The Nucleus

Department of Biological and Environmental Sciences
Cell & Molecular Biology
Dr. David A. Johnson
Biol 405 4 Credits Spring 2017 MWF 11:45-12:50 AM PH 204

The Nucleus
Text Chapter 9

The nucleus is a large, membranous organelle found only in eukaryotes. Its presence makes possible eukaryote-only processes, such as post-transcriptional RNA processing (capping, polyadenylation, editing, splicing). (Splicing is no longer a eukaryote-only process!)
The Nuclear Membrane (Nuclear Envelope): This membrane separating the nucleus from the cytoplasm is actually a double membrane with pores and is attached to the nuclear lamella on the inside. The outer of the two membranes is continuous with the endoplasmic reticulum and has all the characteristics of the ER (including attached ribosomes). The two membranes have the same phospholipid bilayer structure seen in the plasma membrane.
The Nuclear Lamina: Just inside the nuclear membranes is a network of fibrous proteins (lamins) called the nuclear lamina. Lamins are a type of intermediate filament proteins (we will see more examples when we take up the cytoskeleton). These proteins form tertiary and quaternary structure (coiled coil and higher order structures). The lamina binds to the inner nuclear membrane and to chromatin (H2A and H2B histones).
Nuclear Pore Complex: While diffusion of small, uncharged molecules is possible across the phospholipid bilayer, macromolecules, polar molecules, and ions must enter and leave the nucleus through nuclear pores. Nuclear Pore Structure: A nuclear pore joins the inner and outer nuclear membranes and each pore includes numerous (up to 50 in vertebrates) proteins called nucleoporins. With the electron microscope a pattern of eight protein structures in radial symmetry around the central channel can be seen. The pore also has a central ring and a ring on both the cytosolic and nuclear sides of the pore. Protein filaments extend outward from the two surface rings.
Entry and Exit: Entry and exit to and from the nucleus can be passive or active.
Passive Diffusion: Smaller proteins (20-40 kd) and small molecule can freely pass through the open pores in either direction by simple diffusion. Active Transport: Macromolecules (like larger proteins and RNAs) and ribonucleoprotein particles (like preribosomal subunits) must be actively transported into and/or out of the nucleus through the pores. Proteins that must enter the nucleus (they are all made in the cytoplasm) include all of those involved in DNA and RNA metabolism we studied earlier (histones, DNA and RNA polymerases, other replication enzymes, transcription factors, RNA processing enzymes, plus others). While many proteins enter the nucleus, some are shuttled back and forth so must also be able to leave the nucleus. RNAs made by transcription must be able to leave the nucleus.
Protein Entry/Exit: A protein that is destined to enter the nucleus is marked with a short amino acid sequence called a nuclear localization signal (NLS) which is recognized by a nuclear transport receptor (protein). These short nuclear localization signal sequences may be consecutive amino acids or bipartite.
Karyopherins--an Example of a Nuclear Transport Receptor: Karyopherins come in two varieties: importins and exportins (guess which one imports macromolecules into the cell and which one exports). Importins and exportins bind to a protein to be imported or exported and cross the nuclear pore with that protein.

Regulation of Entry/Exit by the Ran Protein: Ran proteins: Ran proteins (RAs-related nuclear proteins) are GTP binding proteins that regulate the entry/exit of other proteins into/out of the nucleus. The GTP can be hydrolyzed to GDP and P by an enzyme on the cytosolic side of the nuclear membrane.
Ran GAP: This hydrolyzing enzyme is called Ran GAP (Ran GTPase-activating protein). Ran GEF: An enzyme that exchanges the GDP bound to Ran for GTP is localized on the inside of the nuclear membrane. This enzyme is called Ran GEF (Ran guanine nucleotide exchange factor). Therefore, Ran-GTP is in high concentration inside the nucleus and Ran-GDP is in high concentration outside the nucleus.
Importins and Exportins: These are also important in this transport process. Protein Import: Import of a protein begins with the binding of an importin to the nuclear localization sequence of the "cargo" protein (the protein to be imported). This complex binds to nuclear filaments on the outside of the nuclear pore and the importin-cargo complex is transported through the pore. Inside the nucleus, Ran-GTP binds to the importin and this binding displaces the cargo, releasing it inside the nucleus. The Ran-GTP-importin complex is then transported back through a pore to the outside where Ran GAP hydrolyzes the GTP to GDP releasing the importin to be reused. (The Ran-GDP itself must be transported by its own transport mechanisms back into the nucleus where it will be quickly converted into Ran-GTP by Ran GEF, replacing its GDP with a GTP.)
Protein Export: Export occurs by a similar mechanism except that inside the nucleus Ran-GTP binding to exportin promotes the binding of the cargo protein. When this Ran-GTP-exportin-cargo complex is transported out of the nucleus, Ran GAP converts the GTP to GDP. This induces the release of the cargo protein and the dissociation of the exportin. (Note: Both entry and exit require GTP, that is, they require energy, that is, they are examples of active transport.)
RNA Entry/Exit: Like the transport of large proteins across the nuclear membrane, the import and export of RNAs in and out of the nucleus is an active (energy-requiring) process. Ran-GTP dependent importins and exportins transport most tRNAs, rRNAs, and snRNAs (similar to protein transport). mRNAs however are transported by other proteins and apparently do not require Ran. snRNAs that are part of snRNPs (used in splicing) are actually transported out of the nucleus, associate with protein, then the completed snRNPs are transported back into the nucleus as a result of nuclear localization signals on the protein. (We will look at rRNA transport below under the nucleolus.)
Organization within the Nucleus: The nucleus, contrary to earlier thinking, is not a homogeneous material nor are the chromosomes randomly distributed in the nucleus. It shows organization and compartmentalization. Chromatin: This is the stuff chromosomes are made of. Chromatin Condensation during Interphase:
Heterochromatin: Chromatin that is condensed during interphase is called heterochromatin. Heterochromatin is of two varieties:
Constitutive heterochromatin is never transcribed and is condensed in all cells (i.e., satellite DNA and centromere DNA). Facultative heterochromatin is condensed in some tissues or at some times but may be decondensed in other tissues or at other times (transcriptionally active DNA is decondensed).
Euchromatin: Chromatin that is not condensed during interphase is called euchromatin.
Localization within the Nucleus: Certain features and processes are not randomly distributed in the nucleus but localized to an area.
Chromatin Localization: Chromatin is apparently not randomly distributed in the nucleus, but the actual patterns vary with tissues, organisms, and time.
Localization of Replication: By visualizing DNA synthesis using bromodeoxyuridine and fluorescent antibodies, it is clear that DNA synthesis occurs in discrete clusters.
Localization of Splicing: Similarly, splicing machinery is found in clusters in the nucleus.
The Nucleolus, rRNA Processing and Transport, Ribosome Assembly and Transport: The nucleolus is the site of 45S pre-rRNA transcription and processing and the site where ribosomes are assembled. The nucleolus is not membrane enclosed.
The NOR: The nucleolus is a structure which is formed by the aggregated tandemly repeated genes for the 45S pre-rRNA. In humans, these genes are on chromosomes number 13, 14, 15, 21, and 22 and make up the nucleolar organizer region (NOR).
45S pre-rRNA Transcription and Cleavage: In the nucleolus, RNA polymerase I transcribes this 45S pre-rRNA and while still in the nucleolus it is cleaved into the 18S, 5.8S, and 28S rRNAs (includes the removal of the spacers: ETS and ITS--external transcribed spacers and internal transcribed spacers; there are also untranscribed spacers between the 45S genes ).
rRNA Chemical Modification: Also in the nucleolus rRNA is chemically modified, especially by methylation.
snoRNAs and snoRNPs: Both the cleavage and chemical modification reactions are catalyzed by protein/RNA complexes called snoRNPs (small nucleolar ribonuclear proteins). The snoRNA components of the snoRNPs include RNAs that cleave the 45S pre-rRNA and RNAs that recognize specific base sequences that are to be methylated.
5S rRNA: 5S rRNAs are transcribed outside of the nucleolus by RNA polymerase III but are assembled into the ribosomes in the nucleolus. rProteins: The proteins of the ribosome are made like all other proteins (transcribed by RNA polymerase II and the transcript is translated in the cytoplasm). They must then enter the nucleus (see above). Ribosome Assembly and Transport: Ribosomal proteins begin to aggregate with the 45S pre-rRNA before its transcription is finished. More than half of them are bound before this rRNA is completely cleaved. When these proteins have bound, the 40S preribosomal subunit (with the 18S rRNA) and 60S preribosomal subunit (with the 28S, 5.8S, and 5S rRNAs) are exported from the nucleus through nuclear pores where they become mature 40S and 60S ribosomal subunits ready to take part in translation.