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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2010.01.001 Byline: Vikram Govind Panse (1), Arlen W. Johnson (2) Abstract: In eukaryotic cells, ribosomes are pre-assembled in the nucleus and exported to the cytoplasm where they undergo final maturation. This involves the release of trans-acting shuttling factors, transport factors, incorporation of the remaining ribosomal proteins, and final rRNA processing steps. Recent work, particularly on the large (60S) ribosomal subunit, has confirmed that the 60S subunit is exported from the nucleus in a functionally inactive state. Its arrival in the cytoplasm triggers events that render it translationally competent. Here we focus on these cytoplasmic maturation events and speculate why eukaryotic cells have evolved such an elaborate maturation pathway. Author Affiliation: (1) Institute of Biochemistry (IBC), ETH Zurich, Schafmattstrasse 18, CH-8093, Zurich, Switzerland (2) Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas, USA

The ribosome stalk is essential for recruitment of translation factors. In yeast, PO and Rpl12 correspond to bacterial L10 and L11 and form the stalk base of mature ribosomes, whereas Mrt4 is a nuclear paralogue of PO. In this study, we show that the dual-specificity phosphatase Yvh 1 is required for the release of Mrt4 from the pre-60S subunits. Deletion of YVH1 leads to the persistence of Mrt4 on pre-60S subunits in the cytoplasm. A mutation in Mrt4 at the protein-RNA interface bypasses the requirement for Yvh1. Pre-60S subunits associated with Yvh1 contain Rpl12 but lack both Mrt4 and PO. These results suggest a linear series of events in which Yvh1 binds to the pre-60S subunit to displace Mrt4. Subsequently, PO loads onto the subunit to assemble the mature stalk, and Yvh1 is released. The initial assembly of the ribosome with Mrt4 may provide functional compartmentalization of ribosome assembly in addition to the spatial separation afforded by the nuclear envelope.

The partitioning of cells by a nuclear envelope ensures that precursors of ribosomes do not interact prematurely with other components of the translation machinery. Ribosomal subunits are assembled in nucleoli and exported to the cytoplasm in a CRM1/Ran--GTP-dependent fashion. Export of the large (60S) subunit requires a shuttling adaptor protein, NMD3, which binds to mature, correctly folded subunits. Immature or defective particles do not bind NMD3 and thus are excluded from the export pathway. This structural proofreading is extended into the cytoplasm, where it is believed that several energy-requiring steps release shuttling factors from the subunit, allowing it to function in translation.

The torpedo model of transcription termination by RNA polymerase II proposes that a 5-3 RNA exonuclease enters at the poly(A) cleavage site, degrades the nascent RNA, and eventually displaces polymerase from the DNA. The results suggest a unified allosteric/torpedo model in which Rat1 is not a dedicated termination factor, but is an integrated component of the cleavage/ polyadenylation apparatus.

Null mutants of XRN1, encoding the major cytoplasmic exoribonuclease in yeast, are viable but accumulate decapped, deadenylated transcripts. A screen for mutations synthetic lethal with xrnl[Delta] identified a mutation in CDC33, encoding eIF4E. This mutation (glutamate to glycine at position 72) affected a highly conserved residue involved in interaction with eIF4G. Synthetic lethality between xrnl and cdc33 was not relieved by high-copy expression of eIF4G or by disruption of the yeast eIF4E binding protein Caf20p. High-copy expression of a mutant eIF4G defective for eIF4E binding resulted in a dominant negative phenotype in an xrn1 mutant, indicating the importance of this interaction in an xrnl mutant. Another allele of CDC33, cdc33-1, along with mutations in CEG1, encoding the nuclear guanylyltransferase, were also synthetic lethal with xrn1[Delta], whereas mutations in PRT1, encoding a subunit of eIF3, were not. Mutations in CDC33, CEG1, PRT1, PAB1, and TIF4631, encoding eIF4G1, have been shown to lead to destabilization of mRNAs. Although such destabilization in cdc33, ceg1, and pab1 mutants can be partially suppressed by an xrn1 mutation, we observed synthetic lethality between xrn1 and either cdc33 or ceg1 and no suppression of the inviability of a pab1 null mutation by xrn1[Delta]. Thus, the inhibition of mRNA turnover by blocking Xrn1p function does not suppress the lethality of defects upstream in the turnover pathway but it does enhance the requirement for [sup.7]mG caps and for proper formation of the eIF4E/eIF4G cap recognition complex.

During ribosome biogenesis in eukaryotes, nascent subunits are exported to the cytoplasm in a functionally inactive state. 60S subunits are activated through a series of cytoplasmic maturation events. The last known events in the cytoplasm are the release of Tif6 by Efl1 and Sdo1 and the release of the export adapter, Nmd3, by the GTPase Lsg1. Here, we have used cryo-electron microscopy to determine the structure of the 60S subunit bound by Nmd3, Lsg1, and Tif6. We find that a central domain of Nmd3 mimics the translation elongation factor eIF5A, inserting into the E site of the ribosome and pulling the L1 stalk into a closed position. Additional domains occupy the P site and extend toward the sarcin-ricin loop to interact with Tif6. Nmd3 and Lsg1 together embrace helix 69 of the B2a intersubunit bridge, inducing base flipping that we suggest may activate the GTPase activity of Lsg1. [ABSTRACT FROM AUTHOR]

In eukaryotic ribosome biogenesis, U3 snoRNA base pairs with the pre-rRNA to promote its processing. However, U3 must be removed to allow folding of the central pseudoknot, a key feature of the small subunit. Previously, we showed that the DEAH/ RHA RNA helicase Dhr1 dislodges U3 from the pre-rRNA. DHR1 can be linked to UTP14, encoding an essential protein of the preribosome, through genetic interactions with the rRNA methyltransferase Bud23. Here, we report that Utp14 regulates Dhr1. Mutations within a discrete region of Utp14 reduced interaction with Dhr1 that correlated with reduced function of Utp14. These mutants accumulated Dhr1 and U3 in a pre-40S particle, mimicking a helicase-inactive Dhr1 mutant. This similarity in the phenotypes led us to propose that Utp14 activates Dhr1. Indeed, Utp14 formed a complex with Dhr1 and stimulated its unwinding activity in vitro. Moreover, the utp14 mutants that mimicked a catalytically inactive dhr1 mutant in vivo showed reduced stimulation of unwinding activity in vitro. Dhr1 binding to the preribosome was substantially reduced only when both Utp14 and Bud23 were depleted. Thus, Utp14 is bifunctional; together with Bud23, it is needed for stable interaction of Dhr1 with the preribosome, and Utp14 activates Dhr1 to dislodge U3. [ABSTRACT FROM AUTHOR]