614 volume 16 number 6 June 2009 nature structural & molecular biology

priming to determine the 3′ end of transcripts would fail to identify uridylated 3′ ends; hence, such mRNAs are not present in EST databases. Circular RT-PCR is necessary to precisely determine the 3′ end of an mRNA, and it was only the application of this technique that allowed the detection of the uridylated RNAs.

1. Rissland, O.S., Mikulaslova, A. & Norbury, C.J. Mol. Cell. Biol. 27, 3612–3624 (2007).

2. Rissland, O.S. & Norbury, C.J. Nat. Struct. Mol. Biol. 16, 616–623 (2009).

3. Trippe, R. et al. RNA 12, 1494–1504 (2006).4. Kwak, J.E. & Wickens, M. RNA 13, 860–867 (2007).5. Li, J., Yang, Z., Yu, B., Liu, J. & Chen, X. Curr. Biol. 15,

1501–1507 (2005).6. Heo, I. et al. Mol. Cell 32, 276–284 (2008).7. Shen, B. & Goodman, H.M. Science 306, 997 (2004).8. Ibrahim, F., Rohr, J., Jeong, W.J., Hesson, J. & Cerutti, H.

Science 314, 1893 (2006).9. Mullen, T.E. & Marzluff, W.F. Genes Dev. 22, 50–65

(2008).10. Song, M.G. & Kiledjian, M. RNA 13, 2356–2365

(2007).11. Ernst, N.L., Panicucci, B., Carnes, J. & Stuart, K. RNA

15, 947–957 (2009).

Other questions concern how the length of the uridine tail is controlled and whether oligouridylation is reversible. Many modifications of macromolecules are reversible; for example, adenosines are removed as well as added in the cytoplasm to alter the length of the poly(A) tail of mRNAs. In trypanosomes, two 3′-to-5′ exonucleases specific for uridines have been described11, and these enzymes have a role in modulating RNA editing. It certainly seems possible that similar enzymes that can remove uridines may be present in other organisms, and that uridine addition may be reversible under some conditions. Thus, some of the monouridylated mRNAs may represent RNAs that were oligouridylated and subsequently deuridylated, leaving one uridine on the mRNA.

This modification has escaped previous detection because methods that use oligo(dT)

and hence these degradation intermediates were not detected.

Many questions remain. Is there specificity for particular mRNAs that undergo oligouridylation? At least three different mRNAs were shown to be uridylated in this study, and there was one example of an mRNA that is not uridylated. Thus, this pathway probably acts on a large proportion of mRNAs. Uridylation can clearly activate decapping and result in degradation. All the mRNAs studied also underwent degradation by deadenylation, so there seem to be two redundant pathways for degrading mRNAs. Cid1 readily adds many uridines to synthetic transcripts when complexes containing Cid1 are purified from cells1 or when Cid1 is expressed in X. laevis oocytes4, although most of the mRNAs isolated from S. pombe contained only a single uridine at the 3′ end.

Apoptosis, a process important for clearing damaged or infected cells, is the induction of cell suicide and can be triggered by either intrinsic cues or activation of the relevant pathways by external ligands. One pathway of extrinsic signaling occurs through Apo2L/TRAIL which activates death receptors (DR) 4 and 5. Such activation triggers cell death through initiation of a cascade involving caspases. A primary responder in this pathway is caspase-8, known to be recruited via its Death effector domains (DEDs) to the activated DRs to form a complex known as the death-induced signaling complex (DISC). In this context, caspase-8 then self-cleaves; its DED remains associated with DISC, whereas the remainder of the protein comprising the catalytically active regions is released and in turn activates downstream executioner caspases in the apoptosis pathway. How the activation of caspase-8 is stably maintained and regulated had not been entirely clear, but Ashkenazi and colleagues (Cell 137, 721–735, 2009) have now found that, upon DR activation, caspase-8 becomes polyubiquitinated (polyUb), a modification key to its activation.

Activation of DR4/5 by Apo2L/TRAIL was found to lead to localization of caspase-8 to membrane and raft/cytoskeletal cell fractions, with ubiquitinated caspase-8 being found largely in the latter compartments. The authors also found that ubiquitinated caspase-8, as well as caspase-8 catalytic activity, primarily associated with high-molecular-weight protein aggregates. To identify the enzyme mediating ubiquitination, the authors looked at the components of DISC using tandem mass spectometry and found that the Ub E3 ligase Cullin-3 (CUL3), co-purifies with DISC in cells stimulated to undergo apoptosis. CUL3 associates with caspase-8, suggesting that it promotes caspase-8 ubiquitination. Indeed, siRNA-mediated knockdown of CUL3 prevented caspase-8 ubiquitination without altering its levels or recruitment to DISC; however, this treatment does

decrease caspase-8 cleavage and activity in high-molecular-weight aggregates, implying that CUL3-mediated ubiquitination is required for the subsequent steps of caspase-8 activation. The deubiquitinating enzyme A20 also associates with caspase-8 and is probably responsible for deubiquitination. Polyubiquitination, rather than monoubiquitination, is key to caspase-8 activation, as artificial constructs encoding polyUb

fused linearly to the enzyme’s C terminus generated a highly active version of the caspase in cells.

What is the precise role of polyUb-caspase-8? The authors found that treatment with MG132, a proteasomal inhibitor, did not increase ubiquitinated caspase-8 levels, suggesting that the modified protein is not being routed for rapid degradation through this

pathway. However they did observe that another protein, the ubiquitin binding protein p62, was also associated with the DISC in cells stimulated to undergo apoptosis in a way that is dependent on CUL3. Although p62 acts downstream of caspase-8 ubiquitination, it is important for its activation and aggregation in cytoplasmic speckles as well as in high-molecular-weight aggregates. Upon co-transfection with CUL3, caspase-8 accumulates in cytoplasmic speckles, which also contain p62 (left), a localization diminished upon siRNA-mediated reduction of p62 (right) or CUL3. Together, these data suggest that, upon DR activation, caspase-8 first localizes to the plasma membrane with DISC, but this is followed by CUL3 modification of caspase-8, allowing p62 to bind the polyubiquitinated caspase and augment its full activation and proteolytic processing. Altogether, this uncovers a mechanism whereby polyUb mediates stabilization and subcellular aggregation of a protein in its active form and provides an additional layer of regulation at the initiating steps of extrinsic apoptosis induction. Sabbi Lall

Activating apoptosis

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