Regulation of ribosome detachment from the mammalian endoplasmic reticulum membrane

In current models, protein translocation in the endoplasmic reticulum (ER) occurs in the context of two cycles, the signal recognition particle (SRP) cycle and the ribosome cycle. Both SRP and ribosomes bind to the ER membrane as a consequence of the targeting process of translocation. Whereas SRP release from the ER membrane is regulated by the GTPase activities of SRP and the SRP receptor, ribosome release from the ER membrane is thought to occur in response to the termination of protein synthesis. We report that ER-bound ribosomes remain membrane-bound following the termination of protein synthesis and in the bound state can initiate the translation of secretory and cytoplasmic proteins. Two principal observations are reported. 1) Membrane-bound ribosomes engaged in the synthesis of proteins lacking a signal sequence are released from the ER membrane as ribosome-nascent polypeptide complexes. 2) Membrane-bound ribosomes translating secretory proteins can access the translocon in an SRP receptor-independent manner. We propose that ribosome release from the ER membrane occurs in the context of protein translation, with release occurring by default in the absence of productive nascent polypeptide-membrane interactions.     

mRNA translation is compartmentalized to the endoplasmic reticulum following physiological inhibition of cap-dependent translation

Eukaryotic cells utilize a cycle of ribosome trafficking on the endoplasmic reticulum (ER) to partition mRNAs between the cytosol and ER compartments. In this process, ribosomes engaged in the synthesis of signal sequence-bearing proteins are trafficked to the endoplasmic reticulum via the signal-recognition particle pathway and are released from the ER upon translation termination. Though the processes governing ribosome trafficking to the ER are well understood, little is known regarding the complementary ribosome release process. In this study, Coxsackie B virus (CBV) infection was used to inactivate the initiation stage of protein synthesis, thereby limiting translation to the elongation and termination stages. Ribosome partitioning between the cytosol and ER compartments was examined to determine the role of termination in ribosome release from the ER. CBV infection resulted in efficient cleavage of eIF4G and PABP, coincident with polyribosome breakdown in the cytosol and ER compartments. Termination resulted in the continued association of ribosomes with the ER compartment, rather than the expected process of ribosome release. Analyses of ribosome/mRNA loading patterns in the cytosol and ER revealed that CBV infection was accompanied by a suppression of mRNA translation in the cytosol and the sustained, although reduced, translation in the ER compartment. Direct biosynthetic labeling experiments demonstrated that protein synthesis on the ER was enhanced relative to the cytosol following CBV infection. In total, these data demonstrate that ribosome and mRNA release from the ER is regulated independent of translation termination and identify the ER as a privileged site for protein synthesis.     

Why are both ends of the polypeptide chain on the outside of proteins?

Protein folding starts before the whole polypeptide has been synthesized by the ribosome. No matter how long the polypeptide is or how intricate the fold, both ends of the chain always end up on the surface. From a topological point of view, this is surprising; one would have expected to find the starting (N-terminal) end inside the core of the folded protein, just as in a ball of yarn. We suggest here that the reason for this apparent paradox is that the first amino acid of the emerging polypeptide chain is gripped during protein synthesis, perhaps by the ribosome, and is not released until the whole polypeptide has been synthesized. This binding would greatly decrease the degrees of freedom for the protein-folding process and could also explain why knots are so rare in proteins. Gripping would also guarantee that the N-terminal is accessible on the protein surface as required for binding of ubiquitin, which regulates the natural degradation of proteins and avoids buildup of protein aggregates, such as those found in Huntington's, Alzheimer's, Parkinson's, and other neurodegenerative diseases.     

Mechanisms of In Vivo Ribosome Maintenance Change in Response to Nutrient Signals

Control of protein homeostasis is fundamental to the health and longevity of all organisms. Because the rate of protein synthesis by ribosomes is a central control point in this process, regulation, and maintenance of ribosome function could have amplified importance in the overall regulatory circuit. Indeed, ribosomal defects are commonly associated with loss of protein homeostasis, aging, and disease (1, 2, 3, 4), whereas improved protein homeostasis, implying optimal ribosomal function, is associated with disease resistance and increased lifespan (5, 6, 7). To maintain a high-quality ribosome population within the cell, dysfunctional ribosomes are targeted for autophagic degradation. It is not known if complete degradation is the only mechanism for eukaryotic ribosome maintenance or if they might also be repaired by replacement of defective components. We used stable-isotope feeding and protein mass spectrometry to measure the kinetics of turnover of ribosomal RNA (rRNA) and 71 ribosomal proteins (r-proteins) in mice. The results indicate that exchange of individual proteins and whole ribosome degradation both contribute to ribosome maintenance in vivo. In general, peripheral r-proteins and those with more direct roles in peptide-bond formation are replaced multiple times during the lifespan of the assembled structure, presumably by exchange with a free cytoplasmic pool, whereas the majority of r-proteins are stably incorporated for the lifetime of the ribosome. Dietary signals impact the rates of both new ribosome assembly and component exchange. Signal-specific modulation of ribosomal repair and degradation could provide a mechanistic link in the frequently observed associations among diminished rates of protein synthesis, increased autophagy, and greater longevity (5, 6, 8, 9).     

Control of rRNA expression in Escherichia coli

The control of ribosome synthesis has been a major focus in molecular biology for over 50 years. As protein synthesis is an essential, yet energetically costly, process, all cells (from bacteria to mammals) devote complex regulatory networks to fine-tune the expression of ribosomal RNA (and therefore ribosome synthesis) to the nutritional environment. In Escherichia coli, ribosomal RNA promoters are among the strongest in the cell and are regulated by trans-acting proteins (Fis and H-NS) and small molecules (guanosine 5'-diphosphate 3'-diphosphate and initiating nucleoside triphosphates). Recent work has dissected many of the molecular mechanisms responsible for the strength and regulation of rRNA promoters.     

In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein

The HIV-2 genomic RNA serves both as a messenger for protein synthesis and as a genome for viral assembly and particle production. Our previous work has shown that the HIV-2 genomic RNA encodes two additional Gag proteins that are N-terminal truncated isoforms of the p57 Gag polyprotein. In this study, by the use of mono- and bicistronic RNAs we show that translation at the three AUGs is driven by three distinct and independent internal ribosome entry segments both in vitro and ex vivo. Furthermore we used the recombinant Gag and HIV-2 protease to show that, in vitro, translation is tightly regulated by these two viral proteins. This regulation is exerted both at the level of protein production and also on the selection of the AUG initiation site which changes the ratio at which the three different Gag isoforms are produced.     

A large presecretory protein translocates both cotranslationally, using signal recognition particle and ribosome, and post-translationally, without these ribonucleoparticles, when synthesized in the presence of mammalian microsomes

Translocation of large presecretory proteins into the mammalian endoplasmic reticulum requires the ribonucleoparticles, signal recognition particle, and ribosome and is tightly coupled to ongoing protein synthesis. We have shown previously that small presecretory proteins can translocate post-translationally in a reaction that does not require these ribonucleoparticles. We now report that one large protein, a synthetic hybrid between preprocecropin A and dihydrofolate reductase, translocates both cotranslationally (with the aid of signal recognition particle and ribosome) and post-translationally (without the involvement of these ribonucleoparticles) during its in vitro synthesis in the presence of dog pancreas microsomes. The distinction between these two modes of translocation was made possible by adding methotrexate to the translocation reaction. Methotrexate can only form a tight complex with those preprocecropin A-dihydrofolate reductase hybrid chains that have completed their synthesis and folded, but in forming this tight complex, this drug prevents translocation of the dihydrofolate reductase domain across the membrane.     

A new face on apoptosis: death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins

Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography-tandem mass spectrometry revealed that the proapoptotic proteins, death-associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways.     

Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly

Two strains of Saccharomyces cerevisiae were constructed that are conditional for synthesis of the 60S ribosomal subunit protein, L16, or the 40S ribosomal subunit protein, rp59. These strains were used to determine the effects of depriving cells of either of these ribosomal proteins on ribosome assembly and on the synthesis and stability of other ribosomal proteins and ribosomal RNAs. Termination of synthesis of either protein leads to diminished accumulation of the subunit into which it normally assembles. Depletion of L16 or rp59 has no effect on synthesis of most other ribosomal proteins or ribosomal RNAs. However, most ribosomal proteins and ribosomal RNAs that are components of the same subunit as L16 or rp59 are rapidly degraded upon depletion of L16 or rp59, presumably resulting from abortive assembly of the subunit. Depletion of L16 has no effect on the stability of most components of the 40S subunit. Conversely, termination of synthesis of rp59 has no effect on the stability of most 60S subunit components. The implications of these findings for control of ribosome assembly and the order of assembly of ribosomal proteins into the ribosome are discussed.     

The ribosome: lifting the veil from a fascinating organelle

It was first suggested that the ribosome is associated with protein synthesis in the 1950s. Initially, its components were revealed as surface-accessible proteins and as molecules of RNA apparently providing a scaffold for subunit shape. Attributing function to the proteins proved difficult, although bacterial protein L11 proved essential for binding one of the decoding protein release factors (RFs). With the discovery that RNA could be a catalyst, interest focussed on the rRNA that, in partnership with mRNA and tRNAs, could potentially mediate the chemical reaction underlying protein synthesis. rRNA interactions and conformational changes were invoked as key elements that facilitated function. The decoding RFs, which are proteins, are exceptions to this rule because they usurp a tRNA function in mediating stop signal recognition. Cryoelectron microscopy and associated image reconstruction technology have now given dramatic snapshots of almost every step of protein synthesis, and X-ray crystallography has revealed, at last, the subunits and monomeric ribosome in exquisite atomic detail.     

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells. A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results. This procedure is readily adaptable to mammalian cells.     

Early rRNA processing is a stress-dependent regulatory event whose inhibition maintains nucleolar integrity

The production of ribosomes is an energy-intensive process owing to the intricacy of these massive macromolecular machines. Each human ribosome contains 80 ribosomal proteins and four non-coding RNAs. Accurate assembly requires precise regulation of protein and RNA subunits. In response to stress, the integrated stress response (ISR) rapidly inhibits global translation. How rRNA is coordinately regulated with the rapid inhibition of ribosomal protein synthesis is not known. Here, we show that stress specifically inhibits the first step of rRNA processing. Unprocessed rRNA is stored within the nucleolus, and when stress resolves, it re-enters the ribosome biogenesis pathway. Retention of unprocessed rRNA within the nucleolus aids in the maintenance of this organelle. This response is independent of the ISR or inhibition of cellular translation but is independently regulated. Failure to coordinately control ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils how the rapid translational shut-off in response to stress coordinates with rRNA synthesis production to maintain nucleolar integrity.     

Ribosomal history reveals origins of modern protein synthesis

The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world.     

Ribosomal protein L6: structural evidence of gene duplication from a primitive RNA binding protein.

In all cells, protein synthesis is coordinated by the ribosome, a large ribonucleoprotein particle that is composed of > 50 distinct protein molecules and several large RNA molecules. Here we present the crystal structure of ribosomal protein L6 from the thermophilic bacterium Bacillus stearothermophilus solved at 2.6 A resolution. L6 contains two domains with almost identical folds, implying that it was created by an ancient gene duplication event. The surface of the molecule displays several likely sites of interaction with other components of the ribosome. The RNA binding sites appear to be localized in the C-terminal domain whereas the N-terminal domain contains the potential sites for protein-protein interactions. The domain structure is homologous with several other ribosomal proteins and to a large family of eukaryotic RNA binding proteins.     

The yeast GTPase Mtg2p is required for mitochondrial translation and partially suppresses an rRNA methyltransferase mutant, mrm2

The assembly of ribosomes involves the coordinated processing and modification of rRNAs with the temporal association of ribosomal proteins. This process is regulated by assembly factors such as helicases, modifying enzymes, and GTPases. In contrast to the assembly of cytoplasmic ribosomes, there is a paucity of information concerning the role of assembly proteins in the biogenesis of mitochondrial ribosomes. In this study, we demonstrate that the Saccharomyces cerevisiae GTPase Mtg2p (Yhr168wp) is essential for mitochondrial ribosome function. Cells lacking MTG2 lose their mitochondrial DNA, giving rise to petite cells. In addition, cells expressing a temperature-sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lowered levels of mitochondrial ribosomal subunits. Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant, mrm2. We propose that Mtg2p is involved in mitochondrial ribosome biogenesis. Consistent with this role, we show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner.