Recognition of subsets of the mammalian A/B-type core heterogeneous nuclear ribonucleoprotein polypeptides by novel autoantibodies

Dangli, Amalia; Plomaritoglou, Angeliki; Boutou, Efrosini; Vassiliadou, N.; Moutsopoulos, Haralampos Μ.; Guialis, Apostolia

doi:10.1042/bj3200761

Cited by 17 publications

(19 citation statements)

References 40 publications

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“…Although hnRNP A3 was not described originally as a component of the core particles identified in HeLa cell nuclei, it is present in multiple isoforms in these cells (47). We have shown that hnRNP A3 is abundant in several tissues, paralleling the tissue distribution of hnRNP A2.…”

Section: Figmentioning

confidence: 67%

Heterogeneous Nuclear Ribonucleoprotein A3, a Novel RNA Trafficking Response Element-binding Protein

S.W.¹,

Moran-Jones²,

Shan³

et al. 2002

Journal of Biological Chemistry

112

138

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The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1 B , hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41-, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41-and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.Establishment of asymmetry in cells requires selective localization of proteins. This may be accomplished by directed protein transport, a well established pathway for plasma membrane and secreted proteins, or by trafficking and subsequent localization of mRNA. Localization of RNA has been intensively studied in Drosophila and Xenopus oocytes (for reviews see Refs. 1-6) and more recently in mammalian somatic cells (7-12).In 1982 Subsequent experiments demonstrated that MBP mRNA is translated close to myelin and the protein rapidly incorporated into the nascent membrane (14 -16) and lead to a model in which MBP mRNA is recruited into RNA transport granules in the perikaryon and then transported, by indirect attachment to the microtubule-bound motor protein kinesin, to the myelin compartment at the cell periphery (10, 17-21). The granules are localized in the myelin compartment, and the RNA cargo is translated, with the MBP being incorporated into the myelin membrane. Deletion studies led to the conclusion that a small element in the 3Ј-untranslated region of the MBP mRNA, the RNA transport sequence (RTS), is sufficient and necessary for this cytoplasmic RNA transport in oligodendrocytes (17). Cytoplasmic trafficking of RNA encoding ␤-actin is also dependent on inclusion in transport granules that are attached to the cytoskeleton. In fibroblasts ␤-actin mRNA transport is microfilament-dependent (9, 22), whereas microtubules are implicated in transport of this mRNA in neurons (23,24).trans-Acting factors have been isolated in pull-down experiments with RTS-labeled magnetic particles. The predominant RTS-binding protein from a number of rat tissues is heterogeneous nuclear ribonucleoprotein (hnRNP) A2 (25), a constituent of...

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Section: Figmentioning

confidence: 67%

Heterogeneous Nuclear Ribonucleoprotein A3, a Novel RNA Trafficking Response Element-binding Protein

S.W.¹,

Moran-Jones²,

Shan³

et al. 2002

Journal of Biological Chemistry

112

138

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“…hnRNP B2 migrates close to hnRNP A1 b and mBx sits on top of hnRNP A2 with the prefix 'm' to indicate the murine origin). The monoclonal 9H10 antibody specifically against hnRNP A1 species clearly established that hnRNPs B2 and A1 b belong to immunologically distinct protein components (Dangli et al, 1996). Through a detailed comparison of the hnRNP A/B proteins in nuclear extracts of human and murine origin, the abundant protein species mBx, which is only present in murine but not in human, has been verified to be an hnRNP A3 ortholog (Plomaritoglou et al, 2000).…”

Section: Ambiguity Of Hnrnp A/b Nomenclaturementioning

confidence: 99%

“…Later, two immunologically related protein B2 and mBx were detected in sera of patients with rheumatic autoimmune disease. Both proteins were identified to be the new members of hnRNP A/B family (Dangli et al, 1996). The mBx protein was subsequently reported to be abundant in murine cells, and the evidences of its molecular characterization revealed it as a new gene product, which is closely related to the Xenopus RNPA3 gene (Plomaritoglou, Choli-Papadopoulou, & Guialis, 2000).…”

Section: Hnrnp A3 Identificationmentioning

confidence: 99%

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Characterization of hnRNP A3 isoforms in The A2RE-proteome

Wei¹

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Polarity is an important prerequisite for cell differentiation. The development of cellular asymmetry and cell polarity can be achieved by either selectively transporting key intracellular proteins or vectorial trafficking mRNAs to a subcellular location for local translation. The "A2RE-mRNA trafficking pathway" fits in the latter category, renowned for moving the myelin basic protein (MBP) RNA molecules from the nucleus to cytoplasmic myelin compartment of oligodendrocytes.This prominent pathway is named after a 11-nucleotide cis-acting sequence of "A2 response element (A2RE)", which is necessary and sufficient for mRNA localization. The universally expressed protein hnRNP A2 was the first identified trans-acting factor bound to the A2RE ciselement directing the A2RE-containing transcripts to their subcellular locations along cytoskeletal components.In addition to hnRNP A2, hnRNP A3 is the second abundant proteins pulled down by the A2RE-sequence. At the beginning of this project, hnRNP A3 had just been identified as a novel key component along with other A2RE-binding proteins. A lot of structural and functional questions arising from the discovery of hnRNP A3 in the A2RE-context await answers.The detailed structural knowledge of hnRNP A3 among the A2RE-binding proteins was essential prior to the investigation of its biological involvement in the A2RE-trafficking pathway. Therefore, the overall aim of this project was to characterize the structure of hnRNP A3 isoforms which were purified by binding to the A2RE-sequence. At the same time, the structural comparison with other A2RE-binding proteins was also carried out, aiming to find out any similar or distinct features between these proteins when they were associated together with the same A2RE-sequence. The investigations in this project were established from aspects of genomics, proteomics and cellular location to characterize the hnRNP A3 isoforms. The experimental data, presented in this thesis, were collected reproducibly and comprise all above aspects.From the genomic aspect, the two splicing forms of hnRNP A3 in rat brain were exclusively identified through the extensive PCR screening. At the protein level, the masses of the intact A2RE-binding proteins were measured by liquid chromatography mass spectrometry (LC/MS) and analyzed. Next, the rat brain A2RE-binding proteins were successfully separated by twodimensional gel electrophoresis (2-DE), showing a more complicated profile of hnRNP A/B isoforms in the A2RE-context. Each binding components were identified by mass spectrometry (MS) based peptide mass fingerprinting. 3Targeting the complexity of hnRNP A3 inside the A2RE-proteome, the post-translational modifications (PTMs) of hnRNP A3 were investigated. Phospho-proteins of the A2RE proteome were detected by blotting and staining. Moreover, the pattern of protein phosphorylation was observed to be isoform-specific between the alternatively spliced isoform pairs. In terms of Arg modifications, the methylation of A3 was confirmed, and six asymmetr...

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“…The A/B-type hnRNP polypeptides (A1/A1b, A2/B1/A2b/B1b, A3/A3a/A3b (Papadopoulou et al, 2012)) are structurally related basic proteins with molecular masses ranging from 30 to 40 kDa (Dangli et al, 1996, Kamma et al, 1999.…”

Section: Hnrnp A/b Subfamilymentioning

confidence: 99%

Role of hnRNP A/B proteins in telomere maintenance and telomerase activity

Saig¹

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Telomere shortening leads to eukaryotic cell senescence, whereas enhanced telomerase activity is associated with telomere expansion and growth of cancer cells. Several studies have suggested hnRNPs are important for telomere maintenance including their ability to bind telomeres, and telomerase. The hnRNPs A/B family are highly abundant multifunctional proteins that bind to single-stranded DNA and RNA and perform many roles in cellular regulation. In this study, affinity pull-down assays, biosensor and UV-cross-linking studies were used to confirm that recombinant hnRNPs A2 and A3, compared to recombinant hnRNP A1, specifically interact with single-stranded telomeric DNA repeat TTAGGG and hexamer repeat. Tandem RRMs are required for strong binding, which is little changed by exclusion of the glycine-rich domain (hnRNPs Δ GRD).Additionally, TRAP assays, that indicate telomerase activity, showed that hnRNPs A1, A2and A3 bind to telomerase in cell extracts. It is not clear whether this association is mediated by binding a specific nucleotide region within telomerase RNA, or a protein component such as dyskerin, or both.In this study I have confirmed and refined the interaction site between the hnRNP A/B proteins and telomerase RNA (hTR). Serial UV-cross-linking RNA electrophoretic mobility shifting assays (REMSA) have shown that hnRNP A2 binds specifically with to a 17nt region within the RNA telomerase template (hTR). Deletion analysis of the hnRNP A/Bs revealed that these proteins bind preferentially to the hTR segment encompassing nucleotides hTR 57-72 mediated mainly through the RRM1 sequence. Importantly, I found that hnRNP A2 can interact simultaneously with telomeric ssDNA repeats. Both hnRNP A1 and hnRNP A3 showed no binding to the short segments of hTR in this assay, yet they could form a complex with full-length hTR. This difference can be explained by the increased insolubility of A1 and A3. Additionally, this study has indicated that both RRMs of hnRNP A2 are essential for either DNA-protein, or RNA-protein interactions, or both for simultaneous binding. In the chromatography experiments, RRM1 and RRM2 on their own were not able to mediate a simultaneous binding with both telomeric DNA and telomerase RNA, while short variant UP2 was successful to maintain this kind of binding feature.The results suggest that hnRNP A2 may help recruit telomerase to the ends of chromosomes.These hnRNP A/B family members are highly abundant in the cell nucleus, perform many roles in the cell and potentially influence telomere biogenesis. From these studies a model was proposed where the hnRNP A2 binding site is adjacent, or close to the template segment of hTR. The close proximity of the hnRNP A2 binding site to the template region on hTR and necessarily, to the end of the ssDNA 3' overhang, supports the proposal that these proteins play an active role in the addition of new telomeric repeats for cellular maintenance. VI Publications included in this thesisNo publications included. Contributions by others to the t...

show abstract

Recognition of subsets of the mammalian A/B-type core heterogeneous nuclear ribonucleoprotein polypeptides by novel autoantibodies

Cited by 17 publications

References 40 publications

Heterogeneous Nuclear Ribonucleoprotein A3, a Novel RNA Trafficking Response Element-binding Protein

Heterogeneous Nuclear Ribonucleoprotein A3, a Novel RNA Trafficking Response Element-binding Protein

Characterization of hnRNP A3 isoforms in The A2RE-proteome

Role of hnRNP A/B proteins in telomere maintenance and telomerase activity

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