Structural Biochemistry/Cell Organelles/Ribosome/Ribosome Synthesis

Background

Ribosome synthesis is a multi-step, error-prone process. The ribosome is comprised of 2 subunits of unequal sizes. These subunits carry out specialized functions within translation such as mRNA decoding for the smaller unit and a peptidyl-transfer reaction for the larger subunit. In eukaryotes, the ribosomes consist of 4 rRNAs and about 80 ribosomal proteins. To synthesis, mature and transport the individual components of ribosomes and assemble them, requires the intervention of approximately 200 protein trans-acting factors as well as numerous amounts of small nucleolar RNAs (snoRNAs). These are involved in the hundreds of individual, error-prone, reactions. There are some functions in processes that don't connect directly to ribosome biogenesis and are then assigned to ribosome synthesis factors such as connections to cell cycle progression, or pre-mRNA splicing and DNA damage response. Because there are so many reactions within a ribosome assembly pathway, the possibility to introduce a mistake with potential deleterious consequences are immense. Such things such as failure to bind or loss of a synthesis factor could lead to a structurally defective ribosome that hold functional consequences in translation. In response to such problems, the cells evolved multiple quality control mechanisms. However, that is not to say that mutations only occur during synthesis as mutations can also occur as a consequence of exposure to genotoxic stress.

Minimizing Defects

An example of creating a way to minimize defects would be in a late assembly step, the cell can bind pre-rRNAs at early nucleolar stages thus committing pre-ribosomes to productive syntheses pathways. Another case would be to bind pre-ribosomes to monitor and tether the structural integrity of ribosomal protein-binding sites. This case would be where there are trans-acting factors with partial homology to ribosomal proteins. This is because defects can delay the binding of trans-acting factors.

Mutations

Cis

Eukaryotes have multiple methods in which they degrade their ribosomes. There are so many reactions in the ribosomal pathways that the possibility of mistakes and mutations are great. Many incidents can occur when mutations occur in cis such as the alteration of rRNA sequencing. Surveillance pathways monitor the structural and functional integrity of RNA. For mutations in the cis conformation, two possible pathways may occur depending on the size and type of the RNA.

There are multiple surveillance pathways that have been described that monitor the integrity of mature RNA molecules, one pathway that monitors mRNA is the "no-go' decay pathway or NGD. This is where mRNAs that induce stalled ribosomes are degraded. LaRivière et al. introduced substitutions in decoding sites and peptidyle transferase center at positions which are necessary in bacteria for ribosome functions. This is done to test if mutations in functionally relevant and conserved ribosomal sites affect the rRNA stability. As a result, the procedure led to the identification of non functional rRNA decay or NRD. In the mRNA NGD, stalled ribosomes triggers initiating endonucleolytic cleavage events on the defective mRNA at the pause site. Then followed by exoribonucleoytic digestion of the 5'- and the 3'- cleaved mRNA products. The RNA exosome digests the defective cleave at the pause site and the 5'->3' exoRNase Xrn1 at the 5'- and 3'- cleaved mRNA. RNA exosome is a conserved multiprotein 3'->5' exoRNase complex active in the synthesis, degradation and surveillance of most classes of cellular RNAs. For NGD, the key components include Dom34 and Hbs1. This however is not the case for 25S NRD, but is true for 18S NRD. As such it indicates two distinct pathways.

In 18S NRD, Dom34 acts together in the same pathway with Hbs1 and they interact in vitro and in vivo. The small molecule inhibitors of translation stabilize the 18S but not the 25S NRD substrates and as a result provides further evidence that 18S NRD activation requires elongating ribosomes and that 18S and 25S NRD are mechanically different. 18S NRD has been linked with cytoplasmic exoRNase Xrn1 and Ski7 as delting both Hbs1 and Ski7 enhances the stabilization of the 18S NRD substrates. Both 18S NRD and mRNA NGD accumulate in the P-bodies that are conserved RNA-protein cytoplasmic granules that contain untranslated mRNA's with a set of repressors. 25S NRD substrates however don't co-localize to P-bodies.

LaRivière's work allowed scientists to discover the "non functional rRNA decay" pathway or NRD. NRD emphasizes on detecting and removing developed ribosomes. NRD is similar to NGD however they both have different kinetics which can be attributed to the higher complexity and compaction of mature ribonucleoprotein particles. NRD has two pathways one in which focuses on the small ribosomal subunits and the other which modifies large ribosomal subunits. There are two main degradation systems for the larger ribosomal subunits: 1. ubiquitin-proteasome system (UPS) and 2. autophagy. UPS targets short lived proteins and involves ubiquitlation and autophagy that degrades long lived proteins.

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Trans

When errors occur in the trans conformation it can signify a failure to bind to or loss of and assembly factor or ribosomal protein. The TRAMP surveillance revolves around the addition of unstructured oligoadenylate tails at the 3'-end of the destructive pre-rRNAs from a poly(A) polymerase activity. Afterwards this method of tagging leads to the degradation performed by the RNA exosome. The addition of these polyA tails allows one to distinguish between normally functioning RNA and then following with a stimulation by exosomal activity. It is suggested that defected RNAs will go through multiple rounds of TRAMP-mediated polyadenylation and then digested by the exosome to increase the degradation effect. There are certain cases that surveillance takes place in a specialized nucleolar domain called "No-body" that contains much TRAMP and exosomal components.

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Pathways of rRNA Decay in Eukaryotes

There are five pathways of rRNA decay described to date.(i)Nucleolar and nuclear pre-40S and pre-60S ribosomal units are monitored actively by the "TRAMP-exosome' pathway. If a misfolded pre-ribosomal unit is identified, TRAMP binds to the molecule followed by the polyadenylation of the 3' ends of defective rRNAs in a step that stimulates both the recruitment and decay of the exosome. It is still not understood how TRAMP detects defective ribosomes. Polyadenylation occurs both at normal and cryptic pre-RNA sites. Differences in RNA Polymerase I activity can activate cryptic cleavage sites. Cytoplasmic mature subunits carrying cis mutations are monitored by the NRD pathway.(ii) During 18S NRD, small subunits with errors along the mRNA are identified by Dom34 and Hbs1. These errors are cleaved endonucleolytically by an unknown activity (thought to be similar to mRNA NGD). The releases products are digested by Xrn1 and the RNA exosome assisted by its cofactor Ski7. (iii) During 25S NRD, defective 60S subunits are targeted for proteasomal degradation by Rtt101-Mms1-mediated ubiquitylation of unidentified associated ribosomal components. During conditions of starvation in the cell, excess ribosomes are turned over to ribophagy and PMN. (iv) Ribophagy is a type of macroautophagy that involves the engulfment of cytosolic fractions to the vacuole. Inside the vacuole, the components are recycled. (v) In PMN, a specific type of microautophagy, a portion of the nuclear envelope is pinched off by the vacuole. This creates a specialized organelle called the NVJ that matures into a vesicle. Finally, the mature vesicle is degraded by resident hydrolases.

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Ribosome surveillance in human health

RNA damage occurs under normal cell growth as well as during stress and in disease situations. Chemical modifications to nucleobases are introduced into RNA and RNPs from exposure to ultraviolet light, oxidation, chlorination, nitration, and alkylation. These alterations all constitute potential physiological triggers to ribosome degradation pathways. Neurodegenerative diseases such as Alzheimer's and Parkinson's have been correlated to damage in rRNA sequences because of exposure to UV light or oxidation. RNA oxidation has been speculated to be involved in disease progression and that RNA susceptibility to oxidative damage is influenced by various factors including the degree of association with protein protection. Mature 5.8S and 25S rRNA are fragmented heavily in yeast cells exposed to high levels of a reactive oxygen species generated by oxidative stress (including exposure to hydrogen peroxide and menadione), chronological aging, and other apoptotic cues. Yeast cells treated with the anti-metabolite and chemotherapeutic agent 5-fluorouracil (5-FU) accumulate polyadenylated pre-rRNAs. This accumulation is exacerbated in exosome mutants, that are hypersensitive to the drug. This suggests that the degradation of 5-FU containing RNAs happens through nucleolar surveillance. Nucleolar dysfunction in cells has been correlated with cancer. Various nucleolar stresses such as drug mediated interference of rRNA synthesis, pre-rRNA processing, or inhibition of ribosome synthesis factor function, lead to nucleolar disruption. Ultimately, the cell cycle becomes defective. An interesting regarding this phenomena is the observation of an increased abundance of polyadenylated rRNA fragments in the gut of western honey bees infected with colony collapse disorder (CCD). The insect guts serves as a primary interference with the environment taking in all the pesticides from outside. CCD has been linked to picorna-like viral infections known to hijack cellular ribosomes. This in addition to the use of pesticides could trigger a ribosome degradation response.

Concluding Remarks

Ribosome synthesis is a major cell activity that can enforce quick energy drain with little regulation. Control is exerted at the level of synthesis, assembly of the pieces, and surveillance of the final product. Ribosome synthesis has evolved to be fully integrated with complex nutrient sensing mechanisms. In the prescence of defective ribosomes, damaged ribosomes, or abundance of mature ribosomes, they are targeted for rapid breakdown and recycling. Many surveillance pathways exists that either select excess or defective ribosomes. The pathways are even intricate enough to survey large or small subunits of the ribosome. The most important concept is that we understand how all these pathways interconnect in the ribosome's synthesis.

References

Lafontaine Denis L.J. "A 'garbage can' for ribosomes: how eukaryotes degrade their ribosomes." <http://www.ncbi.nlm.nih.gov/pubmed/20097077>