Nucleolar Stress - Unit 13 - PDF

© Timoshenko, 2024 Nucleolar stress Page 1 of 8 Unit 13: Nucleolar stress There are four learning objectives of this unit:  Nucleus and nuclear bodies  Nucleoli and their functions  Effects of stress on nucleoli  Activation of...

© Timoshenko, 2024 Nucleolar stress Page 1 of 8 Unit 13: Nucleolar stress There are four learning objectives of this unit:  Nucleus and nuclear bodies  Nucleoli and their functions  Effects of stress on nucleoli  Activation of p53 under nucleolar stress Nucleus and nuclear bodies Nucleus is a large organelle in eukaryotic cells that contains DNA organized into regions with different condensation (eurochromatin and heterochromatin) and nuclear bodies. The electron micrograph shows an interphase nucleus of Bone Marrow Stem Cell. The nucleus is enclosed by a double-membrane structure termed the nuclear envelope, which is interrupted in places by nuclear pore complexes controlling nucleocytoplasmic transport. Nuclear bodies are regions with high concentration of specific proteins and RNAs that form distinct, often roughly spherical structures within the nucleus. Nuclear bodies fill in the interchromatin space and represent multiple structures with different names such as Cajal bodies, clastosomes, histone locus bodies, nuclear speckles, nucleoli, paraspeckles, perinucleolar compartments, PML-nuclear bodies, and Polycomb bodies. You may also remember from the lecture on the heat shock response that a special type of nuclear stress bodies is formed in cells in response to heat stress. These structures appear transiently and are the main site of HSF1 and HSF2 accumulation in stressed human cells. Nucleolus is the largest sub-compartment of the nucleus not bounded by a membrane, which may occupy up to 25% of the nucleus. A cell nucleus can contain several nucleoli, which are assembled on different chromosomes. For example, the fluorescence image on the top-right shows human HeLa cells transfected with a gene for a ribosomal protein fused to GFP. The fluorescent ribosomal protein can be seen in the cytoplasm where it is synthesized and ultimately functions, and in the nucleoli (white arrows), where it is assembled into ribosomes.  © Timoshenko, 2024 Nucleolar stress Page 2 of 8 Functions and internal organization of nucleoli Nucleoli are responsible for ribosome biogenesis in cells and regulating cellular stress responses. Ribosome biogenesis requires three important steps such as transcription of pre- rRNA, processing of pre-rRNA, and assembly of ribosome subunits. All these events are localized in different sub-compartments of nucleoli known as fibrillar center (FC), dense fibrillar component (DFC) and granular component (GC). These compartments are clearly visible on the electron micrographs as regions with different density (light, dark, and grey) and they contain specific molecules. Fibrillar center is where ribosomal RNA synthesis occurs; it associates with DNA sequences that encodes for rRNA, RNA polymerase I machinery, and UBF (upstream binding factor). UBF is an essential component of the RNA Pol I preinitiation complex. Dense fibrillar component contains nascent pre-rRNA transcripts, pre-rRNA processing factors including small nucleolar snoRNAs and snoRNP proteins, e.g. Fibrillarin and Nop58. The pre- RNA precursor undergoes chemical modifications here. Granular component contains ribosomal subunits in various stages of assembly, a specific protein here is NPM1 (nucleophosmin). Precursor ribosomal subunits are ready to be sent to the cytoplasm. Nucleophosmin serves as a nuclear export chaperone and directs the nuclear export of both 40S and 60S ribosomal subunits (Maggi et al, 2008). Nucleolar internal organization can be revealed by fluorescence immunostaining using antibodies against those specific proteins of each sub-compartment. rRNAs and ribosomes Most of the RNA in cells is rRNA (80%). The rest is tRNA (15%) and only 5% of mRNA and non- coding RNA (e.g. miRNA). Ribosomal RNA associates with a set of proteins to form ribosomes. These complex structures, which physically move along an mRNA molecule, catalyze the assembly of amino acids into polypeptide chain. A eukaryotic ribosome is composed of 4 different rRNA molecules and as many as 80 proteins, organized into a large subunit and a small subunit. The ribosomal subunits and the rRNA molecules are commonly designated in Svedberg units (S), a measure of sedimentation rate of macromolecules centrifuged under standard conditions –essentially, it is a logarithmic measure of size. The small ribosomal subunit (40S) contains a single rRNA 18S, which is combined with 33 proteins. The large ribosomal subunit (60S) contains 3 rRNAs (28S, 5.8S, and 5S), which are combined with 47 proteins. The assembled ribosome (80S) consists of 2 subunits (large and small) and is ready to function as a protein-synthesizing machine.  © Timoshenko, 2024 Nucleolar stress Page 3 of 8 Eukaryotic pre-rRNA transcript All cells contain multiple copies of the rRNA genes. Pre-rRNA gene is a single transcription unit which encodes 18S, 5.8S and 28S rRNAs. Red bars on this figure represent regions which encode the 3 rRNA molecules while blue bars are spacers that bind together the rRNA encoding regions. The genes are always arranged in the same 5’  3’ order: 18S  5.8S  28S and the only difference in pre-rRNA transcripts between species are the sizes of the spacers in the pre-rRNA transcript. Pre-rRNA genes are arranged in long tandem arrays (sitting one by one beside each other) and function as nucleolar organizer, this is where nucleolus is localized. The tandem arrays of ribosomal genes for 5.8S, 18S, and 28S rRNAs are clustered in on five different human chromosomes (13, 14, 15, 21, and 22) and presented by appx. 200 copies. Transcription of pre-rRNA is mediated by RNA polymerase I. To note, you might remember that transcription of regular protein-coding genes is mediated by RNA polymerase II. The exception is 5S rRNA genes, which are present as a single tandem array on chromosome 1 and the transcription occurs in nucleoplasm using RNA polymerase III; overall there are ~2000 copies of this gene in cells. Pre-rRNA transcripts can be visualized using electron microscopy. The electron micrograph shows multiple pre-rRNA molecules emerging from two transcription units in nucleolus of a frog oocyte. Transcription goes along the DNA strand and once rRNA is synthesized you see the extension of nascent pre-rRNA strands that look like a feather.  © Timoshenko, 2024 Nucleolar stress Page 4 of 8 Chemical modification and nucleolytic processing of pre-rRNA in DFC Primary rRNA transcript has 3 rRNAs. These fragments need to be separated because 18S is required for the small ribosomal subunit and the other 2 are required for the large ribosomal subunit. This process is complex and associated with chemical modification of the primary rRNA molecules by methylation and pseudouridination, which is eventually needed for recruiting cleavage enzymes. Methylation and pseudouridination of pre-rRNA requires small nucleolar RNAs, which hybridize to pre-rRNAs and bind enzymes that are responsible for the modification. snoRNAs have stem-loop structure and associate with specific proteins to form small nucleolar ribonucleoproteins (snoRNP). There are two major classes of snoRNAs called box C+D snoRNAs and box H+ACA snoRNAs, which are associated with different enzymes. In particular, box C + D snoRNA associates with methyl transferase fibrillarin, which mediates methylation of specific riboses. Box H+ACA snoRNA associates with pseudouridine synthase dyskerin, which converts uridine to pseudouridine; this conversion involves rotation of the pyrimidine ring. Thus, snoRNAs assist in processing pre-rRNAs to yield mature rRNA molecules, which are required to assemble small and large ribosomal subunits. Ribosomal subunit assembly This slide is busy with multiple regulatory molecules involved in the assembly of ribosomes, but we pay attention only to the major steps of this complex mechanism. Initially, specific ribosomal proteins and snoRNAs associate with the nascent pre-rRNA transcript and form precursor Pre-90S complex in nucleolus. Next, the Pre-90S complex is cleaved into two precursor subunits Pre-60S and Pre-40S, which are modified/processed in the nucleoplasm. The maturation and processing of the small subunit is very quick (~5 min) while it takes much longer time for the large subunit (~30 min). The formation of ribosomal subunits requires a lot of energy because uses GTPases and ATPases, especially in the case of the large subunit. Finally, both small and large subunits pass through nuclear pore complexes and move to cytoplasm where mature and functional ribosomes are assembled.  © Timoshenko, 2024 Nucleolar stress Page 5 of 8 Cell cycle-dependent nucleolar dynamics Nucleoli are formed around clusters of ribosomal genes, called Nucleolus Organizer Regions (NORs). These clusters are usually on the short arm of chromosomes which contains genomic repeats/transcription units encoding rRNA. Nucleoli are dynamic structures: each mitosis - nucleolus breaks up as chromosomes condense, and after mitosis - nucleolus reforms from coalesce of tips of several chromosomes (13-14-15- 21-22). The figure depicts characteristic changes of nucleolar architecture in different phases of the cell cycle. In the M-phase, nucleolus disassembles, and many nucleolar proteins associate with the surface of metaphase chromosomes. UBF remains associated with active rDNA repeats, serving as mitotic bookmark and binding platform for the RNA Pol I machinery. In G1-phase, nucleolar association occurs: FC/DFC/DC structures evolve around active transcription units resulting in the formation of a few large nucleoli that contain all active NORs. In S-phase, RNA Pol I may transiently leave active rRNA genes to prevent collisions with the replication machinery which leads to disappearance of the FC (dark green). At this point, cells need to duplicate their genetic material (RNA Pol I not needed) and cells want to focus energy on synthesizing molecules required for cells in the S-phase. In fact, in S-phase cells have sufficient number of ribosomes and want to save energy for protein synthesis rather that to make new ribosomes, which is a very energy-consuming process. In G2-phase, large, fused nucleoli may contain poised/inactive rDNA units, presented as dark green FCs without surrounding DFC. The genes are duplicated but not all of them are active in preparation for mitosis.  © Timoshenko, 2024 Nucleolar stress Page 6 of 8 Stability and stress-induced remodeling of nucleolar architecture Formation of the nucleolus depends on specific types of RNA called aluRNA – these function as a “glue” to form the nucleoli. These RNAs are encoded in the introns of many genes. Alu repeats are the most abundant repetitive intronic elements of primates constituting more than 10% of the human genome. The name “Alu elements” is based on the fact that most of them contain a single recognition site for the restriction enzyme AluI. Alu elements are scattered throughout the human genome at sites where their insertion has not disrupted gene expression. Alu transcripts are synthesized by both RNA polymerase II and III. aluRNA helps to stabilize and form the nucleolus by binding together the proteins which make the nucleolus (e.g. nucleolin and nucleophosmin). If you treat cells with amanitin (a selective inhibitor of RNA polymerase II and III) and induce the depletion of aluRNA, we end up with a dispersed nucleolus. Nucleoli are central parts of cells which are involved in the regulation of cellular responses to stress. Under normal conditions, there is a regular transcription activity and the nucleoli are doing their regular job of making ribosomes. Under stress, there is significant remodeling of nucleoli. Environmental cues such as heat shock or acidosis induce expression of a specific intergenic spacer (IGS) RNA, which targets proteins containing a peptide code, termed the nucleolar detention sequence. Accumulation of IGS transcripts correlates with formation of a large subnucleolar structure, termed Detention Center, structural remodeling, and transcriptional inactivation of nucleoli. The Detention Center occupies the central part of inactive nucleoli, which leads to redistribution of nucleolar factors and movement of FC/DFC structures towards the nucleolar periphery. Detention center tells us that there is relocalization of proteins in the nucleolus under stress. This results in the inhibition of transcriptional activity in the nucleolus which will inhibit the formation of ribosomes.  © Timoshenko, 2024 Nucleolar stress Page 7 of 8 The multifunctional role of nucleolus Nucleoli contain a tremendous number of proteins with different functions, overall about 4500 proteins. Out of these proteins, only 30% are involved in ribosome subunit biogenesis. The other 70% are not related to ribosome biogenesis, however they are important in regulating different aspects of stress signaling. Therefore, the nucleolus is being recognized as a key hub in the cellular stress responses by sensing and reacting to various stimuli. Nucleolar stress signaling pathway rely on the dynamic binding and release of proteins in response to stress stimuli. A central protein of nucleolar stress responses is p53, which controls processes of apoptosis, cell cycle regulation, DNA replication and repair, and more. Transcription factor p53 protein P53 is a transcription factor, also known as “Tumour suppressor gene” or “guardian of genome”. The p53 polypeptide contains several functional domains including 1) two transactivation domains at N-terminus, 2) DNA binding domain (central region), 3) oligomerization domain responsible for tetramerization of p53, and 4) C-terminal domain with nuclear export and nuclear localization signals. P53 is inhibited by protein MDM2 (Mouse double minute 2 homolog), which induces its degradation. The active form of p53 is a tetramer which binds to responsive elements of relevant genes. TP53 and cancer The TP53 gene is the most frequently mutated gene (>50%) in human cancer and most mutations are associated with DNA-binding domain. There is also strong evidence that mutation not only abrogates p53 tumor-suppressive functions, but in some instances can also provide mutant proteins with new oncogenic properties.  © Timoshenko, 2024 Nucleolar stress Page 8 of 8 p53 signaling pathway Normally, p53 level in cells is low because of interactions with E3 ubiquitin ligase MDM2 and fast degradation in proteasomes. Stress stimuli, for instance DNA damage, activate ATM (Ataxia telangiectasia mutated) kinase that phosphorylates and stabilizes p53, which can work now as a transcription factor. Once phosphorylated and activated, p53 tetramers bind response elements in p53-induced genes, which can be involved in processes that induce apoptosis, stop cell division (maintain in G1 and G2), and induce DNA repair. The amount of available p53 is elevated under stress because ATM also induces phosphorylation of MDM2 that inhibits its interaction with p53. There are different nucleolar proteins which can bind MDM2, for example p14ARF, which will also result in the release of p53. The nucleolus plays an essential role in the activation of p53. Nucleolus at normal growth conditions In the nucleolus, the ribosomal DNA repeats are transcribed by RNA Pol I, making rRNAs. There is also production of ribosomal proteins (RP) which interact with rRNAs and assemble into the small and large ribosomal subunits (40S and 60S). Small and large subunits are them released from the nucleus into the cytoplasm where they can bind together and can now translate many proteins in the cytoplasm. P53 is translated in the cytoplasm (constantly synthesized) but goes into the nucleus where it interacts with MDM2, gets ubiquitinated, and then degraded in the cytoplasm that keeps low level of p53 in cells. As a result, p53 target genes are not expressed. Nucleolus and stress response - p53 activation Upon nucleolar stress, nucleolar factors, including ribosomal proteins (RPs) and rRNAs are released from the nucleolus into the nucleoplasm and cytoplasm. Dotted line around the nucleolus represents its disassembly; all of the proteins that were present in the nucleolus are not going to make any more ribosomal subunits. Instead, these proteins are now available to do other jobs considering they have a high affinity to bind MDM2. The binding of MDM2 will result in the release of p53, which is available now to be phosphorylated and induces the activation of p53 target genes. In addition, RPL26 binds p53 mRNA and enhances its translation increasing the levels of p53 in cells.