Grad-seq guides the discovery of ProQ as a major small RNA-binding protein

The functional annotation of transcriptomes and identification of noncoding RNA (ncRNA) classes has been greatly facilitated by the advent of next-generation RNA sequencing which, by reading the nucleotide order of transcripts, theoretically allows the rapid profiling of all transcripts in a cell. However, primary sequence per se is a poor predictor of function, as ncRNAs dramatically vary in length and structure and often lack identifiable motifs. Therefore, to visualize an informative RNA landscape of organisms with potentially new RNA biology that are emerging from microbiome and environmental studies requires the use of more functionally relevant criteria. One such criterion is the association of RNAs with functionally important cognate RNA-binding proteins. Here we analyze the full ensemble of cellular RNAs using gradient profiling by sequencing (Grad-seq) in the bacterial pathogenSalmonella enterica, partitioning its coding and noncoding transcripts based on their network of RNA–protein interactions. In addition to capturing established RNA classes based on their biochemical profiles, the Grad-seq approach enabled the discovery of an overlooked large collective of structured small RNAs that form stable complexes with the conserved protein ProQ. We show that ProQ is an abundant RNA-binding protein with a wide range of ligands and a global influence onSalmonellagene expression. Given its generic ability to chart a functional RNA landscape irrespective of transcript length and sequence diversity, Grad-seq promises to define functional RNA classes and major RNA-binding proteins in both model species and genetically intractable organisms.

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The Interplay of RNA Binding Proteins, Oxidative Stress and Mitochondrial Dysfunction in ALS

Antioxidants ◽

10.3390/antiox10040552 ◽

2021 ◽

Vol 10 (4) ◽

pp. 552

Author(s):

Jasmine Harley ◽

Benjamin E. Clarke ◽

Rickie Patani

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Mitochondrial Dysfunction ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Therapeutic Strategies ◽

Lateral Sclerosis

RNA binding proteins fulfil a wide number of roles in gene expression. Multiple mechanisms of RNA binding protein dysregulation have been implicated in the pathomechanisms of several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Oxidative stress and mitochondrial dysfunction also play important roles in these diseases. In this review, we highlight the mechanistic interplay between RNA binding protein dysregulation, oxidative stress and mitochondrial dysfunction in ALS. We also discuss different potential therapeutic strategies targeting these pathways.

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PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6102-6113.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6102-6113

Author(s):

J T Anderson ◽

M R Paddy ◽

M S Swanson

Keyword(s):

Saccharomyces Cerevisiae ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Uv Light ◽

Binding Protein ◽

Consensus Sequence ◽

Rna Binding Protein ◽

Polyadenylated Rna ◽

Polyadenylated Rnas

Proteins that directly associate with nuclear polyadenylated RNAs, or heterogeneous nuclear RNA-binding proteins (hnRNPs), and those that associate with cytoplasmic mRNAs, or mRNA-binding proteins (mRNPs), play important roles in regulating gene expression at the posttranscriptional level. Previous work with a variety of eukaryotic cells has demonstrated that hnRNPs are localized predominantly within the nucleus whereas mRNPs are cytoplasmic. While studying proteins associated with polyadenylated RNAs in Saccharomyces cerevisiae, we discovered an abundant polyuridylate-binding protein, PUB1, which appears to be both an hnRNP and an mRNP. PUB1 and PAB1, the polyadenylate tail-binding protein, are the two major proteins cross-linked by UV light to polyadenylated RNAs in vivo. The deduced primary structure of PUB1 indicates that it is a member of the ribonucleoprotein consensus sequence family of RNA-binding proteins and is structurally related to the human hnRNP M proteins. Even though the PUB1 protein is a major cellular polyadenylated RNA-binding protein, it is nonessential for cell growth. Indirect cellular immunofluorescence combined with digital image processing allowed a detailed comparison of the intracellular distributions of PUB1 and PAB1. While PAB1 is predominantly, and relatively uniformly, distributed within the cytoplasm, PUB1 is localized in a nonuniform pattern throughout both the nucleus and the cytoplasm. The cytoplasmic distribution of PUB1 is considerably more discontinuous than that of PAB1. Furthermore, sucrose gradient sedimentation analysis demonstrates that PAB1 cofractionates with polyribosomes whereas PUB1 does not. These results suggest that PUB1 is both an hnRNP and an mRNP and that it may be stably bound to a translationally inactive subpopulation of mRNAs within the cytoplasm.

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A potential role for a novel ZC3H5 complex in regulating mRNA translation in Trypanosoma brucei

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014346 ◽

2020 ◽

Vol 295 (42) ◽

pp. 14291-14304

Author(s):

Kathrin Bajak ◽

Kevin Leiss ◽

Christine Clayton ◽

Esteban Erben

Keyword(s):

Gene Expression ◽

Trypanosoma Brucei ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Affinity Purification ◽

Critical Role ◽

Rna Binding Protein ◽

Mrna Translation

In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5′-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of “halfmer” disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames.

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Genome-wide survey of putative RNA-binding proteins encoded in the human proteome

Molecular BioSystems ◽

10.1039/c5mb00638d ◽

2016 ◽

Vol 12 (2) ◽

pp. 532-540 ◽

Cited By ~ 16

Author(s):

Pritha Ghosh ◽

R. Sowdhamini

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Human Proteome ◽

Genome Wide ◽

Genome Wide Survey

We have classified the existing RNA-binding protein (RBP) structures into different structural families. Here, we report ∼2600 proteins with RBP signatures in humans.

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Suppression of C9orf72 RNA repeat-induced neurotoxicity by the ALS-associated RNA-binding protein Zfp106

eLife ◽

10.7554/elife.19032 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 22

Author(s):

Barbara Celona ◽

John von Dollen ◽

Sarat C Vatsavayai ◽

Risa Kashima ◽

Jeffrey R Johnson ◽

...

Keyword(s):

Motor Neurons ◽

Binding Proteins ◽

Rna Binding ◽

Zinc Finger Protein ◽

Rna Binding Proteins ◽

Binding Protein ◽

Affinity Purification ◽

Rna Binding Protein ◽

Severe Motor ◽

Affinity Purification Mass Spectrometry

Expanded GGGGCC repeats in the first intron of the C9orf72 gene represent the most common cause of familial amyotrophic lateral sclerosis (ALS), but the mechanisms underlying repeat-induced disease remain incompletely resolved. One proposed gain-of-function mechanism is that repeat-containing RNA forms aggregates that sequester RNA binding proteins, leading to altered RNA metabolism in motor neurons. Here, we identify the zinc finger protein Zfp106 as a specific GGGGCC RNA repeat-binding protein, and using affinity purification-mass spectrometry, we show that Zfp106 interacts with multiple other RNA binding proteins, including the ALS-associated factors TDP-43 and FUS. We also show that Zfp106 knockout mice develop severe motor neuron degeneration, which can be suppressed by transgenic restoration of Zfp106 specifically in motor neurons. Finally, we show that Zfp106 potently suppresses neurotoxicity in a Drosophila model of C9orf72 ALS. Thus, these studies identify Zfp106 as an RNA binding protein with important implications for ALS.

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Multivalent interactions with RNA drive RNA binding protein recruitment and dynamics in biomolecular condensates in Xenopus oocytes

10.1101/2021.06.21.449303 ◽

2021 ◽

Author(s):

Sarah E Cabral ◽

Kimberly Mowry

Keyword(s):

Protein Interactions ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Dependent Manner ◽

Binding Domains ◽

Multivalent Interactions

RNA localization and biomolecular condensate formation are key biological strategies for organizing the cytoplasm and generating cellular and developmental polarity. While enrichment of RNAs and RNA-binding proteins (RBPs) is a hallmark of both processes, the functional and structural roles of RNA-RNA and RNA-protein interactions within condensates remain unclear. Recent work from our laboratory has shown that RNAs required for germ layer patterning in Xenopus oocytes localize in novel biomolecular condensates, termed Localization bodies (L-bodies). L-bodies are composed of a non-dynamic RNA phase enmeshed in a more dynamic protein-containing phase. However, the interactions that drive the biophysical characteristics of L-bodies are not known. Here, we test the role of RNA-protein interactions using an L-body RNA-binding protein, PTBP3, which contains four RNA-binding domains (RBDs). We find that binding of RNA to PTB is required for both RNA and PTBP3 to be enriched in L-bodies in vivo. Importantly, while RNA binding to a single RBD is sufficient to drive PTBP3 localization to L-bodies, interactions between multiple RRMs and RNA tunes the dynamics of PTBP3 within L-bodies. In vitro, recombinant PTBP3 phase separates into non-dynamic structures in an RNA-dependent manner, supporting a role for RNA-protein interactions as a driver of both recruitment of components to L-bodies and the dynamics of the components after enrichment. Our results point to a model where RNA serves as a concentration-dependent, non-dynamic substructure and multivalent interactions with RNA are a key driver of protein dynamics.

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Combinatorial recognition of clustered RNA elements by a multidomain RNA-binding protein, IMP3

10.1101/398008 ◽

2018 ◽

Cited By ~ 1

Author(s):

Tim Schneider ◽

Lee-Hsueh Hung ◽

Masood Aziz ◽

Anna Wilmen ◽

Stephanie Thaum ◽

...

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Binding Domains ◽

Systematic Strategy ◽

Rna Elements ◽

Target Rna

AbstractHow multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We have established an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, providing a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

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Programmable RNA methylation and demethylation using PUF RNA binding proteins

Chemical Communications ◽

10.1039/c9cc09298f ◽

2020 ◽

Vol 56 (9) ◽

pp. 1365-1368 ◽

Cited By ~ 1

Author(s):

Kouki Shinoda ◽

Akiyo Suda ◽

Kenko Otonari ◽

Shiroh Futaki ◽

Miki Imanishi

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

New Method ◽

Rna Methylation

A new method manipulating local RNA methylation was developed by fusing the programmable RNA binding protein and the m6A demethylase or methyltransferase.

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Capturing RNA–protein interaction via CRUIS

Nucleic Acids Research ◽

10.1093/nar/gkaa143 ◽

2020 ◽

Vol 48 (9) ◽

pp. e52-e52 ◽

Cited By ~ 5

Author(s):

Ziheng Zhang ◽

Weiping Sun ◽

Tiezhu Shi ◽

Pengfei Lu ◽

Min Zhuang ◽

...

Keyword(s):

Mass Spectrometry ◽

Dna Damage ◽

Protein Interactions ◽

Noncoding Rna ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Living Cells ◽

Innovative Methods ◽

Rna Biology

Abstract No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA–protein interactions is very important. Here, we developed a new tool, the CRISPR-based RNA-United Interacting System (CRUIS), which captures RNA–protein interactions in living cells by combining the power of CRISPR and PUP-IT, a novel proximity targeting system. In CRUIS, dCas13a is used as a tracker to target specific RNAs, while proximity enzyme PafA is fused to dCas13a to label the surrounding RNA-binding proteins, which are then identified by mass spectrometry. To identify the efficiency of CRUIS, we employed NORAD (Noncoding RNA activated by DNA damage) as a target, and the results show that a similar interactome profile of NORAD can be obtained as by using CLIP (crosslinking and immunoprecipitation)-based methods. Importantly, several novel NORAD RNA-binding proteins were also identified by CRUIS. The use of CRUIS facilitates the study of RNA–protein interactions in their natural environment, and provides new insights into RNA biology.

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AU-rich RNA binding proteins in hematopoiesis and leukemogenesis

Blood ◽

10.1182/blood-2011-07-347237 ◽

2011 ◽

Vol 118 (22) ◽

pp. 5732-5740 ◽

Cited By ~ 35

Author(s):

Maria Baou ◽

John D. Norton ◽

John J. Murphy

Keyword(s):

Cell Growth ◽

Regulatory Networks ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Growth And Survival ◽

Hematopoietic Malignancies ◽

Gene Regulatory

Abstract Posttranscriptional mechanisms are now widely acknowledged to play a central role in orchestrating gene-regulatory networks in hematopoietic cell growth, differentiation, and tumorigenesis. Although much attention has focused on microRNAs as regulators of mRNA stability/translation, recent data have highlighted the role of several diverse classes of AU-rich RNA-binding protein in the regulation of mRNA decay/stabilization. AU-rich elements are found in the 3′-untranslated region of many mRNAs that encode regulators of cell growth and survival, such as cytokines and onco/tumor-suppressor proteins. These are targeted by a burgeoning number of different RNA-binding proteins. Three distinct types of AU-rich RNA binding protein (ARE poly-U–binding degradation factor-1/AUF1, Hu antigen/HuR/HuA/ELAVL1, and the tristetraprolin/ZFP36 family of proteins) are essential for normal hematopoiesis. Together with 2 further AU-rich RNA-binding proteins, nucleolin and KHSRP/KSRP, the functions of these proteins are intimately associated with pathways that are dysregulated in various hematopoietic malignancies. Significantly, all of these AU-rich RNA-binding proteins function via an interconnected network that is integrated with microRNA functions. Studies of these diverse types of RNA binding protein are providing novel insight into gene-regulatory mechanisms in hematopoiesis in addition to offering new opportunities for developing mechanism-based targeted therapeutics in leukemia and lymphoma.

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