60S ribosomes undergo initial assembly in the nucleolus before export to the cytoplasm and recent analyses have identi®ed several nucleolar pre-60S particles. To unravel the steps in the pathway of ribosome formation, we have puri®ed the pre-60S ribosomes associated with proteins predicted to act at different stages as the pre-ribosomes transit from the nucleolus through the nucleoplasm and are then exported to the cytoplasm for ®nal maturation. About 50 non-ribosomal proteins are associated with the early nucleolar pre-60S ribosomes. During subsequent maturation and transport to the nucleoplasm, many of these factors are removed, while others remain attached and additional factors transiently associate. When the 60S precursor particles are close to exit from the nucleus they associate with at least two export factors, Nmd3 and Mtr2. As the 60S pre-ribosome reaches the cytoplasm, almost all of the factors are dissociated. These data provide an initial biochemical map of 60S ribosomal subunit formation on its path from the nucleolus to the cytoplasm.

Eukaryotic mRNA degradation often occurs in a process whereby translation initiation is inhibited and the mRNA is targeted for decapping. In yeast cells, Pat1, Scd6, Edc3, and Dhh1 all function to promote decapping by unknown mechanism(s). We demonstrate that purified Scd6 and a region of Pat1 directly repress translation in vitro by limiting the formation of a stable 48S pre-initiation complex. Moreover, while Pat1, Edc3, Dhh1 and Scd6 all bind the decapping enzyme, only Pat1 and Edc3 enhance its activity. We also identify numerous direct interactions between Pat1, Dcp1, Dcp2, Dhh1, Scd6, Edc3, Xrn1, and the Lsm1-7 complex. These observations identify three classes of decapping activators that function either to directly repress translation initiation and/or stimulate Dcp1/2. Moreover, Pat1 is identified as critical in mRNA decay by first inhibiting translation initiation, then serving as a scaffold to recruit components of the decapping complex, and finally activating Dcp2.

No abstract

The mechanism by which miRNAs inhibit translation has been under scrutiny both in vivo and in vitro. Divergent results have led to the suggestion that miRNAs repress translation by a variety of mechanisms including blocking the function of the cap in stimulating translation. However, these analyses largely only examine the final output of the multistep process of translation. This raises the possibility that when different steps in translation are rate limiting, miRNAs might show different effects on protein production. To examine this possibility, we modeled the process of translation initiation and examined how the effects of miRNAs under different conditions might be explained. Our results suggest that different effects of miRNAs on protein production in separate experiments could be due to differences in rate-limiting steps. This analysis does not rule out that miRNAs directly repress the function of the cap structure, but it demonstrates that the observations used to argue for this effect are open to alternative interpretations. Taking all the data together, our analysis is consistent with the model that miRNAs may primarily repress translation initiation at a late step.

Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system.How to cite this article: WIREs RNA 2014, 5:747–763. doi: 10.1002/wrna.1244