S. cerevisiae
As described in chapter 3.1, She2p can bind to non-specific stem-loop containing RNAs. Surprisingly, the difference in affinity between specific bud-localizing mRNAs and non- specific unrelated RNAs was only moderate. The protruding helix E and the very C-terminus of She2p were shown to participate in ASH1-mRNA binding, and thus, provide RNA-binding specificity. Furthermore, bud-localizing mRNAs are bound by an extended surface region on She2p tetramers provided by the distinct RNA-binding motifs. Selective, but moderate reduction of ASH1-mRNA binding completely abolishes formation of a cytoplasmic transport complex and prevents mRNA localization in vivo. Thus, I conclude that She2p mediates the specificity for the interaction with ASH1 mRNA.
However, it is absolutely clear that the complex of She2p and bud-localizing mRNAs must be stabilized during mRNP assembly in vivo. This assumption is supported by the observation that complexes consisting of only She2p and RNA are rather transient in vitro (this study). It is likely that the She2p:mRNA association is stabilized already in the nucleus. She2p is proposed to bind ASH1 mRNA co-transcriptionally (Du et al., 2008), which might result in the formation of an unstable priming complex (Figure 36). Potential candidates for joining such a priming complex are the proteins Loc1p, Puf6p, and Khd1p. All of them are found in the nucleus, are reported to bind to ASH1 mRNA, and are required for translational silencing of
ASH1 (Deng et al., 2008; Du et al., 2008; Gu et al., 2004; Irie et al., 2002; Long et al., 2001; Paquin et al., 2007; Shen et al., 2009). However, none of these proteins is known to bind to all four ASH1-localization elements. Thus, it remains unclear how the She2p:mRNA interaction might be stabilized in the nucleus. Preliminary studies indicate that the strictly nucleolar protein Loc1p interacts with all ASH1 zipcodes (this study, data not shown). Loc1p in turn,
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was recently identified to interact with She2p in vivo (Shen et al., 2009) and in vitro (this study, data not shown). Thus, one can assume that Loc1p might stabilize the interaction of She2p with its target mRNA, potentially by remodeling the mRNP complex. Puf6p binds to the
ASH1-E3 zipcode as well as to She2p (Gu et al., 2004; Shen et al., 2009), whereas Khd1p binds to sequences adjacent to the ASH1-E1 zipcode (Irie et al., 2002). Their potential nuclear role in stabilization of She2p:mRNA complexes, however, remains unknown. Both proteins are also supposed to accompany the cytoplasmic translocation complex, thereby preventing premature ASH1 translation (Gu et al., 2004; Paquin et al., 2007). By this means, Khd1p and Puf6p could positively affect the complex stability of She2p and the E1 and E3 zipcode, respectively (Figure 36).
In the cytoplasm, the adapter protein She3p is proposed to strengthen the She2p:mRNA complex through interaction of its C-terminal domain with She2p (Figure 36) (Böhl et al., 2000; Long et al., 2000). However, a stabilizing effect of She3p on mRNA binding by She2p remains to be demonstrated in vitro. Unfortunately, any attempt to purify stable She3p fragments for corresponding in vitro studies failed due to rapid protein degradation (data not shown). Likewise, short She3p peptides, which were apparently identified to interact with She2p by screening a She3p-peptide library, could not be bound to She2p (data not shown). Thus far, the She3p-interacting region of She2p is unknown. It was suggested previously that She3p interacts with a heterologous region consisting of the hydrophobic upper surface of She2p and mRNA (Gonsalvez et al., 2003; Niessing et al., 2004). This idea is based on the identification of a yeast strain encoding a She2p variant with an exchange of amino acid leucine 130 into serine (Gonsalvez et al., 2003). The She2p mutant failed to interact with She3p in vivo. Consistently with this, the She2p-L130Y mutant showed reduced ASH1-E3 binding (Niessing et al., 2004 and this study). However, with the identification of She2p- tetramer formation through the upper surface region, I conclude that the previously observed functional defects directly resulted from a disrupted oligomerization state of She2p. Thus, the upper surface of She2p is extremely unlikely to interact directly with She3p.
Besides the upper surface, there is only one additional small hydrophobic patch remaining that could be involved in She3p binding. Interestingly, this region is located on the lower half of She2p and involves parts of the recently identified non-classical nuclear localization signal (Figure 4) (Shen et al., 2009). In principle, She2p could interact through a similar surface region both with the importin α Srp1p and She3p, since both associations would be restricted to different time points during mRNP assembly. In this scenario, cargo-free She2p is imported
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into the nucleus via its interaction with Srp1p. In the nucleus, She2p selectively recognizes bud-localizing mRNAs, traverses the nucleolus, and is exported to the cytoplasm in an mRNA cargo-dependent manner. In the cytoplasm, the She2p:mRNA complex is stabilized by interaction with She3p through a hydrophobic patch at the lower half of She2p. However, it is also possible that She3p interacts with a different region in She2p. Likewise, She3p might recognize a heterologous surface of She2p and mRNA, which may be located not on the top of She2p, but rather on the side.
The idea that an RNA-priming complex might form upon mRNA recognition might also apply to Drosophila Staufen. In a fundamental study, Staufen was reported to bind to RNA with extensive secondary structures, such as U1 and U2 RNAs, adenoviral VA1 RNA, and the 3’ UTR of bicoid mRNA (St Johnston et al., 1992). RNAs without any predicted secondary structures were not bound. Interestingly, comparable attributes also apply to She2p (chapter 3.1). In vivo injection experiments in Drosophila embryos using a variety of RNAs showed that Staufen-containing mRNPs only form and localize efficiently upon injection of the bicoid
3’ UTR, the target mRNA of Staufen (Ferrandon et al., 1994). Injection of highly concentrated heterologous stem-loop RNAs resulted in the formation of a few, yet immobile particles.
Figure 36: Sequential ASH1-mRNP assembly in S. cerevisiae. Step 1: She2p binds ASH1 mRNA co- transcriptionally to form a priming complex. Step 2: The priming complex is passed through the nucleolus and is read-out by the trans-acting factors Loc1p, Khd1p, and Puf6p, which results in assembly of a translationally silent mRNP. Before the pre-mRNP is exported into the cytoplasm, Loc1p dissociates from the complex and remains in the nucleolus. Step 3: In the cytoplasm, the ASH1 pre-mRNP associates with She3p, which stabilizes the complex. She3p itself is bound to Myo4p, which then transports the stable, translationally repressed mature mRNP to the bud tip.
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Based on this finding the authors concluded that the interaction of Staufen with bicoid is specific and that the interaction of Staufen with non-specific RNAs occurs in a fundamentally distinct way. Thus, it might be possible that also Staufen forms an initial and specific priming complex, which is interpreted and stabilized by accessory factors during mRNP assembly.