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. 2003 Feb 18;100(4):2100-5.
doi: 10.1073/pnas.0334355100.

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

Massimo Mallardo  1 , Anke Deitinghoff, Juliane Müller, Bernhard Goetze, Paolo Macchi, Christopher Peters, Michael A Kiebler
Affiliations

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

Massimo Mallardo et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Figures

Figure 1
Figure 1
Identification of soluble Staufen complexes. (A) Fractionation behavior of Staufen complexes on centrifugation. Rat forebrain homogenates (H) were differentially centrifuged. Equal protein amounts of both pellets and supernatants were resolved by SDS/PAGE and blotted against Staufen-specific antibodies. The majority of both Stau1 and Stau2 proteins were detected in the P100. However, a small fraction of both Staufen proteins was routinely found in the S100. Please note: S100 contains 3-fold more total protein than P100. (B) Five different Staufen complexes of various sizes were detected by GF. S100 was applied to a commercial GF column, 3-ml fractions were collected, and aliquots were processed for Western blotting. Blots were immunodecorated for Stau1, Stau2, calnexin, L7a, β-tubulin, KHC, and synaptophysin. Five different Staufen complexes were detected: (i) high-molecular weight granules for Stau1 (fractions 90–96) and (ii) Stau2 (fractions 99–105), that cofractionate with ER and ribosomal markers, respectively; (iii) particles for Stau1 (peak in fraction 120), which cofractionate with KHC, and (iv) Stau2 (peak in fraction 138), which do not contain both ER and ribosomal markers; (v) free Stau1 (peaks in fractions 168 and 189/192).
Figure 2
Figure 2
Stau1 particles but not Stau1 granules are sensitive to RNase treatment. (A) RNase pretreatment of S16 supernatants released Stau1 from the pellet into the S100. Homogenates (H) were prepared and the resulting S16 was treated with RNase A or mock-treated. Samples were processed as described in Fig. 1. (B) RNase A pretreatment partially disassembled Stau1 particles. RNase A pretreated or mock-treated S100 was analyzed by GF. RNase pretreatment selectively disassembled Stau1 particles (marked by bars) but did not affect the large Staufen granules that contain ribosomes and ER.
Figure 3
Figure 3
Staufen particles are enriched in specific mRNAs. (A) Selective enrichment of mRNAs within Stau1 particles. Several GF were performed and the different Staufen complexes were pooled. The associated RNA was detected by radioactive reverse transcription with oligo(dT) primers. Whereas Staufen particles contain only a small amount of total RNA (data not shown), there is a 15.1- and 4.4-fold enrichment for mRNA in both Stau1 and Stau2 particles compared with the free cytosolic mRNA (monomers, pool 5). (B) The same RNAs from A were visualized by autoradiography. Staufen particles contain a specific subset of small RNA fragments (lanes 3 and 4) compared to the Staufen granules (lanes 1 and 2) and the control (Ctr). (C and D) The dendritically localized RNA BC1 is highly enriched in Stau1 particles. RNA from single GF fractions were analyzed by Northern blot (C) or RT-PCR (D). Whereas 18S rRNA is predominantly found in the Stau2 granule pool overlapping with the position of ribosomes, the BC1 transcript is highly enriched in the Stau1 particle pool. In addition, the dendritically transported CaMKIIα, but not cell body-restricted RNAs, e.g., GAPDH and β-actin could be detected in the Stau1 particle pool. Stau1 G, Stau1 granules; Stau1 P, Stau1 particles; *, negative control for PCR.
Figure 4
Figure 4
CaMKIIα and BC1 RNA colocalize with Stau1–HA in the same particles within dendrites of transfected hippocampal neurons. In situ hybridization for CaMKIIα, BC1, and GAPDH RNAs was combined with immunocytochemistry for Stau1–HA detected with anti-HA antibodies. Phase contrast (PC) as well as corresponding fluorescent images of the same neurons are shown for each RNA. High magnification images of selected dendrites as well as the merge (within the computer image) are shown (Insets). (Magnifications: ×40.)
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Comment in

  • Insights into mRNA transport in neurons.
    Roegiers F. Roegiers F. Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1465-6. doi: 10.1073/pnas.0630376100. Epub 2003 Feb 10. Proc Natl Acad Sci U S A. 2003. PMID: 12578967 Free PMC article. No abstract available.

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