A Novel Murine Staufen Isoform Modulates the RNA Content of Staufen Complexes

Abstract

Mouse Staufen (mStau) is a double-stranded RNA-binding protein associated with polysomes and the rough endoplasmic reticulum (RER). We describe a novel endogenous isoform of mStau (termed mStau i ) which has an insertion of six amino acids within dsRBD3, the major double-stranded RNA (dsRNA)-binding domain. With a structural change of the RNA-binding domain, this conserved and widely distributed isoform showed strongly impaired dsRNA-binding ability. In transfected cells, mStau i exhibited the same tubulovesicular distribution (RER) as mStau when weakly expressed; however, when overexpressed, mStau i was found in large cytoplasmic granules. Markers of the RER colocalized with mStau i -containing granules, showing that overexpressed mStau i could still be associated with the RER. Cotransfection of mStau i with mStau relocalized overexpressed mStau i to the reticular RER, suggesting that they can form a complex on the RER and that a balance between these isoforms is important to achieve proper localization. Coimmunoprecipitation demonstrated that the two mStau isoforms are components of the same complex in vivo. Analysis of the immunoprecipitates showed that mStau is a component of an RNA-protein complex and that the association with mStau i drastically reduces the RNA content of the complex. We propose that this new isoform, by forming a multiple-isoform complex, regulates the amount of RNA in mStau complexes in mammalian cells.RNA transport and localization provide an efficient way to distribute genetic information and to allow different portions of the cell to establish their own biochemical fates (12,20,32,44). Examples of this process have been described in many different organisms and cell types. RNA localization and/or localized translation are linked to different biological processes such as asymmetric cell division (7, 28, 39, 45), long-term potentiation (30), synaptic transmission (40), cell motility (23), and axis formation in oocytes (44). It is now apparent that many determinants of RNA localization are conserved among these systems. The process of mRNA localization is initiated by association of RNA with one or more RNA-binding proteins (RBPs) through a targeting signal most commonly located in the 3Ј untranslated region (3Ј UTR) of the transcripts. This association results in the formation of large ribonucleoprotein complexes (RNPs) (1, 13, 24). These complexes then migrate along the cytoskeleton to their final destination, where they are anchored and translated. For the entire localization process, translation of localized mRNAs needs to be tightly regulated. Specific signals are important for repressing translation during mRNA transport and derepressing translation once RNPs are properly localized (15,21,22). The crucial role played by the cytoskeleton in many steps of transport, anchoring, and translation (2, 34, 41, 47) is another feature which appears to be conserved in numerous RNA localization systems. The multistep process of RNA localization is dependent on specific trans-acting proteins. T...

Staufen Recruitment into Stress Granules Does Not Affect Early mRNA Transport in Oligodendrocytes

“…A 55-kDa Staufen 1 and a 53-kDa Staufen 2 isoforms are expressed in these cells, in agreement with previously reported data in other cell types (Wickham et al, 1999;Kiebler et al, 1999;Brizard et al, 2000;Duchaine et al, 2000Duchaine et al, , 2002Monshausen et al, 2001). We found that both, Staufen 1 and Staufen 2 form granules that associate with the cytoskeleton in the tubulin as well as in the actin-rich domains of the myelinating processes.…”

“…How Staufen molecules may contribute to stress granule structure, function or dynamics is unknown. Given their ability to form granules by binding to multiple RNA targets and oligomerization via the dsRBDs (Ferrandon et al, 1994;Micklem et al, 2000;Duchaine et al, 2000), one possibility is that Staufen molecules mediate SG assembly or stability. Moreover, stress granules do not form in the absence of microtubules (Loschi and Boccaccio, unpublished results; Ivanov et al, 2003), suggesting the participation of tubulindependent motors to gather the otherwise disperse RNPs.…”

“…A different type of RNA granules known as stress granules (SGs) appears transiently upon induction of cellular stress. SGs are large ribonucleoparticles (RNPs) and are thought to be in dynamic equilibrium with translating polysomes (Kedersha et al, 2000Anderson and Kedersha, 2002).The double-stranded RNA-binding protein Staufen emerges as a relatively ubiquitous RNA granule-forming factor (Ferrandon et al, 1994;Duchaine et al, 2000; Kiebler and DesGrosseillers, 2000;Micklem et al, 2000). This protein was initially described in Drosophila oocytes, where it is found in granules involved in microtubule-dependent localization of maternal mRNAs to define the anterior-posterior axis of the embryo (Lasko, 1999;Kloc et al, 2002).…”

Insights into mRNA transport in neurons

“…Finally, the isolation of Staufencontaining particles by Mallardo et al (19) has confirmed the previous suggestion that there are two pools of both Staufen 1 and 2 in mammalian neurons (24,25). One pool fractionates with endoplasmic reticulum (ER) in their biochemical experiments and may represent Staufen observed in large granules that colocalize with the rough ER within the soma or at synaptic terminals (22,26,28,32,33 …”

Genome wide identification of Staufen2-bound mRNAs in embryonic rat brains

“…All further manipulations of the brain lysates were performed at 4 o C. Cell lysates were centrifuged at 9,300 g for 10 min to remove nuclei and cell debris. After centrifugation, supernatants were incubated with rabbit polyclonal anti-Stau2 antibodies or pre-immune serum (4) for 2 hours at 4 o C, then with a 50% protein A-sepharose slurry for 2 hours at 4 o C as described before (26), except that Escherichia coli tRNA (Sigma-Aldrich) was added to a final concentration of 200 mg/ml. Immune complexes were washed five times with the lysis buffer and eluted from the resin by heating at 95 o C for 5 minutes in elution buffer (100 mM Tris-HCl pH 7.4, 200 mM DTT and 4% SDS).…”