Poly(A)-binding proteins and mRNA localization: who rules the roost?

RNA-binding proteins are often multifunctional, interact with a variety of protein partners and display complex localizations within cells. Mammalian cytoplasmic poly(A)-binding proteins (PABPs) are multifunctional RNA-binding proteins that regulate multiple aspects of mRNA translation and stability. Although predominantly diffusely cytoplasmic at steady state, they shuttle through the nucleus and can be localized to a variety of cytoplasmic foci, including those associated with mRNA storage and localized translation. Intriguingly, PABP sub-cellular distribution can alter dramatically in response to cellular stress or viral infection, becoming predominantly nuclear and/or being enriched in induced cytoplasmic foci. However, relatively little is known about the mechanisms that govern this distribution/relocalization and in many cases PABP functions within specific sites remain unclear. Here we discuss the emerging evidence with respect to these questions in mammals.

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Quantitative Proteomics Links the LRRC59 Interactome to mRNA Translation on the ER Membrane

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra120.002228 ◽

2020 ◽

Vol 19 (11) ◽

pp. 1826-1849

Author(s):

Molly M. Hannigan ◽

Alyson M. Hoffman ◽

J. Will Thompson ◽

Tianli Zheng ◽

Christopher V. Nicchitta

Keyword(s):

Protein Synthesis ◽

Steady State ◽

Membrane Protein ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Translation ◽

Integral Membrane Protein ◽

Translation Factors ◽

Er Membrane

Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61β (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61β and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [35S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation.

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Single-molecule imaging reveals translation of mRNAs localized to stress granules

10.1101/2020.03.31.018093 ◽

2020 ◽

Author(s):

Daniel Mateju ◽

Bastian Eichenberger ◽

Jan Eglinger ◽

Gregory Roth ◽

Jeffrey A. Chao

Keyword(s):

Single Molecule ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cellular Stress ◽

Mrna Translation ◽

Stress Granules ◽

Living Cells ◽

Single Molecule Imaging ◽

Direct Role

SUMMARYCellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively nontranslating mRNA. Here we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. While our data confirms that non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence for translation of mRNA localized to SGs. Our data indicate that SG-associated translation is not rare and that the entire translation cycle (initiation, elongation and termination) can occur for these transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results argue against a direct role for SGs in inhibition of mRNA translation.

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Faculty Opinions recommendation of Recognition of a bicoid mRNA localization signal by a protein complex containing Swallow, Nod, and RNA binding proteins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1011561.181214 ◽

2003 ◽

Author(s):

Elizabeth Gavis

Keyword(s):

Protein Complex ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Localization ◽

Localization Signal

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RNA Binding Proteins and Regulation of mRNA Translation in Erythropoiesis

Frontiers in Physiology ◽

10.3389/fphys.2018.00910 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 8

Author(s):

Kat S. Moore ◽

Marieke von Lindern

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Translation

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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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Targeting of transcripts encoding membrane proteins in polarized epithelia: RNA–protein binding studies of the SGLT1 3′-UTR

Biochemical Society Transactions ◽

10.1042/bst0360525 ◽

2008 ◽

Vol 36 (3) ◽

pp. 525-527 ◽

Cited By ~ 2

Author(s):

Christopher M. Pedder ◽

Dianne Ford ◽

John E. Hesketh

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Protein Localization ◽

Complex Structure ◽

Mrna Translation ◽

Drosophila Embryo ◽

Binding Studies ◽

Polarized Epithelia ◽

Apical Localization

mRNA stability, mRNA translation and spatial localization of mRNA species within a cell can be governed by signals in the 3′-UTR (3′-untranslated region). Local translation of proteins is essential for the development of many eukaryotic cell types, such as the Drosophila embryo, where the spatial and temporal localization of bicoid and gurken mRNAs, among others, is required to establish morphogen gradients. More recent studies have suggested that mRNA localization also occurs with transcripts coding for membrane-based or secreted proteins, and that localization at organelles such as the endoplasmic reticulum directs translation more efficiently to specific subdomains, so as to aid correct protein localization. In human epithelial cells, the mRNA coding for SGLT1 (sodium–glucose co-transporter 1), an apical membrane protein, has been shown to be localized apically in polarized cells. However, the nature of the signals and RNA-binding proteins involved are unknown. Ongoing work is aimed at identifying the localization signals in the SGLT1 3′-UTR and the corresponding binding proteins. Using a protein extract from polarized Caco-2 cells, both EMSAs (electrophoretic mobility-shift assays) and UV cross-linking assays have shown that a specific protein complex is formed with the first 300 bases of the 3′-UTR sequence. MFold predictions suggest that this region folds into a complex structure and ongoing studies using a series of strategic deletions are being carried out to identify the precise nature of the motif involved, particularly the role of the sequence or RNA secondary structure, as well as to identify the main proteins present within the complex. Such information will provide details of the post-transcriptional events that lead to apical localization of the SGLT1 transcript and may reveal mechanisms of more fundamental importance in the apical localization of proteins in polarized epithelia.

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Two Yeast La Motif-containing Proteins Are RNA-binding Proteins that Associate with Polyribosomes

Molecular Biology of the Cell ◽

10.1091/mbc.10.11.3849 ◽

1999 ◽

Vol 10 (11) ◽

pp. 3849-3862 ◽

Cited By ~ 36

Author(s):

Suzanne G. Sobel ◽

Sandra L. Wolin

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Polymerase Iii ◽

Mrna Translation ◽

Protein Synthesis Inhibitors ◽

La Protein ◽

Evolutionarily Conserved ◽

Synthetically Lethal ◽

Type Strains

We have characterized two Saccharomyces cerevisiaeproteins, Sro9p and Slf1p, which contain a highly conserved motif found in all known La proteins. Originally described as an autoantigen in patients with rheumatic disease, the La protein binds to newly synthesized RNA polymerase III transcripts. In yeast, the La protein homologue Lhp1p is required for the normal pathway of tRNA maturation and also stabilizes newly synthesized U6 RNA. We show that deletions in both SRO9 and SLF1 are not synthetically lethal with a deletion in LHP1, indicating that the three proteins do not function in a single essential process. Indirect immunofluorescence microscopy reveals that although Lhp1p is primarily localized to the nucleus, Sro9p is cytoplasmic. We demonstrate that Sro9p and Slf1p are RNA-binding proteins that associate preferentially with translating ribosomes. Consistent with a role in translation, strains lacking either Sro9p or Slf1p are less sensitive than wild-type strains to certain protein synthesis inhibitors. Thus, Sro9p and Slf1p define a new and possibly evolutionarily conserved class of La motif-containing proteins that may function in the cytoplasm to modulate mRNA translation.

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A protein aggregation-dependent scaling-free synaptic facilitation rule for neural network simulations

10.1101/031856 ◽

2015 ◽

Author(s):

Michele Sanguanini ◽

Antonino Cattaneo

Keyword(s):

Protein Aggregation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Translation ◽

Cellular Level ◽

Long Term Memory ◽

Differential Model ◽

Synaptic Facilitation ◽

The One

The regulation of mRNA translation at synaptic level is believed to be fundamental in memory and learning at cellular level. A family of RNA binding proteins (RBPs) which emerged to be important during development and in adult neurons is the one of Cytoplasmic Polyadenylation Element Binding proteins (CPEBs). Drosophila Orb2 (homolog of vertebrate CPEB2 protein and of the neural isoform of Aplysia CPEB) has been found to be involved in the translation of plasticity-dependent mRNAs and has been associated to Long Term Memory (LTM). Orb2 protein presents two main isoforms, Orb2A and Orb2B, which form an activity induced amyloid-like functional aggregate, which is thought to be the translation-inducing state of the RBP. Here we present a two-states continuous differential model for Orb2A-Orb2B aggregation and we propose it, more generally, as a new synaptic facilitation rule for learning processes involving protein aggregation-dependent plasticity (PADP).

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

Life Science Alliance ◽

10.26508/lsa.201800187 ◽

2018 ◽

Vol 1 (5) ◽

pp. e201800187 ◽

Cited By ~ 8

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.

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