Structural Biochemistry/Cell Organelles/Ribosome/Maturation

Intro
Ribosomes are present in both eukaryotic and prokaryotic cells. However between the two exists differences in the functions and characteristics of the ribosomes. In eukaryotic cells ribosomes are assembled in the nucleus and transferred to the cytoplasm where they finish maturation. Maturation includes trans acting shutting factors, transport factors, incorporating the rest of the proteins in ribosomes, and the final step in rRNA step process. Recent research, for example on the large ribosomal subunit has confirmed that 60S subunit is transported from the nucleus using an inactive state. After the subunits reaches the cytoplasm it leads to events that cause it to be transitionally competent.

Biogenesis
In cells the ribosome is responsible for the last step in decoding information from genes in proteins. Ribosomes are made of subunits, which are complex and made of RNA and proteins. The two subunits each have a distinct job they are responsible for. Small 40S subunit is used to decode and the large 60S is responsible for polypeptide syntheses. Using structural analyses of prokaryotic ribosomes provides detailed insights for the mechanisms of the ribosome functions, however what we know about the vivo assembly of ribosomes is still not as well known and is still rudimentary. Biogenesis begins with the transcription of pre-RNA that undergoes co-transcriptional folding, modification and assembly with r-proteins forming the two subunits. In bacteria assembly of the subunits need a few trans-acting factors. However in eukaryotic cells it is a complicated process which requires all three RNA polymerase and over 200 trans acting factors. Thus helping the maturation and intercellular transport if the subunits. Ribosomes in eukaryotic cells are assembled in the nucleolus at the rRNA transcription. The released pre-ribosomes although seeming preassembled required maturation steps in the nucleoplasm and the cytoplasm. In the 1970s Planta and Warner's work led to the identification of the first pre-ribosome, 90S particle. 90S is processed to give the smaller 66S and 43S particles. These being the precursors to the mature 60S and 40S subunits. The particles contain pre-rRNA, r-proteins and various amount of trans acting factors. In the early 1990s applying genetic approaches in buddying yeast allowed for the identification of various trans acting factors which led to a better and more clear understanding of the high ordered steps in the processes involved for rRNA. Even though these advances were accomplished, the composition and make up of pre-ribosomal particles still remains unknown and a mystery until the recent decade. With tandem affinity purification, TAP, in combination with mass spectrometry has allowed us to isolate and analyse the composition of maturing pre 60S and pre 40S in buddying yeast.The analyses helped in the ordering of the pre-ribosomal particles in the 60S and 40S pathways, allowing us to picture the highly complex assembly process.

In yeast, RNA polymerase I in the nucleolus helps transcribe 35S. The transcribed rRNA is methylated, pseudo-uridylated, and loaded with r-protein and trans acting factors which allows it to form 90S. 90S particle has r-proteins and trans acting factors which are important for the 40S biogenesis pathway. The cleavage of the rRNA at the A2 site thus releases the pre 40S particle which the maturation and biogenesis are independent of the 60S. Once pre 40S is released the remaining pre-rRNA assembles with large subunit r-proteins and the biogenesis factors form the pre 60S particles. Differently, pre 40S particles undergo few compositional changes as they move from the nucleoplasm. Compared to the pre 60S, pre 40S are exported to the cytoplasm. In contrary pre 60S particles are associated with about 100 trans acting factors along the biogenesis pathway and also change in composition as they move through the nucleoplasm to the nuclear pore complex.

In biogenesis there are different stages that take part in the nucleus and the cytoplasm. In the stages the trans acting factors are released from pre-ribosomal particles and are recycled for the new rounds of biogenesis. The events are caused by enzymes that consume energy that are associated with maturing pre-ribosomal particles. The site of these events of the enzymes and the precise function of them in ribosome maturation still remains unknown to us. Research in the field has shown that two large AAA-ATPases Rix7 and Rea1 implicates in maturation of the pre 60S subunit. Rix7 seems to strip Nsa1 fromt he subunit in the nucleolar transition, and Rea1 is believed to drive pre 60S particles to export competence by the removal of Rsa4. The AAA-ATPases is this believed to contribute directly to the sequential reduction of the complexity of the pre-ribosomal particles, before they are removed from the nucleus.

Nuclear export of pre-ribosomal subunits
Once pre-ribosomal subunits are produced in the nucleus, they are transported into the cytoplasm through the NPC (Nucleaer Pore Complex). The NPC is the largest protein complex in the cell an dis responsible for the protected exchange of components between the nucleus and cytoplasm. It also serves to prevent the transport of material not destined to cross the nuclear envelope. It is found that for the nuclear export of r-subunits, unique nucleoporins are needed to make the export of both subunits to happen. Nucleoporins are simply the proteins of the NPC. Also, researcher have discovered that Ran GTP-GDP cycle is required. Pre-40S and pre-60S particles are exported independently of each other, however, they both need to have the general nuclear export factor Xpo1 or Crm1 that directly recognizes nuclear export sequences. Nuclear export sequences are amino acid sequenecs that label a protein for export into the cytoplasm from the nucleus. Nmd3 is the only known Crm1 adapter for the pre-60S particle. However, there are at least three NES-containing trans-acting factors that function as Crm1 adapters in pre-40S export. Those trans-acting factors include Ltv1 (in humans), DIM2 and RIO2. An interesting fact is that there is an redundancy in 40S export adapters as the nature of Ltv1 is almost non-essential. In order to achieve efficient export of pre-ribosomal subunits requires multiple receptors. For example, in budding yeast, pre-60S particles use additional factors that assist the nuclear pore complex for its export business.

Maturation of pre-ribosomal subunits at the cytoplasmic level
During the early stage of biogenesis, many of the trans-acting factors that are related with pre-ribosomal particles are released from the nucleus and can be recycled back. This process happens before nuclear export. There is a few factors remain related to to the pre-ribosomal subunits as they go into the cytoplasm. We will look at the maturation of the pre-60S subunit and pre-50S subunit independently.

Maturation of the pre-60S subunit
Pre-60S suniunits enter the cytoplams with a entourage of non-ribosomal factors that must be released by unique factors in the cytoplasm. It should be emphasized that several ribosomal proteins are needed to add functionality to the subunits. The steps in the maturation of the pre-60S subunit are shown as below.
 * 1) Pre-60S particles exit the nucleus and enter the cytoplasm
 * 2) In the cytoplasm, a third essential AAA-ATPase is introduced to pre-60S particles

Maturation of pre-ribosomal subunits
The trans acting factors that are associated with pre-ribosomal particles in early biogenesis are released and recycled to the nuclease before nuclear exportation, but some factors are still associated with the particles as they enter the cytoplasm. By releasing and recycling the factors as well as the assembly of the remainder of the r-proteins and the finals processing of RNA constitute the "cytoplasmic maturation steps" in the ribosome biogenesis pathway. The steps needed are not only crucial for complete maturation of subunits but also because if it fails to recycle a factor the nucleus it leads to its depletion from the nucleolar, thus inducing lays in pre-rRNA processing, defects in assembly, and impaired nuclear export.

'Pathway' of cytoplasmic maturation
ATPases and GTPases do not dependent on each other and therefore the events driven by ATPases and GTPases can occur without a specific order. However, some evidence shows that coupling of these events could happen. Drg1 a type of AAA-ATPase, blocks Rlp24, Nog1, and Arx1 from recycling and also blocks Tif6 from recycling to an extent. With this information, it is possible to start ordering the events. First, Drg1 releases Rlp24, and the loading of Rlp24 into subunits brings Rei1. Arx1 is then released when Rei1 works with Jjj1 and Ssa1/Ssa2. Arx1 being in the subunit eventually hinders Tif6 from being released. Starting with Drg1 releasing Rlp24, a series of events takes place and eventually it gets to the release of Tif6.

Conclusion
Since it is extremely crucial that translation of the genetic code done correctly, it is reasonable to speculate that quality-control mechanisms are evolved by eukaryotic cells to monitor ribosome biogenesis. Few research proved that cytoplasmic maturation steps in 40S and 60S biogenesis pathways are done by activation of subunits. This is accomplished when inhibitory factors are removed and functionality is added. Transfer of only the functional ribosomal subunits is guaranteed with the control of these cytoplasmic maturation steps.