In particular, a series of properties unique to the URUAY motif suggest that URUAY-type sequences act as sites of competition between unknown RNA-binding proteins and the intrinsically disordered region of ECT2. We also show that the abundance of many ECT2/3 mRNA targets is decreased in meristematic cells devoid of ECT2/3/4-activity. In contrast, loss of ECT2/3/4 activity has no effect on polyadenylation site usage in ECT2/3 targets, consistent with the exclusive cytoplasmic localization of ECT2 observed by super-resolution confocal microscopy. Our study reconciles conflicting results between genetic observations on N6-adenosine methylation and ECT2/3/4 function on the one side, and ECT2 target identification on the other, and point to regulation of cytoplasmic mRNA function, including abundance, as a mechanism of plant YTHDF action.

scholarly journals Matrin3: Disorder and ALS Pathogenesis

2022 ◽  
Vol 8 ◽  
Author(s):  
Ahmed Salem ◽  
Carter J. Wilson ◽  
Benjamin S. Rutledge ◽  
Allison Dilliott ◽  
Sali Farhan ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Binding Proteins ◽  
Rna Binding ◽  
Nuclear Dna ◽  
Neurodegenerative Disorder ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Disordered Regions

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of both upper and lower motor neurons in the brain and spinal cord. ALS is associated with protein misfolding and inclusion formation involving RNA-binding proteins, including TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS). The 125-kDa Matrin3 is a highly conserved nuclear DNA/RNA-binding protein that is implicated in many cellular processes, including binding and stabilizing mRNA, regulating mRNA nuclear export, modulating alternative splicing, and managing chromosomal distribution. Mutations in MATR3, the gene encoding Matrin3, have been identified as causal in familial ALS (fALS). Matrin3 lacks a prion-like domain that characterizes many other ALS-associated RNA-binding proteins, including TDP-43 and FUS, however, our bioinformatics analyses and preliminary studies document that Matrin3 contains long intrinsically disordered regions that may facilitate promiscuous interactions with many proteins and may contribute to its misfolding. In addition, these disordered regions in Matrin3 undergo numerous post-translational modifications, including phosphorylation, ubiquitination and acetylation that modulate the function and misfolding of the protein. Here we discuss the disordered nature of Matrin3 and review the factors that may promote its misfolding and aggregation, two elements that might explain its role in ALS pathogenesis.

scholarly journals A unique ribonucleoprotein complex assembles preferentially on ecdysone-responsive sites in Drosophila melanogaster.

1993 ◽  
Vol 13 (9) ◽  
pp. 5323-5330 ◽  
Author(s):  
S A Amero ◽  
M J Matunis ◽  
E L Matunis ◽  
J W Hockensmith ◽  
G Raychaudhuri ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Polytene Chromosomes ◽  
Cell Extract ◽  
Sequence Motifs ◽  
Drosophila Cell ◽  
Rnase Digestion

The protein on ecdysone puffs (PEP) is associated preferentially with active ecdysone-inducible puffs on Drosophila polytene chromosomes and contains sequence motifs characteristic of transcription factors and RNA-binding proteins (S. A. Amero, S. C. R. Elgin, and A. L. Beyer, Genes Dev. 5:188-200, 1991). PEP is associated with RNA in vivo, as demonstrated here by the sensitivity of PEP-specific chromosomal immunostaining in situ to RNase digestion and by the immunopurification of PEP in Drosophila cell extract with heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As revealed by sequential immunostaining, PEP is found on a subset of chromosomal sites bound by the HRB (heterogeneous nuclear RNA-binding) proteins, which are basic Drosophila hnRNPs. These observations lead us to suggest that a unique, PEP-containing hnRNP complex assembles preferentially on the transcripts of ecdysone-regulated genes in Drosophila melanogaster presumably to expedite the transcription and/or processing of these transcripts.

scholarly journals Inferring Sequence-Structure Preferences of RNA-Binding Proteins with Convolutional Residual Networks

2018 ◽  
Author(s):  
Peter K. Koo ◽  
Praveen Anand ◽  
Steffan B. Paul ◽  
Sean R. Eddy
Keyword(s):  
Rna Structure ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Sequence Data ◽  
Synthetic Data ◽  
Sequence Motifs ◽  
Negative Effects ◽  
Interpretable Model ◽  
Saliency Analysis

AbstractTo infer the sequence and RNA structure specificities of RNA-binding proteins (RBPs) from experiments that enrich for bound sequences, we introduce a convolutional residual network which we call ResidualBind. ResidualBind significantly outperforms previous methods on experimental data from many RBP families. We interrogate ResidualBind to identify what features it has learned from high-affinity sequences with saliency analysis along with 1st-order and 2nd-orderin silicomutagenesis. We show that in addition to sequence motifs, ResidualBind learns a model that includes the number of motifs, their spacing, and both positive and negative effects of RNA structure context. Strikingly, ResidualBind learns RNA structure context, including detailed base-pairing relationships, directly from sequence data, which we confirm on synthetic data. ResidualBind is a powerful, flexible, and interpretable model that can uncovercis-recognition preferences across a broad spectrum of RBPs.

scholarly journals SEQing: web-based visualization of iCLIP and RNA-seq data in an interactive python framework

2019 ◽  
Author(s):  
Martin Lewinski ◽  
Yannik Bramkamp ◽  
Tino Köster ◽  
Dorothee Staiger
Keyword(s):  
Binding Sites ◽  
Binding Proteins ◽  
Target Genes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Seq ◽  
Web Based ◽  
Genome Wide ◽  
Functional Relevance ◽  
Nucleotide Resolution

AbstractBackgroundRNA-binding proteins interact with their target RNAs at specific sites. These binding sites can be determined genome-wide through individual nucleotide resolution crosslinking immunoprecipitation (iCLIP). Subsequently, the binding sites have to be visualized. So far, no visualization tool exists that is easily accessible but also supports restricted access so that data can be shared among collaborators.ResultsHere we present SEQing, a customizable interactive dashboard to visualize crosslink sites on target genes of RNA-binding proteins that have been obtained by iCLIP. Moreover, SEQing supports RNA-seq data that can be displayed in a diffrerent window tab. This allows, e.g. crossreferencing the iCLIP data with genes differentially expressed in mutants of the RBP and thus obtain some insights into a potential functional relevance of the binding sites. Additionally, detailed information on the target genes can be incorporated in another tab.ConclusionSEQing is written in Python3 and runs on Linux. The web-based access makes iCLIP data easily accessible, even with mobile devices. SEQing is customizable in many ways and has also the option to be secured by a password. The source code is available at https://github.com/malewins/SEQing.

scholarly journals m6A-binding YTHDF proteins promote stress granule formation by modulating phase separation of stress granule proteins

2019 ◽  
Author(s):  
Ye Fu ◽  
Xiaowei Zhuang
Keyword(s):  
Phase Separation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Super Resolution ◽  
Stress Granule ◽  
Granule Formation ◽  
Intrinsically Disordered ◽  
Condensate Formation ◽  
Resolution Imaging

AbstractDiverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of m6A-modified mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are crucial for SG formation. Super-resolution imaging further reveals that YTHDF proteins are in a super-saturated state, forming clusters that reside in the periphery of and at the junctions between SG core clusters, and promote SG phase separation by reducing the activation energy barrier and critical size for condensate formation. Our results reveal a new function and mechanistic insights of the m6A-binding YTHDF proteins in regulating phase separation.

scholarly journals The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Laura Arribas-Hernández ◽  
Sarah Rennie ◽  
Michael Schon ◽  
Carlotta Porcelli ◽  
Balaji Enugutti ◽  
...  
Keyword(s):  
Indirect Effects ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Profiles ◽  
Alternative Polyadenylation ◽  
Target Mrna ◽  
Target Mrnas ◽  
Yth Domain ◽  
Target Sets

Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3 and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

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