Pasos a la construcción de relaciones de confianza

The exosome is involved in the maturation and degradation of many different RNAs and it soon became obvious that it needs specific cofactors which help to distinguish between all these RNA species. In the last years a diverse array of activators and cofactors have been identified that activate the exosome on defined classes of transcripts. The most important activators in the context of a yeast cell are shown in figure 27.

6.4.1 Mtr4 and the TRAMP Complex in the Nucleus

To access the active site of the exosome, RNA substrates have to pass through a narrow pore that makes it impossible for double-stranded or structured RNA to be degraded. The DExH-box RNA helicase Mtr4 (also known as Dob1) has nucleic acid dependent ATPase activity, can unwind duplex RNA in the 3' → 5' direction and binds to single stranded RNA, especially to short poly(A) substrates (Bernstein et al., 2008). Mtr4 was shown to be required for most nuclear activities of the exosome (LaCava et al., 2005; Vanacova et al., 2005). Mtr4 is believed to assist the exosome as it moves through structured regions of its RNA substrates and thereby enables the RNA to reach the active site behind the exosome pore.

Figure 27: Activation of the exosome by different cofactors in a yeast cell. Many different substrates and activating cofactors have been identified to activate the exosome in the nucleus and the cytoplasm of a yeast cell. The various pathways are described in chapter 6.4 (adapted from Houseley et al., 2006).

The action of Mtr4 on some RNAs occurs in the context of the TRAMP complexes, which consists of a Poly(A) polymerase (Trf4 or Trf5), a zinc-knuckle protein (Air1 or Air2) and the Mtr4 protein (LaCava et al., 2005; Wyers et al., 2005; Vanacova et al., 2005; Kadaba et al., 2004 and 2006). The TRAMP complex is thought to bind RNA through the zinc-knuckle, putative

RNA-binding domains that are present in Air1 and Air2. Mtr4 might then actively recruit the exosome to the RNA, as its depletion leads to RNA hyper-adenylation in vivo, which indicates that polyadenylation has been uncoupled from degradation (Houseley and Tollervey, 2006). The Trf4/5 protein adds short poly(A) tails to the substrate, thereby forming a favorable substrate for the exosome, and Mtr4 helps to dissolve RNA secondary structures.

Mtr4 processes a diverse array of pre-RNAs together with the exosome: maturation of the 5.8S ribosomal RNA and processing of snRNAs such as the U4 small nuclear RNA (Allmang et al., 1999). As part of the TRAMP complex it is additionally involved in the degradation of defective tRNAs and precursor ribosomal RNAs before they are exported to the cytoplasm.

Two distinct forms of TRAMP complexes, TRAMP4 and TRAMP5 have been identified in yeast. They show clear substrate preferences in vivo, but their mode of selectivity is still unclear. Both the exosome and the TRAMP4 complex interact with an additional RNA binding protein complex called the nuclear pre-mRNA down-regulation complex 1 (Ndr1) (Vasiljeva and Buratowski, 2006). This complex consists of the RNA helicase Sen1 and the proteins Ndr1 and Nab3 (nuclear polyadenylated RNA-binding) that recognize specific sequence motifs on RNAs (Steinmetz and Brow, 1998; Carroll et al., 2004). This complex is required for transcription termination of snRNA and snoRNA genes (Steinmetz et al., 2001). In vitro the Nrd-complex can directly stimulate exosome degradation of substrates with Ndr1- and Nab3-binding motifs. In vivo it probably helps to bring the exosome to specific RNA substrates.

6.4.2 The Ski Complex

In the cytoplasm the exosome needs the specific cofactor Ski7, a GTPase with homology to translation factors for most of its activities (Araki et al., 2001). In addition, the Ski2, Ski3 and Ski8 proteins from a complex known as the Ski complex are involved in most of the cytoplasmic activities of the exosome:

The ski (superkiller) genes were identified via mutations that cause overexpression of a killer toxin encoded by the endogenous double-stranded RNA (Toh et al., 1978). It could be shown that not only the exosome, but the proteins Ski2, Ski3 and Ski8 are required for the 3'-mRNA degradation (Anderson and Parker, 1998). Ski2p is a putative RNA helicase with homology to Mtr4 (Widner and Wickner, 1993), Ski3p is a tetratricopeptide repeat (TPR) protein (Rhee et al., 1989) and Ski8p contains five WD-40 (beta-transducin) repeats (Matsumoto et al., 1993). The

three Ski proteins form a stable complex, which is localized in the cytoplasm (Brown et al., 2000). Mutations in the three genes inhibit 3' → 5' mRNA decay, but do not affect other functions of the exosome (Anderson and Parker, 1998). Therefore the Ski-complex was proposed to be the cofactor of the exosome in the degradation of mRNAs in yeast.

Ski7, another member of the Ski proteins, is permanently associated with the exosome in the cytoplasm. The ski7 gene was initially identified as one of the ski gene family (Benard et al., 1999). It was shown that Ski7 is also required for 3' → 5' mRNA degradation and acts in the same pathway as the Ski-complex, because deletion of the ski7 gene caused impaired 3'-mRNA decay similar to ski2, ski3 or ski8 deletions (van Hoof et al., 2002). When introducing a mutation in one of the genes of the Ski-complex, the complex did not assemble any more. In contrast, the Ski complex was still intact in the ski7 mutant, suggesting that Ski7 is not a member of the Ski complex and is not required for the formation of the complex (Brown et al., 2000). Therefore it seems that the role of Ski7 in mRNA degradation differs from the rest of the Ski complex.

The Ski complex together with the exosome and Ski7 function in the nonsense-mediated decay and the non-stop decay mRNA surveillance pathways (Mitchell and Tollervey, 2003; Lejeune et al., 2003; van Hoof et al., 2002).

6.4.3 Sequence Specific Cofactors

Some cofactors were identified that recruit the exosome to RNA substrates with specific sequences. They include the Nrd1 protein (see 6.4.1), which enhanced the RNA degradation activity of the exosome in vitro and in vivo (Arigo et al., 2006; Vasiljeva and Buratowski; 2006). The precursors of many RNA species contain Ndr1-binding sites, which probably function as targets for exosome-mediated degradation (Steinmetz et al., 2001). It is likely that during normal processing of RNAs with Ndr1 binding site, this binding site is removed and hence only correctly processed RNAs are protected from exosome-mediated degradation.

In human cells, ARE (AU-rich elements) mediated degradation is an important mRNA turnover pathway that involves the recruitment of the exosome (Mukherjee et al., 2002). AREs are found in the 3'-untranslated region of many mRNAs that code for proto-oncogenes, nuclear transcription factors and cytokines. They represent the most common determinant of RNA stability in mammalian cells and are known to target mRNAs for rapid degradation (Barreau et al., 2005). Mukherjee et al. (2002) suggested that certain subunits of the human exosome specifically bind to AREs causing an ARE-dependent degradation of mRNAs. Other studies have

shown that several ARE-binding proteins, for instance TTP (Tristetraprolin) and KSRP (KH splicing regulatory protein), are physically associated in vitro with the exosome and are required for preferential degradation of ARE-containing mRNAs by the exosome (Chen et al., 2001; Gherzi et al., 2004). Tran et al. (2004) proposed the involvement of the RNA helicase RHAU. RHAU displaces mRNA stabilizing proteins from the ARE and then recruits the exosome to the RNA to facilitate mRNA decay. ARE-mediated decay can also occur in yeast, where the activity requires a TTP homolog – the role of the exosome has not yet been reported.

It is still not much known about the way all these cofactors are able to activate the exosome. However, a physical recruitment of the exosome to specific RNA substrates seems to play a key role in stimulating RNA degradation both in the nucleus and the cytoplasm.