Part III Cell Structure and Funtion BL424 Chapter 9: the nucleus Nucleus is principal feature that distinguishes eukaryotic from prokaryotic cells. Nucleus is site of replication of DNA, transcription of DNA, and RNA processing (Fig. 9.1); Translation occurs in cytoplasm; separation of processes permits regulation Student learning outcomes: 1*. Explain the general structure of the nuclear envelope (connected to ER), the nuclear lamina, and the nuclear pore complex. 2*. Explain concisely the movement of proteins and RNA molecules between the nucleus and the cytoplasm, describing briefly the selectivity, regulation and energy-requirements. 3. Describe the internal organization of the nucleus. 4*. Describe the structure and function of the nucleolus. Important Figures: 1*, 4, 5*, 6, 8, 9*, 10, 11*, 13, 15, 25*, 29*, 30, 31* Important Tables: 1 1*. Nuclear envelope and traffic between the nucleus and cytoplasm. [ The double-layer nuclear envelope (Fig. 1*) separates the contents of nucleus and cytoplasm: The outer and inner phospholipid bilayer membranes are joined at nuclear pores: The outer membrane connects to the ER and can have ribosomes bound = rough ER (synthesis of proteins destined for modification, transport to organelles or secretion) The inner membrane has distinctive proteins: (Fig. 9.5*) The nuclear lamina (mesh) composed of lamin proteins underlines this membrane and binds chromatin thru H2A/H2B. Lamins are intermediate filaments that associate as dimers and higher-order structures (Fig. 9.4), Humans have 3 genes -> about 7 different proteins. Lamins often prenylated (Fig. 8.34) and bind membrane; Also bind membrane proteins: lamin B receptor (LBR), emerin Mutations in lamin genes can cause human disease Hutchinson-Gilford Progeria results from mutations LMNA gene (premature aging syndrome) Erery-Dreifuss muscular dystrophy due to mutations in: Emerin (X-linked, inner membrane protein) LMNA (non-X-linked, affects lamins A and C proteins)The nuclear pore complex is a complex structure (channel) that controls transport (Figs. 9.5, 9.6, 9.8): It has 8-fold symmetry, and 30-50 different proteins: Nucleoporins Rings on cytoplasmic and nuclear side, 8 spokes; About 30 times bigger than ribosome – 120 nm Central channel about 10-40 nm Small molecules diffuse freely through (proteins < 20 KD); Macromolecules (proteins, RNAs) are selectively transported, In specific directions, which requires energy. (Fig. 9.6); Ex. mRNA goes out, transcription factors go in Selective transport of proteins to and from the nucleus uses amino acid sequences present in the protein. Nuclear localization sequences (NLS) are recognized by receptors that transport these proteins into nucleus ex. SV40 virus, T antigen has one very basic sequence; mutated sequence and protein stayed cytoplasm; add to other protein and it goes to nucleus. Nuclear export sequences are present on proteins targeted for export from the nucleus. Proteins that shuttle have both sequences. Translocation through the nuclear pore complex requires Ran, a small GTP-binding protein (Fig. 9.10) (like Ras) Ran has different conformations when binding GTP or GDP. Ran/GTP is higher in the nucleus – determines transport (because of differential localization of enzymes GAP (GTpase-activating protein) and GEF (G exchange factor) Nuclear transport receptors (karyopherins) (Table 1): Receptors have specificity for their cargo, specific sequences. Importins bind NLS, take molecules into the nucleus (Fig. 9.11): Ran-GTP dissociates cargo, takes importin to cytoplasm Exportins take molecules to the cytoplasm (Fig. 12): Exportin binds NES, Ran-GTP helps exit; Ran-GAP hydrolyzes GTP, releases cargo & exportin The nuclear import of some proteins, (Fig. 9.13). such as transcription factors, can be regulated: by binding of other molecules (ex. NF-kB bound to IkB has NLS masked); or (de)phosphorylation (Pho4 inactive with PO4, imported after de-PO4)RNAs are transported out of the nucleus as ribonucleoprotein particles (Fig. 9.15*). ex. rRNA plus its proteins (Fig. 9.31); mRNA (processing proteins, exportins) some small RNAs (snRNAs, snoRNAs) (Fig. 9.15) go to cycloplasm, and then return to function in nucleus with their proteins 2. Internal organization of the nucleus includes matrix of lamins. Interphase chromosomes have tightly condensed (fig. 9.16) Heterochromatin (transcriptionally inactive); And decondensed euchromatin (active). Nucleolus is site of rRNA synthesis Chromatin is organized as looped domains bound to lamins (Figs. 9.17-9.20) Some nuclear components are localized (sequestered) to discrete subnuclear structures: DNA replication (Fig. 9.21) speckles with splicing machinery (Fig. 9.22) PML bodies have transcription factors (Fig. 9.23) 3*. The nucleolus is site of rRNA synthesis and processing, tRNA processing and ribosome assembly (Figs. 9.25, 26, 28). The nucleolar organizing region = site of genes for rRNA. Numerous copies of RNA genes are present (Fig. 9.25) Each has 18S, 5.8S and 28 S genes + spacers: Transcribed into pre-RNA that gets processed (Fig. 9.29, 7.43). Size of nucleolus depends on level of gene activity of cells Since many ribosomes are needed; Nucleolus is not seen during mitosis SnoRNAs (small nucleolar, SnoRNPs) mediate processing of pre-rRNA , other small RNAs (Fig. 9.30). Ribosome assembly in nucleolus (Fig. 31) requires: import of ribosomal proteins from cytoplasm, association of 5S RNA and other rRNA, export of the subunits to cytoplasm. Review: relevant questions at end of chapter are all. Read the molecular medicine connection – nuclear lamina genes Consider how we know the roles of the particular modifications of proteins or of the different receptors and Ran protein. Particular mutant cells might be constructed and analyzed… consider how these experiments could be