Presentation on theme: "The Nucleus Nuclear Organization Nuclear Envelope and Molecular Trafficking Nucleolus and rRNA Processing The nucleus is one of the main features that."— Presentation transcript:
1The Nucleus Nuclear Organization Nuclear Envelope and Molecular Trafficking Nucleolus and rRNA ProcessingThe nucleus is one of the main features that distinguishes eukaryotic cells from prokaryotic cells.-You might even say that the nucleus serves as the regulatory center or the cell.-It serves several function including;-storage of DNA-site of DNA synthesis, transcription and RNA processing – gene expression is regulated by post-transcriptional modifications in the nucleus. HOW?-transport by way of nuclear envelope-processing of rRNA
2The Structure and Function of the Nuclear Envelope The nuclear envelope has a complex structure consisting of two nuclear membranes, an underlying nuclear lamina, and nuclear pore complexes.Nuclear membranes are a system of two concentric membranes (inner and outer) that surround the nucleus.The nuclear envelope separates the contents of the nucleus from the cytoplasm and provides the structural framework of the nucleus.The nuclear envelope separates the contents of the nucleus from the cytoplasm and creates a structural framework of the nucleus and acts as a barrier that prevents the free passage of molecules between the nucleus and the cytoplasm. This helps to maintain the integrity of the nucleus as a distinct biochemical compartment.-It consists of 2 nuclear membranes, a lamina and nuclear pore complexes.
39.1 The nuclear envelopeThe selective traffic of proteins and RNAs through the nuclear pore complexes establish the internal composition of the nucleus and also play a critical role in regulating eukaryotic gene expression.cell4e-fig jpg In this electromicrograph you can see the dual membrane that surrounds the nucleus. You can also see a nuclear pore complex and how the membrane of the endoplasmic reticulum integrates with the nuclear membrane. Both of these things become important when we talk about transport.-The selective traffic of proteins and RNAs through the nuclear pore complexes helps to establish the composition of the nucleus. It also serves to regulate gene expression in eukaryotic cells.-The nuclear pore serves to transport molecules into and out of the nucleus and the ER serves to process and transport protein to the cell surface.
4Structure of the Nuclear Envelope The critical function of the nuclear membranes is to act as a barrier that separates the contents of the nucleus from the cytoplasm.The nuclear lamina underlies the inner nuclear membrane and is a fibrous meshwork that provides structural support to the nucleus.Lamins are 60- to 80- kilodalton fibrous proteins that make up the nuclear lamina.Let’s talk about the specifics of the structure of the nuclear membrane. As I mentioned before, it consists of 2 nuclear membranes, nuclear pore complexes and an underlying nuclear lamina.-The nucleus is surrounded by 2 concentric membranes called the inner and outer membranes. The outer membrane is integrated with the endoplasmic reticulum which allows the space btw. The inner and outer membrane is connected with the lumen of the ER. This membrane is similar in structure to the ER membrane. It has ribosomes on its cytoplasmic surface.-The inner membrane carries only proteins that are need inside the nucleus…..for example proteins that bind to the nuclear lamina.-The nuclear lamina is the structure located on the inside surface of the inner nuclear membrane. It is a fibrous network that serves to create the nuclear structure This fibrous network is composed of kDalton fibrous proteins called lamins. Groups of these lamins form the fibers you see on the inside surface of the nuclear membrane.
5Structure of the Nuclear Envelope Lamins are composed of intermediate filament proteins which combine with each other to form higher-order structures.Attachment of Lamins to the nuclear envelope is mediated by prenylation and binding to specific inner nuclear membrane proteins such as emerin and the lamin B receptor.Nuclear Lamina diseases: Hutchinson-Gilford progeria-Lamins are composed of intermediate filament proteins….these proteins also serve to maintain the structure of the cytoskelaton.-Most mamallian cells have 3 lamin genes, A, B and C, that code for 7 different lamin proteins. These 7 lamin proteins combine with each other to form the structure of the lamina.-How do they form the complex structure of the lamina? Two lamin proteins interact forming a dimer. Two alpha helical regions of 2 polypeptide chains join together forming these coiled coils. Polymers of these dimers make up the nuclear lamina. Often this occurs by the head to tail association of dimers which leads to formation of linear polymers and side by side association of polymers--How do these lamins associate with the inner nuclear membrane? This attachment is mediated by the posttranslational addition of lipid (prenylation of C-terminal residues) on the lamins. Also, Lamins bind to specific inner nuclear membrane proteins such as emerin and the lamin B receptor, mediating their attachment to the nuclear envelope and localizing and organizing them within the nucleus.-The lamina also attaches to chromatin by way of histones H2A and H2B.-The nuclear lamina really creates a mesh network throughout the nucleus…..many of the proteins necessary for DNA replication, transcription and RNA processing bind to the lamina. So in essence the lamina serves to facillitate many of the events that occur in the nucleus.-What happens if there are mutations in one of the lamin genes? Defects in production of the lamin A protein results in the disease Hutchinson-Guilford progeria.
6Nuclear Lamina Defects X-linked Emery-Dreifuss muscular DystrophyResults form mutations in the nuclear envelope protein, emerin.Non-Sex Linked Emery Dreifuss Muscular DystrophyResults from mutations in the LMNA gene which is a single gene that encodes lamin A and C.Hutchinson-Guilford Progeria DiseaseResults from a 150 bp deletion within the lamin A gene.-Both of the Emery Dreifuss Muscular Dystrophies are characaterized by stiff knees, elbows, neck and heels and electrical defects in the heart. Generally these symptoms occur by age 10. By age 20, it progresses to serious heart defects which may require a pacemaker. These progresses to gradual wasting and weakness of the upper arm and lower leg muscles.-Hutchinson-Guilford Progeria disease is characterized by premature aging.-It was originally thought that these sorts of nuclear lamina defects would cause generalized structural defects in rapidly dividing cells. This does not seem to be the case. There appears to be a tissue specific expression pattern of these mutated proteins. It is not clear why this is the case.
7The Nuclear Pore Complex A nuclear pore complex consists of a structure with eightfold symmetry organized around a large central channel.Nuclear pore complexes are the only channels that are present in the nuclear membrane to allow small polar molecules , ions and macromolecules (proteins and RNAs) which permit the transport from the nucleus to the cytoplasm.-These pore complexes consist of eight structural subunits that surround a central channel. It is a very large structure with a mass of about 125 million daltons. For comparative sake, it is about 30 larger than a ribosome.-In vertebrates, the nuclear pore complex consists of different pore proteins called nucleoporins.-The nuclear pore complex plays a very important role in the physiology of cells….it regulates what gets into and out of the nucleus of a cell.
8The Nuclear Pore Complex Nuclear pore complexes are the only channels through which small polar molecules, ions, and macromolecules can travel between the nucleus and the cytoplasm.Depending on their size, molecules can travel through the nuclear pore complex by one of two different mechanisms.Passive transportActive transport –energy dependentThe structure of the nuclear pore complex: There are 8 spokes arranged around a central channel. The spokes are connected to rings at the nuclear and cytoplasmic surfaces. The spoke ring assembly is anchored within the nuclear envelope at sites of fusion between the inner and ourter nuclear membranes. Protein filaments extend from the cytoplasmic and nuclear rings, forming a distinct basketlike structure on the nuclear side.The movement of molecules into and out of the nucleus is critical to normal eukaryotic cell function.-RNA is synthesized in the nucleus and must be exported to the cytoplasm for protein synthesis.-Also, proteins that perform nuclear functions such as transcription factors must be transported into the nucleus.-The nuclear pore complex allows proteins to be continuously shuttled between the nucleus and cytoplasm.-The size of the molecule that must be transported into or out of the nucleus, one of two mechanism is typically utilized:-If the molecules are less than kDaltons in size, then they pass freely through the pore in either direction. These molecules diffuse freely through open aqueous channels (9 nm wide) in the pore complex. This type of transport is called passive transport.-Most proteins and RNAs are too large to be transported through these open channels. These macromolecules pass through the nuclear pore complex by an active or energy dependent means of transport. They are selected transported across the nuclear membrane through the use of energy.
9Selective Transport of Proteins to and from the Nucleus Nuclear export signals are specific amino acid sequences that target proteins for export from the nucleus.Protein import through the nuclear pore complex begins when a specific importin binds to the nuclear localization signal of a cargo protein in the cytoplasm.How are proteins transported across the nuclear membrane?-Selective transport of proteins to and from the nucleus is generally mediated by proteins or portions of protien. Many protiens contain nuclear localization and nuclear export signals or sequences that are needed to allow entry into or out of the nucleus.- In addtion there are protiens such as importins and exportins that functions to shuttle molecules into and out of the nucleus.
10Selective Transport of Proteins to and from the Nucleus Nuclear localization signals are specific amino acid sequences that are recognized by transport receptors and direct the transport of proteins through the nuclear pore complex.Nuclear localization signals have been identified in many proteins.There are lots of proteins that are essential to the normal functioning of the cell. Since all proteins are synthesized in the cytoplasm by the ribosome, many of these proteins must be transported to the nucleus inorder to drive processes such as transcription and replication. These are protiens such as DNA polymerases, RNA polymerases, histones and transcription factors.-How do they get into the nucleus? They enter the nucleus by way of the nuclear pore complex and are targeted to the nuclear pore complex by a nuclear localization signal that is present on most nuclear proteins. They have specific amino acid sequences called NLS that serve to send the proteins into the nucleus. These sequences are recognized by transport receptors that transport the proteins through the nuclear pore complex.-There have been numerous localization signals identified to date. The first nuclear localization signal was identified in the simian 40 (SV40) T antigen which is a virus encoded protein that initiates viral DNA replication in infected cells. It is 7 AA in length.-Sometimes the localization signals are short stretches of basic amino acids called classic NLS and in other cases the NLS consists of amino acids that are close together, but not adjacent to each other(called bipartite NLS).
11Selective Transport of Proteins to and from the Nucleus Nuclear transport receptors are proteins that recognize nuclear localization signals and mediate transport across the nuclear envelope.Karyopherin is a nuclear transport receptor.Importins transport macromolecules to the nucleus from the cytoplasm.Exportins transport macromolecules from the nucleus to the cytoplasm.How do the nuclear localization sequences serve to send a protein into the nucleus? They are recognized by proteins that function as nuclear transport receptors. Most of these receptors belong to the Karyopherin protein family.-Members of the Karyopherin family can function as importins that transport substances into the nucleus or exportins that transport molecules out of the nucleus.-After binding to these Karyopherin receptors, the are then transported through the nuclear pore complex.
129.10 Distribution of Ran/GTP across the nuclear envelope Ran is one of several types of small GTP-binding proteins that regulate movement of macromolecules through the nuclear pore.cell4e-fig jpg Movement of these substances through the nuclear pore complex is mediate by a protein called Ran. It mediates both nuclear import and export. Ran is one of several types of small GTP- binding proteins whose conformation and activity are regulated by GTP binding and hydrolysis.-There are actually lots of cellular proteins that are regulated in this manner such as Ras, the elongation factors that we discussed in chapter 8, Arf, Rac, Rho, etc.-How does Ran work? The enzymes that stimulate GTP hydrolysis to GDP are localized to the cytoplasmic side of the nuclear envelope, while the enzymes that trigger the exhange of GDP for GTP are localized to the nuclear side of the nuclear envelope. As a result there is an unequal distribution of Ran/GTP across the nuclear pore. The location of the higher concentration of Ran/GTP determines the directionality of the nuclear transport.-So how does Ran manage to regulate what passes through the nuclear pore complex? Ran regulates the movement through the nuclear pore by controlling the activity of the nuclear transport receptors. Transport though the pore complex begins when a specific importin nuclear transport receptor binds to the nuclear localization signal of the cargo protien in the cytoplasm. The cargo protein-importin complex binds to specific nuclear pore proteins in the cytoplasmic filaments. By sequential binding to more interior nuclear pore proteins, the complex is translocated through the nuclear pore.-Once inside the nucleus, the cargoprotein/importin complex is disrupted by the binding of Ran/GTP to the importin. This causes a change in conformation to the importin and displaces the cargo protein from the complex and releases it in the nucleus. Then, the importin- Ran/GTP complex is transported back through the pore to the cytoplasm where the GTPase activating protein hydrolyzes the GTP on the Ran to GDP and releases the importin into the cytoplasm where it is free to bind new cargo proteins and participate in a new round of transport.
139.12 Nuclear exportcell4e-fig jpg -In a similar manner, proteins are targeted for export from the nucleus by specific amino acid sequences called nuclear export signals. These sequences are recognized by receptors within the nucleus called exportins that direct protien transport through the nuclear pore complex to the cytoplasm.-They bind to Ran/GTP promotes the formation of stable complexes between exportins and their cargo proteins. This is the opposite of the import process. Exportins form stable complexes with their cargo protiens in association with Ran/GTP within the nucleus.-Following transport to the cytosolic side of nuclear envelope, GTP hydrolysis and release of Ran/GDP leads to the dissociation of the cargo protien which is release into the cytoplasm.
14Regulation of Nuclear Protein Import The transport of proteins to the nucleus is yet another level at which the activities of nuclear proteins can be controlled.In one mechanism of regulation, transcription factors associate with cytoplasmic proteins that mask their nuclear localization signals.The activity of nuclear protiens can be regulated by their transport. For example transcription factors are functional only when they are present in the nucleus. There by controlling their import to and export from the nucleus is one way of regulating gene expression.-We will talk more about this type of regulation in Chapter 15 when we discuss cell signaling, but for now it is important to understand that regulated nuclear import of both transcription factors and protein kinases has an important role in controlling cell response to an external stimuli. It provides a mechanism for signals received at the cell surface to be transmitted to the nucleus.-One mechanism of regulation is for transcription factors to associate with cytoplasmic proteins to mask their nuclear localization signals….causing these proteins to remain in the cytoplasm. A good example of this is the trancription factor NfKB.-NF-kb is maintained as an inactive complex with Ikb which serves to hide or mask its nuclear localization signal, keeping Nfkb in the cytoplasm. However, in response to certain extracellular signals, Ikb is phosphorylated and degraded by ubiquitin-mediated proteolysis. This exposes the NLS on Nf-kb, allowing it to be imported into the nucleus by combining with the proteins Importin. This allows for the Nf-kb to interact with target genes and activate gene expression of those targets.-A similar process happens with the yeast protein Pho4, except it is maintained in the cytoplasm when the protein is phosphorylated on a residue within the NLS. The phosphate group function to mask the NLS and keeps the Pho4 in the cytoplasm. Signal mediated dephosphorylation allows the protein to be imported into the nucleus.
15Most RNAs are exported from the nucleus to the cytoplasm. Transport of RNAsMost RNAs are exported from the nucleus to the cytoplasm.RNAs are transported across the nuclear envelope as ribonucleoprotein complexes, or RNPs.RNA are transported in the oppposite direction of most proteins. Proteins are generally transported from the cytoplasm to the nucleus , while most RNAs are generated in the nucleus are must be exported to the cytoplasm to participate in translation.-Once again this is an important step in the regulation of gene expression, without tRNA, rRNAs or mRNA translation or protein synthesis is prevented.-Much like protein import, export of RNAs to the cytoplasm is an energy dependent process requiring nuclear transport recepotors (Karyopherins…exportins) to interact with the nuclear pore complex. They transport most tRNAs, rRNAs and snRNAs in a Ran/GTP dependent manner.-RNAs are transported across the nuclear envelope as ribonucleoprotein complexes (RNPs). Their export from the nucleus is mediated by nuclear export signals present on proteins within the subunit complex. Pre-mRNAs and mRNAs are associated with a set of at least 20 proteins throughout their processing in the nucleus and transport to the cytoplasm-There are small RNAs, such as snRNAs and snoRNAs, function within the nucleus as components of the RNA processing machinery. These molecules are recycled during transport so that they can be reused during successive transport events.
16Internal Organization of the Nucleus The nucleus has an internal structure that organizes the genetic material and localizes nuclear functions.Chromatin within the nucleus is organized into large loops of DNA, and specific regions of these loops are bound to the lamin matrix by lamin- binding proteins in the chromatin.Lets switch gears a little and talk about the organization within the nucleus. The internal structure of the nucleus serves to organize the genetic material and localize nuclear functional processes.-The loosely organized matrix of the nuclear lamins extend from the nuclear lamina into the interior of the nucleus. The lamins serve as sites for chromatin attachment and organize proteins into functional nuclear bodies.-Chromatin is organized into large loops of DNA and specific region s of these loops are boun to the lamin matrix by lamin binding proteins in the chromatin. Other nuclear proteins form lamin-dependent complexes that form nuclear bodies that have roles in DNA repair, chromatin organization , gene regulation and signal transduction.-All of these things occur on the nuclear lamina, which is probably the basis of lamin-related genetic diseases.
179.16 Heterochromatin in interphase nuclei The chromosomes in interphase chromatin are actually arranged in an organized fashion.Heterochromatin is condensed, transcriptionally inactive chromatin.Euchromatin is decondensed, transcriptionally active interphase chromatin that is distributed throughout the nucleus.cell4e-fig jpg -Chromatin becomes highly condensed during mitosis to form the compact metaphase chromosomes that are distributed to daughter nuclei.--During interphase, some of the chromatin (heterochromatin) remains highly condensed and is transcriptionally inactive, while the remainder of the chromatin (euchromatin) is decondensed and distributed throughout the nucleus.-Cells contain 2 types of heterochromatin:-Constituitive heterochromatin which contains DNA sequences that are generally not transcribed such as the satellite sequences present at centromeres-Facultative heterochromtin which contains sequences that are not transcribed in the cell being examined but are transcribed in other cell types. This type of heterochromatin varies depending on the cell type.
18Chromosomes and Higher-Order Chromatin Structure Individual chromosomes also occupy distinct territories within the nuclei of mammalian cells.The chromatin in interphase nuclei is organized into looped domains of about 50 to 100 kb of DNA.-The last slide gives you the sense that interphase chromatin appears to be uniformly distributed, but is not the case.-In reality, the chromosomes in interphase chromatin are actually arranged in an organized fashion and divided into discrete functional domains that play an important role in regulating gene expression. Rather than randomly winding around one another, each chromosome occupies a discrete region of the nucleus.-This is illustrated in this diagram. In part A, probes to repeated sequences on chromosome 4 identified by yellow flourescence, occupy distinct regions within the nucleus. In part B there is a model of chromosome organization. It shows you that chormosomes are occupying discrete territories that are separated by intrachromasomal domains in which RNA processing and transport are thought to occur.-If you think about the numerous processess that must occur in the nucleus (transcription, regulation of expression, RNA processing and editting and DNA repair), it makes sense that the nucleus must have a highly ordered structure.-Much like the DNA in the metaphase chromosomes, the chromatin in interphase nuclei is organized into looped domains contianing about kb of DNA. These chromatin domains seem to represent discrete functional units that independently regulate gene expression.
19Sub-Compartments within the Nucleus Many proteins of the nucleus are localized to discrete, sub-nuclear bodies that have a low-density, sponge-like structure that allows macromolecules from the rest of the nucleus to move in and out.The nuclei of mammalian cells contain clustered sites of DNA replication within which the replication of multiple DNA molecules takes place.As part of the highly ordered structure of the nucleus, there are sub-nuclear bodies or compartments that carry out particular functions. Most nuclear processes occur in distinct regions of the nucleus.-Many enzymes an other important proteins of the nucleus are localized to discrete subnuclear bodies that have a low-density, sponge-like structure that allows macromolecules from the rest of the nucleus to move in and out. Many of these sub-nuclear structure have not been fully characterized. It is not clear the functional role of many of these discrete regions within the nucleus.-In mammalian cells, the nuclei contian clustered regions or sites of DNA replication. In this micrograph, newly replicated DNA was labeled by brief exposure of the cells to bromodeoxyuridine which incorporates into the DNA in place of thymidine. This technique allows visulization of newly synthesized DNA by immunflourescence following staining with an anitbody against bromodeoxyurindine. Using this technique you can see that the newly replicated DNA is present in discrete clusters distributed throughout the nucleus.-Why do these two cells have different levels of immunoflourescence? One is early in DNA synthesis and the other is late in DNA synthesis ( more DNA present). At the beginning of DNA synthesis, the newly replicated DNA was detected in about 20 discrete sites mostly clustered around the nucleolus, but later in DNA synthesis the process is spread to hundreds of sites located all over the nucleus.-Based on this type of staining, it is thought that DNA replication seems to take place in large structures that contain multiple replication complexes that are organized into distinct functional bodies. These regions of DNA synthesis are referred to as replication factories
20Sub-Compartments within the Nucleus Components of the mRNA splicing machinery are concentrated in discrete nuclear bodies termed nuclear speckles.Nuclei contain several other types of distinct structures, such as PML bodies and cajal bodies. The function of PML bodies remains largely unknown.The nucleolus is the most prominent nuclear body and is the site of rRNA transcription and processing as well as aspects of ribosome assembly-In addition to the replication factories that exist for DNA synthesis within the nucleus, there are other subnuclear bodies with specific functions. Here you can see a model of nuclear structure-Nuclear speckles: Unlike actively transcribed genes that are found throughout the nucleus, components of the mRNA splicing machinery are concentrated in discrete nuclear bodies called nuclear speckles. They are located in discrete structures that serve to store the splicing components (snRNPs and splicing factors) which can be recruited to actively transcribe genes for pre-mRNA processing as needed.-PML bodies: In addition to the nuclear speckles there are other types of distinct bodies within the nucleus besides the nucleolus. There are typically 5-20 PML bodies within the nucleus of mamalian cells. These bodies are known to interact with chromatin and are sites of accumulation of trasncription facotrs and chromatin modifying proteins (HDACs). They were first identified as sites of localization of a transcriptional regulatory protein involved in acute promyelocytic leukemia. The full function of these bodies is still not clear.-Cajal bodies: These discrete structures contain the protein coilin and are rich in small RNPs. They are thought to serve as sites of RNP assembly especially in the final steps of snRNP processing. Again, there exact functional role within the nucleus is not fully clear.-Nucleolus: The largest of the subnuclear compartments is the nucleolus and it is the site of rRNA transcription and processing. Also, some ribosomal assembly occurs within the nucleolus.
21Ribosomal RNA Genes and the Organization of the Nucleolus The nucleolus is associated with the chromosomal regions that contain the genes for the 5.8S, 18S, and 28S rRNAs.To meet the need for transcription of large numbers of rRNA molecules, all cells contain multiple copies of the rRNA genes.-As we have discussed before, an actively growing cells requires a large number of ribosomes to meet its need for protein synthesis. As such these cells must have a means of producing enough ribosomes to synthesize enough protein for a cell to not only survive, but to be able to reproduce itself.-The nucleolus serves as a ribosome production center for the regulated production of rRNAs and assembly of the ribosomal subunits.-Recent evidence suggests that nucleoli also have a more general role in RNA modification and that several types of RNA move in and out of the nucleolus at specific stages during their processing.-The nucleolus is not surrounded by a membrane and is associated with the chromosomal regions that contain genes for 5.8S, 18S and 28S rRNAs. These rRNA are transcribed as a single unit within the nucleolus by RNA polymerase I which produces a 45S pre-rRNA. The 45S pre-rRNA is processed to the 18S rRNA of the 40S (small) ribosomal subunit and to the 5.8S and 28S rRNAs of the 60S (large) ribosomal subunit.-Remember that we said that the demand for ribosomes is high in a transcriptionally active cell…..To meet the needs for transcription of large numbers of rRNA molecules, most cells contain several copies of the rRNA genes. For example, the human genome contains about 200 copies of the gene that encodes the 5.8S rRNA. The genes for the 5.8S, 18S and 28S rRNA are clustered in tandem arrays on 5 different human chromosomes (13, 14, 15, 21, 22).
22Ribosomal RNA Genes and the Organization of the Nucleolus Morphologically, nucleoli consist of three distinguishable regions: the fibrillar center, dense fibrillar component, and granular component.Nucleolar organizing regions are where nucleoli become associated with the chromosomal regions that contain the 5.8S, 18S, and 28S rRNA genes.Now that we have discussed the purpose or functional role of the nucleoli, lets talk a little about the structure of the nucleoli.-This electron micrograph show you the detail of a single nucleoli. Keep in mind that a cell may have more than one nucleoli. It consists of 3 distinct regions: the fibrillar center, dense fibrillar component and granular component. These regions are thought to represent sites of progressive stages of rRNA transcription, processing and ribosome assembly.-Following each cell division, nucleoli become associated with the chromosomal regions that contain 5.8S, 18S and 28S rRNA genes. These regions are called nucleolar organizing regions.-The size of the nucleoli depends on the metabolic activity of the cell. Larger nucleoli are found in cells that are actively synthesizing proteins The change in size is primarily due to the granular component since this is the region of ribosome assembly.
23Transcription and Processing of rRNA Each nucleolar organizing region contains a cluster of tandemly repeated rRNA genes separated from each other by nontranscribed spacer DNA.In higher eukaryotes the primary transcript of the rRNA genes is the large 45S pre-rRNA, which contains the 18S, 5.8S, and 28S rRNAs as well as transcribed spacer regions.-The nucleolar organizing regions contains a cluster of tandemly repeated rRNA genes separated from each other by nontranscribed spacer DNA. These genes are actively transcribed by RNA polymerase I.-This can be visualized using electron microscopy. Here you see 3 rRNA genes separated by nontranscribed spacer DNA. Each rRNA gene is surrounded by an array of densely packed growing RNA chains….this give a sort of Christmas tree appearance.-In human and other eukaryotes, the primary transcript of the rRNA genes is the large 43S pre-rRNA. This contains the 18S, 5.8S and 28S rRNAs as well as the spacer regions seen here.
24Transcription and Processing of rRNA The processing of pre-rRNA involves a substantial amount of base modification resulting both from the addition of methyl groups to specific bases and ribose residues and from the conversion of uridine to pseudouridine.Small nucleolar RNAs, or snoRNAs, are complexed with proteins and function in pre-rRNA processing.-These primary 45S transcript contains external spacers at the 5’ and 3’ ends of the pre-rRNAs and 2 internal transcribed spacers btw. The 18S, 5.8S and 28S rRNA sequences. During processing several cleavage steps take place to remove these spacer sequences and produce mature 18S, 5.8S and 28S rRNAs.-In addition to cleavage, the processing of pre-rRNA involves some base modification that results from the addition of methyl groups to specific bases and ribose residues as well as the conversion of uridine to pseudouridine. In most animal cells, pre-rRNA processing involves the methylation of about 100 ribose residues and 10 bases and the formation of about 100 pseudouridines.
25Transcription and Processing of rRNA Small nucleolar RNAs or snoRNAs complex with proteins to generate snoRNPs which regulate processing of rRNAs.Most snoRNAs function as guide RNAs to direct the specific base modifications of pre-rRNA, including methylation of specific ribose residues and the formation of pseudouridines.These processing events that must occur to pre-rRNA requires the actions of both proteins and RNAs that are localized to the nucleolus. The nucleoli contains more than 300 proteins and numerous small nucleolar RNAs (snoRNAs) that are needed to process pre-rRNA.-Much like the spliceosomal snRNAs, the snoRNAs are complexed with proteins to form snoRNP (small nucleolar ribonucleoproteins). These snoRNPs consist of a single snoRNA associated with 8-10 proteins. The snoRNPs assemble on the pre-rRNA to form processing complexes. This process is very similar to the assembly and action of the spliceosomes used to process pre-mRNA.-The snoRNAs contain short sequences complementary to rRNA. Complimentary base pairing between snoRNAs and pre-rRNAs targets the enzymes that catalyze base modifications (methylation, etc) to the appropriate site on pre-rRNAs.-Some snoRNAs are responsible for the cleavages of pre-rRNA into 18S, 5.8S and 28S products. The most abundant snoRNA is U3 (there are about 200,000 copies per cells) is required for cleavage of pre-rRNA within the 5’ external transcribed spacer sequences.-Most of the snoRNAs do not function in this manner. They serve as a guide RNAs to direct the specific base modification of pre-rRNA, including the mehtylation of specific ribose residues and the formation of pseudouridines. The regions of complementarity include the sites of base modification in the rRNA. By base pairing with specific regions of the pre-rRNA, the snoRNAs act as guide RNAs that target the enzymes responsible for ribose mehtylation or pseudouridinationto the correct site on the pre-rRNA molecule.
26Ribosome AssemblyThe formation of ribosomes involves the assembly of the ribosomal precursor RNA with both ribosomal proteins and 5S rRNA.The association of ribosomal proteins with rRNA begins while the pre-rRNA is still being synthesized.The final stages of ribosome maturation follow the export of pre-ribosomal particles to the cytoplasm, forming the active 40S and 60S subunits of eukaryotic ribosomes.-The formation of ribosomes involves the assembly of the ribosomal precursor RNA with both ribosomal proteins and 5S rRNA. The ribosomal proteins are transcribed outside the nucleolus and translated in the cytoplasm. These proteins then must be imported from the cytoplasm to the nucleolus so that they can be assembled with rRNAs to form pre-ribosomal particles.-This diagram provide an overview of ribosome assembly. Ribosomal proteins are imported to the nucleolus from the cytoplasm and begin to assemble on pre-rRNA while the pre-rRNA is still being synthesized. This occurs prior to any rRNA processing (cleavage, etc.) with about ½ of the ribosomal proteins complexed to the pre-rRNA before any cleavage event has occured. As the pre-rRNA is processed, additional ribosomal proteins and the 5S rRNA assemble to form preribosomal particles. Relatively early in the ribosome assembly process, the 2 nascent ribosomal subunits separate. This allow the process of these two precursor subunits to occur separately.-Ribosome assembly is concluded when the pre-ribosomal particles are exported to the cytoplasm, yielding the 40S and 60S ribosomal subunits.The association of ribosomal proteins with rRNA begins while the pre-rRNA is still being synthesized, and more than half of the ribosomal proteins are complexed with the pre-rRNA before its cleavage.