Primary structure of human nuclear ribonucleoprotein particle C proteins: conservation of sequence and domain structures in heterogeneous nuclear RNA, mRNA, and pre-rRNA-binding proteins

In the eucaryotic nucleus, heterogeneous nuclear RNAs exist in a complex with a specific set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). The C proteins, C1 and C2, are major constituents of hnRNPs and appear to play a role in RNA splicing as suggested by antibody inhibition and immunodepletion experiments. With the use of a previously described partial cDNA clone as a hybridization probe, full-length cDNAs for the human C proteins were isolated. All of the cDNAs isolated hybridized to two poly(A)+ RNAs of 1.9 and 1.4 kilobases (kb). DNA sequencing of a cDNA clone for the 1.9-kb mRNA (pHC12) revealed a single open reading frame of 290 amino acids coding for a protein of 31,931 daltons and two polyadenylation signals, AAUAAA, approximately 400 base pairs apart in the 3' untranslated region of the mRNA. DNA sequencing of a clone corresponding to the 1.4-kb mRNA (pHC5) indicated that the sequence of this mRNA is identical to that of the 1.9-kb mRNA up to the first polyadenylation signal which it uses. Both mRNAs therefore have the same coding capacity and are probably transcribed from a single gene. Translation in vitro of the 1.9-kb mRNA selected by hybridization with a 3'-end subfragment of pHC12 demonstrated that it by itself can direct the synthesis of both C1 and C2. The difference between the C1 and C2 proteins which results in their electrophoretic separation is not known, but most likely one of them is generated from the other posttranslationally. Since several hnRNP proteins appeared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as multiple antigenically related polypeptides, this raises the possibility that some of these other groups of hnRNP proteins are also each produced from a single mRNA. The predicted amino acid sequence of the protein indicates that it is composed of two distinct domains: an amino terminus that contains what we have recently described as a RNP consensus sequence, which is the putative RNA-binding site, and a carboxy terminus that is very negatively charged, contains no aromatic amino acids or prolines, and contains a putative nucleoside triphosphate-binding fold, as well as a phosphorylation site for casein kinase type II. The RNP consensus sequence was also found in the yeast poly(A)-binding protein (PABP), the heterogeneous nuclear RNA-binding proteins A1 and A2, and the pre-rRNA binding protein C23. All of these proteins are also composed of at least two distinct domains: an amino terminus, which possesses one or more RNP consensus sequences, and a carboxy terminus, which is unique to each protein, being very acidic in the C proteins and rich in glycine in A1, and C23 and rich in proline in the poly(A)-binding protein. These findings suggest that the amino terminus of these proteins possesses a highly conserved RNA-binding domain, whereas the carboxy terminus contains a region essential to the unique function and interactions of each of the RNA-binding proteins.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6102 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6102-6113 ◽

Cited By ~ 42

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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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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Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.164-171.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 164-171

Author(s):

M J Matunis ◽

W M Michael ◽

G Dreyfuss

Keyword(s):

Hela Cells ◽

Mammalian Cells ◽

Binding Proteins ◽

Rna Binding ◽

Consensus Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Predicted Amino Acid Sequence ◽

Cdna Clones ◽

Hnrnp Proteins

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.

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Matrin3: Disorder and ALS Pathogenesis

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.794646 ◽

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.

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PUB1: a major yeast poly(A)+ RNA-binding protein

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6114-6123.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6114-6123

Author(s):

M J Matunis ◽

E L Matunis ◽

G Dreyfuss

Keyword(s):

Rna Polymerase Ii ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Uv Light ◽

Binding Protein ◽

Gene Replacement ◽

Yeast Saccharomyces Cerevisiae ◽

Binding Domains ◽

Development And Differentiation

The expression of RNA polymerase II transcripts can be regulated at the posttranscriptional level by RNA-binding proteins. Although extensively characterized in metazoans, relatively few RNA-binding proteins have been characterized in the yeast Saccharomyces cerevisiae. Three major proteins are cross-linked by UV light to poly(A)+ RNA in living S. cerevisiae cells. These are the 72-kDa poly(A)-binding protein and proteins of 60 and 50 kDa (S.A. Adam, T.Y. Nakagawa, M.S. Swanson, T. Woodruff, and G. Dreyfuss, Mol. Cell. Biol. 6:2932-2943, 1986). Here, we describe the 60-kDa protein, one of the major poly(A)+ RNA-binding proteins in S. cerevisiae. This protein, PUB1 [for poly(U)-binding protein 1], was purified by affinity chromatography on immobilized poly(rU), and specific monoclonal antibodies to it were produced. UV cross-linking demonstrated that PUB1 is bound to poly(A)+ RNA (mRNA or pre-mRNA) in living cells, and it was detected primarily in the cytoplasm by indirect immunofluorescence. The gene for PUB1 was cloned and sequenced, and the sequence was found to predict a 51-kDa protein with three ribonucleoprotein consensus RNA-binding domains and three glutamine- and asparagine-rich auxiliary domains. This overall structure is remarkably similar to the structures of the Drosophila melanogaster elav gene product, the human neuronal antigen HuD, and the cytolytic lymphocyte protein TIA-1. Each of these proteins has an important role in development and differentiation, potentially by affecting RNA processing. PUB1 was found to be nonessential in S. cerevisiae by gene replacement; however, further genetic analysis should reveal important features of this class of RNA-binding proteins.

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RNA-binding proteins with prion-like domains in health and disease

Biochemical Journal ◽

10.1042/bcj20160499 ◽

2017 ◽

Vol 474 (8) ◽

pp. 1417-1438 ◽

Cited By ~ 152

Author(s):

Alice Ford Harrison ◽

James Shorter

Keyword(s):

Phase Transitions ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Low Complexity ◽

Fused In Sarcoma ◽

Heterogeneous Nuclear Ribonucleoproteins ◽

Health And Disease ◽

Lateral Sclerosis

Approximately 70 human RNA-binding proteins (RBPs) contain a prion-like domain (PrLD). PrLDs are low-complexity domains that possess a similar amino acid composition to prion domains in yeast, which enable several proteins, including Sup35 and Rnq1, to form infectious conformers, termed prions. In humans, PrLDs contribute to RBP function and enable RBPs to undergo liquid–liquid phase transitions that underlie the biogenesis of various membraneless organelles. However, this activity appears to render RBPs prone to misfolding and aggregation connected to neurodegenerative disease. Indeed, numerous RBPs with PrLDs, including TDP-43 (transactivation response element DNA-binding protein 43), FUS (fused in sarcoma), TAF15 (TATA-binding protein-associated factor 15), EWSR1 (Ewing sarcoma breakpoint region 1), and heterogeneous nuclear ribonucleoproteins A1 and A2 (hnRNPA1 and hnRNPA2), have now been connected via pathology and genetics to the etiology of several neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Here, we review the physiological and pathological roles of the most prominent RBPs with PrLDs. We also highlight the potential of protein disaggregases, including Hsp104, as a therapeutic strategy to combat the aberrant phase transitions of RBPs with PrLDs that likely underpin neurodegeneration.

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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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Cataloguing and Selection of mRNAs Localized to Dendrites in Neurons and Regulated by RNA-Binding Proteins in RNA Granules

Biomolecules ◽

10.3390/biom10020167 ◽

2020 ◽

Vol 10 (2) ◽

pp. 167 ◽

Cited By ~ 3

Author(s):

Ohashi ◽

Shiina

Keyword(s):

Synaptic Plasticity ◽

Cell Fate ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Memory Formation ◽

Long Term Memory ◽

Local Translation ◽

Rna Granules

Spatiotemporal translational regulation plays a key role in determining cell fate and function. Specifically, in neurons, local translation in dendrites is essential for synaptic plasticity and long-term memory formation. To achieve local translation, RNA-binding proteins in RNA granules regulate target mRNA stability, localization, and translation. To date, mRNAs localized to dendrites have been identified by comprehensive analyses. In addition, mRNAs associated with and regulated by RNA-binding proteins have been identified using various methods in many studies. However, the results obtained from these numerous studies have not been compiled together. In this review, we have catalogued mRNAs that are localized to dendrites and are associated with and regulated by the RNA-binding proteins fragile X mental retardation protein (FMRP), RNA granule protein 105 (RNG105, also known as Caprin1), Ras-GAP SH3 domain binding protein (G3BP), cytoplasmic polyadenylation element binding protein 1 (CPEB1), and staufen double-stranded RNA binding proteins 1 and 2 (Stau1 and Stau2) in RNA granules. This review provides comprehensive information on dendritic mRNAs, the neuronal functions of mRNA-encoded proteins, the association of dendritic mRNAs with RNA-binding proteins in RNA granules, and the effects of RNA-binding proteins on mRNA regulation. These findings provide insights into the mechanistic basis of protein-synthesis-dependent synaptic plasticity and memory formation and contribute to future efforts to understand the physiological implications of local regulation of dendritic mRNAs in neurons.

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