Biomedical subjects

Michael A Kiebler

Publications and source records attributed to Michael A Kiebler.

10 recordsLinked to original sources

PubMed2006

Neuronal RNA granules: movers and makers.

RNA localization contributes to cell polarity and synaptic plasticity. Evidence will be discussed that RNA transport and local translation in neurons may be more intimately linked than originally thought. Second, neuronal RNA granules, originally defined as intermediates involved in mRNA transport, are much more diverse in their composition and functions than previously anticipated. We focus on three classes of RNA granules that include transport RNPs, stress granules, and P bodies and discuss their potential functions in RNA localization, microRNA-mediated translational regulation, and mRNA degradation.

Animals↗

PubMed2006

Dendritic localization of the translational repressor Pumilio 2 and its contribution to dendritic stress granules.

Pumilio (Pum) protein acts as a translational inhibitor in several organisms including yeast, Drosophila, Xenopus, and mammals. Two Pumilio genes, Pum1 and Pum2, have been identified in mammals, but their function in neurons has not been identified. In this study, we found that Pum2 mRNA is expressed during neuronal development and that the protein is found in discrete particles in both the cell body and the dendritic compartment of fully polarized neurons. This finding indicates that Pum2 is a novel candidate of dendritically localized ribonucleoparticles (RNPs). During metabolic stress, Pum2 is present in stress granules (SGs), which are subsequently detected in the somatodendritic domain. It remains excluded from processing bodies under all conditions. When overexpressed in neurons and fibroblasts, Pum2 induces the formation of SGs that also contain T-cell intracellular antigen 1 (TIA-1)-related protein, eukaryotic initiation factor 4E, poly(A)-binding protein, TIA-1, and other RNA-binding proteins including Staufen1 and Barentsz. This induction of SGs is dependent on the RNA-binding domain and a glutamine-rich region in the N terminus of Pum2. This glutamine-rich region behaves in a similar manner as TIA-1 and prion protein, two molecules with known roles in protein aggregation. Pum2 downregulation in neurons via RNA interference (RNAi) interferes with the formation of SGs during metabolic stress. Cotransfection with an RNAi-resistant portion of the Pum2 mRNA restores SG formation. These results suggest a role for Pum2 in dendritic RNPs and SG formation in mammalian neurons.

Animals↗

PubMed2006

The brain-specific double-stranded RNA-binding protein Staufen2 is required for dendritic spine morphogenesis.

Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of beta-actin mRNA and fewer dendritic beta-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.

Actins↗

PubMed2006

Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants.

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

Amino Acid Sequence↗

PubMed2005

A putative nuclear function for mammalian Staufen.

In addition to its role in rRNA processing and ribosome assembly, the nucleolus plays a part in the assembly of non-ribosomal ribonucleoprotein particles (RNPs) that are destined for cytoplasmic RNA delivery. Recent evidence indicates that mammalian Staufen2, a brain-specific RNA-binding protein involved in RNA localization, can--at least transiently--enter the nucleolus. Therefore, the assembly of Staufen2 into transport-competent RNPs might occur in the nucleus before their export into the cytoplasm. This could provide new insights into the mechanisms of subcellular RNA localization.

Active Transport, Cell Nucleus↗

PubMed2004

The brain-specific double-stranded RNA-binding protein Staufen2: nucleolar accumulation and isoform-specific exportin-5-dependent export.

The mammalian double-stranded RNA-binding proteins Staufen (Stau1 and Stau2) are involved in RNA localization in polarized neurons. In contrast to the more ubiquitously expressed Stau1, Stau2 is mainly expressed in the nervous system. In Drosophila, the third double-stranded RNA-binding domain (RBD3) of Staufen is essential for RNA interaction. When conserved amino acids within the RBD3 of Stau2 were mutated to render Stau2 defective for RNA binding, the mutant Stau2 proteins accumulate predominantly in the nucleolus. This is in contrast to wild type Stau2 that mostly localizes in the cytosol. The nuclear import is dependent on a nuclear localization signal in close proximity to the RBD3. The nuclear export of Stau2 is not dependent on CRM1 but rather on Exportin-5. We show that Exportin-5 interacts with the RBD3 of wild type Stau2 in an RNA-dependent manner in vitro but not with mutant Stau2. When Exportin-5 is down-regulated by RNA interference, only the largest isoform of Stau2 (Stau2(62)) preferentially accumulates in the nucleolus. It is tempting to speculate that Stau2(62) binds RNA in the nucleus and assembles into ribonucleoparticles, which are then exported via the Exportin-5 pathway to their final destination.

Animals↗

PubMed2003

Coupling the iron-responsive element to GFP--an inducible system to study translation in a single living cell.

Local protein synthesis in a cell represents an elegant mechanism to achieve important biological phenomena such as cell migration, body axis formation during embryonic development and establishment of cell polarity. A prerequisite to studying translation in a restricted cellular compartment is the ability to unambiguously discriminate between proteins that arise through local protein synthesis and those that reach the site of interest by diffusion or transport. To tackle this problem, we set up a green fluorescent protein (GFP)-based reporter system that allows one to uncouple the translation of reporter gene mRNA from its subcellular localization. The system is based on the iron-responsive element, which regulates the translation of both endogenous ferritin and transferrin transcripts in response to changes in iron concentration. Translation of the reporter messenger RNA (mRNA) is thus dependent on iron in the medium; both its transcription and localization, however, are unaffected. Known targeting sequences can be used to direct the mRNA transcript to a subcellular compartment of interest. For instance, the full-length 3'-untranslated region of calcium/calmodulin-dependent protein kinase IIalpha mRNA can be added to the construct, after the stop codon of the GFP sequence, to selectively target the transcript into the dendrites of transiently transfected hippocampal neurons. This novel fluorescent assay will allow us to address a number of important biological questions in living mammalian cells.

Amino Acid Sequence↗

PubMed2003

Isolation and characterization of Staufen-containing ribonucleoprotein particles from rat brain.

Localized mRNAs are thought to be transported in defined particles to their final destination. These particles represent large protein complexes that may be involved in recognizing, transporting, and anchoring localized messages. Few components of these ribonucleoparticles, however, have been identified yet. We chose the strategy to biochemically enrich native RNA-protein complexes involved in RNA transport to identify the associated RNAs and proteins. Because Staufen proteins were implicated in intracellular RNA transport, we chose mammalian Staufen proteins as markers for the purification of RNA transport particles. Here, we present evidence that Staufen proteins exist in two different complexes: (i) distinct large, ribosome- and endoplasmic reticulum-containing granules preferentially found in the membrane pellets during differential centrifugation and (ii) smaller particles in the S100 from rat brain homogenates. On gel filtration of the S100, we identified soluble 670-kDa Staufen1-containing and 440-kDa Staufen2-containing particles. They do not cofractionate with ribosomes and endoplasmic reticulum but rather coenrich with kinesin heavy chain. Furthermore, the fractions containing the Staufen1 particles show a 15-fold enrichment of mRNAs compared with control fractions. Most importantly, these fractions are highly enriched in BC1, and, to a lesser extent, in the alpha-subunit of the Ca(2+)/calmodulin-dependent kinase II, two dendritically localized RNAs. Finally, both RNAs colocalize with Staufen1-hemagglutinin in particles in dendrites of transfected hippocampal neurons. We therefore propose that these Staufen1-containing particles may represent RNA transport intermediates that are in transit to their final destination within neurons.

Animals↗

PubMed2003

A GFP-based system to uncouple mRNA transport from translation in a single living neuron.

An inducible fluorescent system based on GFP is presented that allows for the uncoupling of dendritic mRNA transport from subsequent protein synthesis at the single cell level. The iron-responsive element (IRE) derived from ferritin mRNA in the 5'-UTR of the GFP reporter mRNA renders translation of its mRNA dependent on iron. The addition of the full-length 3'-UTR of the Ca(2+)/calmodulin-dependent protein kinase II alpha (CaMKIIalpha) after the stop codon of the GFP reading frame targets the reporter mRNA to dendrites of transfected fully polarized hippocampal neurons. As we show by time-lapse videomicroscopy, iron specifically turns on GFP reporter protein synthesis in a single transfected hippocampal neuron. We investigate whether GFP expression is affected--in addition to iron--by synaptic activity. Interestingly, synaptic activity has a clear stimulatory effect. Most importantly, however, this activity-dependent protein synthesis is critically dependent on the presence of the full-length 3'-UTR of CaMKIIalpha confirming that this sequence contains translational activation signals. The IRE-based system represents a new convenient tool to study local protein synthesis in mammalian cells where mRNA localization to a specific intracellular compartment occurs.

5' Untranslated Regions↗

PubMed2002

Staufen2 isoforms localize to the somatodendritic domain of neurons and interact with different organelles.

Mammalian Staufen2 (Stau2) is involved in mRNA transport in neurons. Here, we report that Stau2 is a double-stranded RNA-binding protein that is mainly expressed in the brain. We show that Stau2 is found in the somatodendritic compartment of neurons. In dendrites, Stau2 is aligned on individual tracts and colocalizes with microtubules. Stau2 is expressed as at least three splice isoforms, which can be observed in several subcellular complexes. Although a 62 kDa isoform (Stau2(62)) fractionates in ribosome-free fractions of light density, Stau2(59) and Stau2(52) are found in high-density complexes. These complexes are resistant to EDTA and to non-ionic detergent. For the first time, we also provide evidence for an interaction of some Stau2 isoforms with ribosomes, thus pointing to an interesting new role for Stau2 in translation. EDTA treatment, which dissociates ribosome subunits, does not release Stau2 from the subunits, suggesting that Stau2-ribosome associations are not mediated mainly by mRNA intermediates. Although Stau2 has many features in common with its paralogue Stau1, it does not colocalize with Stau1-containing particles, indicating that these proteins are components of different complexes in dendrites. Our findings suggest that members of the Staufen family share evolutionarily conserved properties and highlight the complexity of Staufen-mediated RNA transport in neurons.

Alternative Splicing↗