Exam 3 - Cell Bio

Translation Basics

During translation, nucleotide is read in triplets = “codons” from 5’-3’ (synthesizing protein from N terminus → C terminus)

• Start codon is slightly downstream of 5’ end (UTR) of mature mRNA. ⇒ AUG (methionine) 

• Stop codon is slightly upstream of 3’ end (UTR) of mature mRNA. ⇒ UAA, UAG, UGA

  • Does not code for amino acid. 

• ORF: Open reading frame -> region that codes for amino acids

tRNA

• Anticodon sequence is 3’-5’ ⇒ antiparallel to the mRNA transcript. 

• Modified bases: stability. 

• 3-Dimensional structure w/ 3’ single-stranded overhang ⇒ amino acid attachment gene. 

  • pre-tRNAs are made by RNA Pol III and undergo processing steps → mature tRNAs. 

tRNA - Activator

• Aminoacyl tRNA synthetases (enzyme) recognize both the tRNA and the amino acid → example: histidyl tRNA synthetase for histidine. 

  • ATP hydrolysis at the amino acid → aminoacyl AMP → attaches amino acid to tRNA’s 3’ overhang. 

  • Creates a charged tRNA that can complementary base pair with the mRNA. 

  • At least one enzyme/amino acid. 

• Can be recycled.

tRNA modification

Modification of tRNA can result in modified nucleotides that end up in the anticodon ⇒ Unusual base pairing in codon-anticodon recognition. 

• Relates to redundancy of genetic code. 

• When tRNA is attached to mRNA by ribosome → 2 nucleotide pairs are really tight, whereas the third position complementary codons not as close → nucleotide freer to move (wobble effect). 

• G-U can base pair due to weaker H bonding. 

• Modified inosine in 1st position of anticodon → can base pair with uridine, cytosine, and adenine. 

Wobble hypothesis: base in the first position on tRNA (5’ end of anticodon, 3’ of codon) = usually an abnormal base like inosine, pseudoeuridine, tyrosine.

Structure of ribosomes

Ribosomes help form peptide bonds (covalent, dehydration reaction) between amino acids. 

• Direction of synthesis: Amino terminus → carboxyl terminus

• Reads one amino acid at a time. 

Parts of 80S ribosome ⇒ composed of multiple polypeptides and ribosomal RNAs. 

• Large subunit (60S): where the polypeptide forms between P and A sites (catalyze peptide bond formation) ⇒ 28S, 5.8S, 5S rRNAs 

  • Main role: The RNAs in the large subunit are the enzymatic parts (NOT THE PROTEINS) 

• Small subunit (40S): Recognizes AUG ⇒ 18S rRNA

• NOTE: 28S, 18S, 5.8S (nucleolus, RNA Pol I); 5S (nucleus, RNA Pol III). 

pre-rRNA processing

Processing of pre-rRNA transcript is processed via cleavage → mature rRNAs. 

• Then, other modifications ⇒ for stability and structure. 

• Finally, they are complexed with ribosomal proteins 

• Location: Nucleolus.

Prokaryotic mRNA

Poly-cistronic: one mRNA can be translated to produce multiple proteins.

• Lac operon: 3 different mRNAs back to back in 1 transcript w/o 5’ methyl capping and 3’ poly-A (translation and transcription occurs in the same area in bacteria). 

  • 3 diff protein coding sequences w/ multiple translation start sites. 

• Small subunit of ribosome recognizes AUG & has complementary base sequence to sequence upstream of AUG. ⇒ Shine-Dalgarno sequence.

Eukaryotic mRNA

One gene → one mRNA → one protein (Mono-cistronic) 

• Small subunit recognizes the 5’ methyl cap and scans downstream for the AUG. 

  • In contrast, prokaryotic mRNA translation DOES NOT require scanning → directly identify the S-D sequence.

Translation: Initiation

Starts: the cytosol

• Requires: eukaryotic Initiation Factors (eIFs) 

  • eIF4E: specifically binds the 5’ cap ⇒ inspector: ensures mRNA is stable. 

  • Poly-A Binding Protein (PABP): binds the poly-A tail.

    • Poly-A tail is required for translation because PABP is part of the initiation complex. 

    • PABP recruits eIF4G (component of eIF4 complex) 

• W/ the complex, the small subunit binds at the 5’ UTR and scans to find the start codon. 

  • ATP is required for proteins to “flatten/unwind” the mRNA.  

• Large subunit is recruited: 

  • eIF5B (also a GTP-binding protein) uses energy from GTP to piece the ribosome complex together. 

  • eIF2 binds initiator: charged methionyl transfer RNA and plug it into the ribosome → start initiation (energy: GTP → GDP) 

    • Regulated by binding GTP/GDP. 

Exceptions for translation

• Exception: 

  • Some viral & eukaryotic mRNAs have internal ribosome entry sites (IRES) closer to the AUG → where translation can initiate independently of the 5’ methyl cap. 

    • Viruses enable their own mRNA to be transcribed more quickly > our endogenous mRNA. 

    • Eukaryotic mRNAs tend to be used in stressful conditions ⇒ saves energy.

Translation: Elongation

• Polypeptide chain elongates by successively adding amino acids. 

• Requires: eukaryotic Elongation Factors (eEFs) 

• Parts of large subunit: 

  • P (peptidyl) site

  • A (aminoacyl) site

  • E (exit) site) 

• Process:

  • Start codon at the P site. 

  • Loading of new tRNA at A site → plugged in by eEF1α w/ GTP → GDP. 

    • Peptide bond 

  • eEF2 (GTP → GDP) leads to translocation ⇒ shifts the chain over by 1 codon. 

    • tRNA at the P site is now at the E site and is released; tRNA at A site is now at P and carries the chain.

Translation: Termination

• Requires: Release Factors → proteins that have a similar shape to tRNA and recognize the stop codons.

  • Bind to stop codon at the A site & stimulate hydrolysis of bond between tRNA and polypeptide at P site. 

  • Disrupt the process of translation ⇒ polypeptide is released (to fold) and the ribosome dissociates. 

G-binding protein life cycle

Sustains translation

• Example: eEF1α hydrolyzes GTP w/ GTPase (cutting GTP) activity → once hydrolysis is complete eEF1α is bound to GDP and needs to be refreshed with GTP. 

  • Generally: Exchange of GDP for GTP requires guanine exchange factor (GEF) 

    • Knocking off the GDP → new GTP will preferentially bind (in excess) to activate the eEF1α. 

• GAP (GTPase-activating protein): accelerates the hydrolysis of GTP → GDP cuz G proteins are relatively slow w/ hydrolysis.

  • Inhibits GTP-binding proteins.

Translational regulation of specific mRNAs

• Translational repressors inhibit translation by binding to 5’ or 3’ UTR sequences of mRNA. 

• Regulation of Poly-A tail length

• RNAi

Translational repressors at 5’ UTR

• Example: the regulation of Ferratin translation in response to iron lvls

  • Ferratin: synthesized for iron storage 

  • IRE (Iron-response element) in the 5’ UTR is an RNA sequence that allows for the binding of ribosome elements. 

    • When iron is adequate: binding of 40s (small) ribosomal subunit → translation proceeds. 

    • Iron scarce: IRP (iron-regulatory protein) binds to IRE = blocks the initiation of translation. 

  • Faster process to block the transcriptional level ⇒ increase adaptability. 

Translational repression at 3’ UTR

translational repressor binding at 3’ UTR interferes with PABP ⇒ also affects initiation factor binding.

Poly-A regulation

• Poly-A regulates translation rate via PABP (which binds eIF4G) 

• Length of poly-A can be regulated by proteins that bind 3’ UTR → extension or shortening.

RNAi

Can be used to block gene expression: 

• siRNAs produced from double-stranded RNAs by Dicer (nuclease) → Synthetic siRNAs can be transfected to target mRNA of interest → associated with RISC → targets specific mRNA. 

• miRNA (endogenous): pri-mRNA → Drosha → pre-miRNA → Dicer → miRNA duplex → association with RISC & unwinding of miRNA strands:

  • Perfect pairing w/ target mRNA = mRNA cleavage 

  • Mismatched pairing:  repression of translation, deanylation & mRNA degradation. 

→ regulation depends on how “good” of a match they are

Global regulation of translation

Purpose: translation is energy-intensive, therefore turning off = allows for energy conservation in stressed/starved conditions.

Targeting eIF2/eIF4 

• GDP-bound eiF2 and eiF4 are inactive

Global regulation of translation: eiF2

• eiF2: attaches tRNA carrying methionine to initiate translation. 

  • GEF (GTP-Exchange Factor) protein exchanges GDP for GTP 

    • eIF2B exchanges GDP for GTP on eIF2 ⇒ the cycle continues if cells are healthy and we have growth factors. 

  • Stress/growth factor starvation: causes phosphorylation of eIF2 and eIF2B by regulatory kinases = allosteric modulation. 

    • Exchange of GTP for GDP is blocked. 

Global regulation of translation: eiF4E

• eiF4E: binds the 5’ methyl cap 

  • Growth factors: activation of kinases → phosphorylate 4E-BP (binding protein) so eiF4 complex can form ⇒ translation proceeds. 

  • In absence of growth factors: non-phosphorylated 4E-BPs bind to elF4E & inhibits translation. 

Protein folding: denatured protein

• Denatured protein can (although it takes time) reform its native shape on its own: each amino acid inherently has properties based on R-group tendencies. 

  • Experiment: Denatured RNase could reform native RNase on its own ⇒ amino acid sequence alone determines structure.

Chaperones

Chaperones speed up the process of DNA folding

• Does not determine the final 3-D structure of protein (is determined by primary sequence of amino acids) ⇒ chaperones play the helping role to accelerate this pre-determined formation. 

• Belong to Hsp70 family: Heat-shock proteins → elevated expression in response to high temperatures. 

  • Allows cell to compensate for denaturing effect of higher temperatures. 

Mode of action: Preventing aggregation

Stabilize unfolded polypeptide chains to prevent aggregation.

• Chaperones help the folding of protein as translation is occurring. 

• Chaperones recognize and bind to the hydrophobic amino acid R-groups that are protruding 

  • Hydrophobic R-groups = lipophilic amino acids = more sticky & easier to aggregate → bad in cell bcz of high concentration of other proteins. 

• Can also help stabilize unfolded polypeptide chains to prevent aggregation during transport into organelles 

  • Transfer polypeptide chain → mitochondria (cytosolic chaperone → mitochondrial chaperone.) 

    • Matrix is more basic (pH ~8) than cytosol → so can protonate certain amino acids and cause the protein to change shape. → so need different chapterons.

Mode of action: Provide isolated environment

• Purpose: isolated environment for folding to occur.

• Key class of chaperones: Chaperonin

• ATP required to transfer chaperonin both in & out of the chaperonin. 

  • Chaperones can play a role in transferring partially folded intermediates into this chaperonin. 

Diseases with protein misfolding/aggregation

Alzheimer’s disease, Parkinson’s, and Type 2 diabetes. 

• Native proteins experience both misfolding & aggregation of these misfolded proteins ⇒ making high-order structures called amyloids (β-sheet structures)

  • Lead to cell death because they cannot be removed 

  • Alzheimer’s: Amyloid-β and tau (associated with cognitive abilities) aggregated 

    • Drug: Adycanumab ⇒ antibody that targets amyloid-β aggregates ⇒ increase in cognitive abilities. 

• In pathology: increase in expression of misfolded proteins and decrease in recycling/degradation machinery → especially as we age. 

Early-onset Alzheimer’s: AD Protective variant

• APOE3 (APOE Christchurch) and reelin (RELN-COLBOS) ⇒ delaying the disease compared to other members of family w/o them = protective factor. 

  • So while the individual carries a mutation that predicts early-onset of AD (amyloid, Tau deposits), they also have variants of these protective alleles.

Chaperones: Protein Disulfide Isomerase (PDI)

Protein Disulfide Isomerase: PDI

• Cysteine (amino acid) has sulfur groups that can form disulfide bonds. 

• Crucial enzyme in the ER ⇒ involved in formation, breakage, and rearrangement of disulfide bonds in nascent proteins. 

Chaperones: Peptidyl Prolyl Isomerase (PPI)

• Proline breaks the alpha-helix because it binds back into the central carbon ⇒ making ring-structure = not as free to rotate between cis → trans conformation. 

  • In contrast, other amino acids are more free to do so. 

• PPI catalyzes the isomerization of peptide bonds that involve proline between cis and trans conformation. 

  • 90% of proline bonds are in trans conformation, but cis conformation often functionally relevant. 

Proteolysis

Proteolytic processing of insulin at the translational level: 

• Pre-proinsulin → cleavage of signal sequence at N’ terminus (during transfer to ER) and disulfide bond formation → forms proinsulin that is not active because of connecting polypeptide sequence. 

• When glucose is active: 

  • Removal of connecting polypeptide by protease → frees the insulin (to activate pathways that break down glucose.) 

  • A and B polypeptide chains are joined by disulfide bonds in insulin

• Important so insulin can react quickly (protein already made, just requires activation) 

Protein modification: Addition of carbohydrates

• Glycoproteins: Glycosylation adds carbohydrate chains to proteins. 

  • Carbohydrate moieties play an important role in protein folding in ER → targeting proteins for transport; recognition sites cell-cell interactions 

    • Most glycoproteins are for secretion or incorporation into plasma membrane.

    • Location: ER and Golgi apparatus

  • One sugar/time

Types of carbohydrate additions to proteins

• Types: 

  • N-linkage: occurring in asparagine residues ⇒ in ER before translation is completed

    • Sugar: N-acetylglucosamine

  • O-linkage: binds to oxygen atom in serine or threonine residues ⇒ added within Golgi apparatus. 

    • N-acetylgalactosamine

Protein modification: Addition of lipids - External

• modification frequently target & anchor these proteins to plasma membrane

  • Outer surface of plasma membrane: Modified in ER by addition of lipids linked to oligosaccharides to carboxy terminus 

    • Name: GPI anchors → glycolipids that attaches proteins to cell membrane.

Protein modifications: Addition of lipids - Internal

• Lipid to the cytosolic face of plasma membrane proteins (inner surface): amino terminus

  • N-myristoylation: initiating methionine is removed = glycine at the N-terminus of polypeptide chain. Myristic acid (14-carbon fatty acid) added. 

  • Prenylation: Fatty acids to cysteine residues. 

  • Palmitoylation: Palmitic acid (16-C fatty acid) added to side chain of internal cysteine residue. 

Binding of small molecule - Mechanisms

• Regulation of enzymes w/ inhibitory molecules that compete w/ substrate to bind active site. 

• Binding of molecules to allosteric site to change enzymatic conformation & other proteins 

  • I.e. transcription factors like E. coli lac repressor & lactose or steroid hormones in eukaryotes)

  • Translation factors eEF1alpha and eIF2 (G proteins) regulated by GPT/GDP binding → GTP = active conformation. 

  • Ras oncogene (G protein): in active GTP-bound form = can interact w/ target molecule to signal cell division but also be inactivated in GDP conformation; mutation = locks Ras in active conformation ⇒ uncontrolled proliferation of cancer cells. 

Protein kinases

catalyzing phosphorylation of protein

•  Transfer a phosphate group (PO42-) from ATP to a protein’s hydroxyl group (serine, threonine, or tyrosine residues.) 

• Two types: PKA and ERK 

• Purpose: Change chemical nature of side chain. 

  • Polar hydroxyl group replaced with negatively charged phosphate group ⇒ can disrupt interactions of side chain and/or cause new interactions.

Specificity determined by: 

• Recognition motifs 

• Additional regulatory proteins can bind both kinase & target protein to guide assembly. 

• Direct interactions between kinase and allosteric site & recognition motif of protein. 

Recognition motif

Sequence of amino acids in the vicinity of phosphorylation site (recognition motifs) ⇒ directly interact with kinase’s catalytic site ⇒ “lock-and-key” fit. 

• Consensus motif: generalized representation of common features found in recognition motifs → describes essential amino acids for recognition & allows for some variability. 

  • X = amino acid; / = alternatives. 

Each kinase can have multiple targets.

ERK kinase: allosteric interaction

• ERK kinase has catalytic site & distal docking site w/ nonpolar negative amino acids → interact with target proteins like Elk-1 w/ docking motif of nonpolar, positive amino acids. 

  • Binding = facilitates interactions between ERK catalytic site and recognition motif of target protein ⇒ once in complex, kinase initiates phosphorylation at recognition site.

Glycogen phosphorylase

Homodimeric enzyme 

• Catalyze breakdown of glycogen to glucose-1-phosphate 

• Regulated by phosphorylation at serine-14 of each its subunits. 

  • When serine-14 is not phosphorylated, the enzyme subunits adopt a conformation in which their catalytic sites are inaccessible for substrate binding. 

  • Phosphorylation = activates the enzyme ⇒ catalytic sites accessible for substrate binding.

Protein phosphatases

Removes the phosphate group (hydrolysis of phosphorylated amino acid residues) ⇒ serine, threonine, or tyrosine residues.

• Rely on the recruitment of phosphatases to target proteins by regulatory proteins (vs. recognition motifs in phosphorylated residue) 

Other modification of proteins with small molecules

• Histone modification:

  • Acetylation of lysine residues

  • Methylation of lysine and arginine residues 

• Nitrosylation (addition of NO groups) to cysteine residues.

Other non-small molecule modifications

• Covalent attachment of polypeptides: 

  • Ubiquitin = 76 amino-acid polypeptide that attaches to amino groups in side chain of lysine residues. 

    • Purpose: protein degradation, proteins involved in intracellular events (gene expression, repair, endocytosis, vesicle trafficking) 

Protein-protein interactions and regulatory role

Many proteins form & function as multimeric complexes. → noncovalent interactions between polypeptide chains regulate protein activity. 

Example: Protein kinase A regulation

• Enzyme PKA in inactive state has 2 regulatory and 2 catalytic subunits 

• cAMP binds to allosteric site of regulatory subunits = conformational change ⇒ dissociation from catalytic subunits

  • cAMP = allosteric regulator 

• The free catalytic subunits are enzymatically active. 

Regulating protein level

1. Regulating rate of synthesis through genetic expression. 

2. Regulating rates of degradation = proteolysis

• Ubiquitin-proteasome pathway: mediator of regulated protein degradation in eukaryotes

  • Location: cytoplasm and nuclesome

Structure of proteasome

• Large protein complex w/ ~60 subunits that assemble into central barrel w/ hollow core. 

  • Core lined with proteolytic enzymes and two identical lid complexes ⇒ controls what enters the central barrel for degradation. 

    • Also contains one or two regulatory particles 

• Proteasome is covalently modified with ubiquitin (expressed ubiquitously in most cells) → ubiquitin signals unwanted proteins for destruction. 

  • Targets: misfolded, damaged or regulatory proteins.

Process: Ubiquitin-proteasome pathway

• Ubiquitin activated through linkage with E1 enzyme. 

• Ubiquitin transferred to E2 enzyme (ubiquitin-conjugating enzyme)

• E2 complexed with E3 (ubiquitin ligase) ⇒ transferring ubiquitin to a lysine residue on the target protein. 

  • Some E3 can help mediate selective ubiquitination by forming protein-protein complexes w/ E2 and target

  • Proteins targeted for degradation marked by addition of multiple ubiquitins = polyubiquitin chain. 

• Polyubiquitin chain recognized by subunits of proteasome lid = triggers ATP-mediated unfolding of target protein & translocation into the central barrel. 

  • Proteolyzed into short peptides. 

  • Lid complexes deubiquitinates & remove ubiquitin = recycled.

Cyclin degradation in cell cycle

• Cyclin B: regulatory subunit for Cdk1 protein kinase 

  • After synthesis in interphase ⇒ forms complex with Cdk1 → active cycline-B-Cdk1 complex 

    • Cdk1 kinase activation initiates mitosis events. 

  • Cdk1 also activates ubiquitin ligase ⇒ Ubiquitination of cyclic B = targets it for proteasomal degradation at the end of mitosis. 

  • Degradation of cyclin B = inactivates Cdk1 = cell exits mitosis and re-enter interphase. 

• Mechanism: controls timing & progression of cell division phases in eukaryotic cells.

Structure of inner and outer membrane of nucleus

Two phospholipid bilayers around the nucleus and the perinuclear space containing the lumen. 

• Perinuclear space is continuous w/ the lumen of the ER 

• Inner side: nuclear lamina ⇒ holding the “circular/spherical” shape of nucleus.

Nuclear lamina

Made up of lamins (fibrous, intermediate-filament proteins)

1. Lone polypeptide w/ two different ends 

2. The lamin polypeptide can form a dimer ⇒ alpha-helical regions wind around each other = coiled coil

3. Head-to-tail association of dimers and side-by-side association ⇒ form higher structure of lamina

Mutations in lamin gene

Hutchinson-Gilford progeria disease: genetic disorder characterized by rapid aging in children & often shortens life expectancy ⇒ caused by mutations in LMNA gene (encodes lamin A and lamin C proteins)

• Mutation = 150-base-pair deletion in exon 11 because of aberrant splicing. 

• Proteins important to nuclear envelope structure 

• In HGPS = truncated lamin A (progerin) accumulates & leads to nuclear instability = affects cellular functions.

How nuclear lamina is anchored to inner nuclear membrane

Protein-protein interactions: interacting with nuclear lamins

• Emerin

• Lamin B receptor (LBR)

• SUN proteins: bind w/ KASH proteins in outer surface of nuclear membrane ⇒ constructing the LINC complex 

  • LINC complex connects nuclear lamina to the cytoskeleton ⇒ gives the nucleus a position in the cell. 

Addition of lipids (prenylation)

Nuclear lamin and chromatin

Nuclear lamins bind chromatin indirectly via interactions between lamin-associated proteins & histones H2A and H2B ⇒ can change accessibility of genes.

Nuclear pore complex

Only channels for transport in/out of nucleus. → large so can be seen in electron microscopy. 

• Eightfold symmetry with a central channel 

  • Composed of ~30 nucleoproteins (NUPS) 

    • FG-NUPs forms the central channel: rich in phenylalanine and glycine ⇒ filter for molecules passing in and out. 

  • Eight cytoplasmic filaments on cytoplasmic ring ⇒ outer nuclear membrane

  • Nuclear ring attached to the nuclear basket ⇒ inner nuclear membrane

Mode of transport in/out of nucleus

Passive diffusion: Small molecules/proteins ⇒ no energy, down concentration gradient to reach equilibrium. 

Energy-dependent transport: Larger proteins and RNAs are selectively transported; requires energy (active transport) 

• requires nuclear transport receptors (karyopherins)
  

Nuclear proteins: NLS features

• Nuclear proteins contain nuclear localization signals (NLS) 

  • Origin: first discovered in mutated T antigen

    • T antigen that wasn’t arriving at the nucleus ⇒ the mutation occurred at the NLS ⇒ NLS is required for nuclear localization

    • Transfer of T antigen NLS to cytosolic proteins ⇒ results in THAT new protein’s nuclear localization ⇒ NLS alone is sufficient for nuclear localization of any protein. 

• NLS can be bipartite: amino acid sequences separated by non-NLS amino acids. 

• NLS are rich in basic amino acids (Lysine, arginine, etc.) 

• Similar principles for localization of proteins to subcellular locations (like mitochondria, ER, nucleus)

Importin

Family of receptor proteins that bind NLS and transport nuclear proteins (and itself) into nucleus

• GTP-binding protein Ran controls nuclear transport by importins

  • Life cycle: 

    • Ran in nucleus = associated with GTP (high Ran-GTP levels; low Ran-GDP levels) 

    • Ran in cytoplasm = high Ran-GDP levels. 

    • Ran-GDP → Ran-GTP by Ran GEF in the nucleus ⇒ facilitates export of Ran-GTP into cytoplasm. 

    • Ran-GTP’s hydrolysis is accelerated by Ran GAP attached to the cytosolic filaments of the pore. ⇒ forms Ran-GDP. 

Process of transport of proteins into nucleus

1. So importin-protein complex enters the nucleus through the nuclear pore complex 

2. Importin bind to Ran/GTP to dislodge cargo protein 

3. Ran-GTP transports importin out of nucleus

4. Conversion of Ran-GTP → RanGDP release importin.

Process of export of proteins out of nucleus

• Proteins destined for export contain nuclear export signal (MES) 

• NES recognized and bound by exportins. 

• The binding of Ran-GTP stabilizes the complex and transports out of nucleus. 

• Conversion of Ran/GTP → Ran/GDP by RanGAP releases cargo protein. 

• Ran-GDP is transported by NTF2 back into the nucleus

mRNA export from nucleus

 Cytosol: independent of Ran & does not need karyopherins. 

• Distinct transporter complex moves the mRNA through nuclear pore. ⇒ ribonucleoprotein particles. 

  • Includes Poly-A binding protein that stays associated with the mRNA. 

• Helicase on the cytoplasm side releases the mRNA and ensures unidirectional transport. → “strips” mRNA of the proteins. 

Transport of snRNAs between nucleus and cytoplasm

• snRNA is transported out into the cytoplasm and binds to proteins → become snRNPs. 

• Snurportin brings snRNPs back into the nucleus, where they can function to splice pre-mRNAs.

Regulation of nuclear transport: Protein-protein interactions

• Protein-protein interactions mask NLS from importins

  • Example: NF-κB: Sequestering transcription factor IκB. 

    • In absence of appropriate stimuli, protein-protein interactions between NF-κB and IκB masks the NLS (nuclear localization signal) of NF-κB

    • Extracellular stimuli causes phosphorylation of IκB: 

      • Targets IκB for degradation

      • Proteolysis → Releases NF-κB = exposing its NLS 

    • NF-κB imported into the nucleus by importin = activates transcription 

Regulation of nuclear transport: Phosphorylation

• Phosphorylation blocks NLS-importin interactions

  • Pho4: regulated directly by phosphorylation. 

    • Pho4’s NLS is blocked by phosphate at serine (adjacent to it) 

    • Dephosphorylation of Pho4 by phosphatase = allows importin to bind to Pho4 → entering nucleus & activates transcription of target genes. 

Chromosome conformation capture (3C)

• Purpose:

  • Study 3D organization of chromatin inside the nucleus

  • Detects DNA regions that interact physically, even if far apart in linear sequence of DNA ⇒ in proximity when chromosome is folded in the nucleus. 

• Steps: 

  • Cross-linking: fix nearby DNA segments together in place. 

  • Digestion: cut DNA into fragments with restriction enzymes. 

  • Ligation: join the ends of cross-linked fragments that were close in 3D space. → produce contiguous sequences. 

    • Ligation in dilute conditions to favor ligation of fragments that are close in 3D space.

  • Reverse crosslinks & sequence: identify interacting regions. 

    • PCR analysis can amplify ⇒ amount of product can qantify the frequency of interaction of specific DNA segments 

Chromatin localization

Chromatin: Lighter regions → distributed throughout the nucleus. 

• Actively transcribed genes = situated at periphery of chromosome territories → topologically associated domains (TADs) 

  • Their boundaries are formed by CTCF and cohesin 

Heterochromatin: darker regions → mostly associated with the nuclear envelope and the nucleolus. 

• DNA creates a structure that surrounded the nucleolus, where rRNAs are transcribed, processed, and ribosomal subunits are assembled. 

  • This region is called nucleolus-associated domains (NADs) → DNA sequences in NAD and LADs overlap.

• Heterochromatin localized to the periphery of the nucleus ⇒ anchors chromatin to nuclear lamina → lamina-associated domains (LADs)

Chromatin structure is DYNAMIC! ⇒ change in coordination with changes in gene expression.

Flourescence in situ hybridization (FISH)

• Purpose: visualizes specific DNA or RNA sequences within intact cells or chromosomes → typically to study gene expression (active or inactive regions) & chromosomal location

• Flourescence with probes for gene-rich chromosomes (example: Chr 19) appear more open and dispersed, whereas gene-poor ones are compact and inactive (Chr-18).

• Can target entire chromosomes or specific exons.  

Replication factories

Most nuclear processes occur in distinct regions. → DNA replication takes place in replication factories. 

• Staining shows that replication occurs in the same place/clusters ⇒ DNA is drawn to these factories, rather than the factories moving around. 

  • BrdU: where newly incorporated DNA nucleotides are. 

  • PCNA: clamp protein.

Transcription hubs

Transcription also occurs in transcription factories that contain newly synthesized RNA. 

• Transcription hub: clustered sites of mediator complexes, RNA polymerases, and transcription factors. 

• Co-regulated genes (i.e. immunoglobulin genes) from different chromosomes may be transcribed in the same factory.

Nuclear bodies

Nucleolus	rRNA transcription, processing, and ribosome assembly.
Cajal body	snRNP assembly
Clastosome	Proteasomal proteolysis
Histone locus body	Transcription and processing of histone pre-mRNAs
Speckle	Storage of pre-mRNA splicing factors
PML Body	Transcriptional regulation, DNA repair
Polycomb body	Gene silencing

Function of nucleoli

1. rRNA synthesis → transcription of 5.8s, 18s, and 28s (RNA Pol I); NOTE: 5S is transcribed in nucleus. 

2. rRNA processing

3. Assembly of ribosomal subunits. 

Function of nucleoli: Assemblage of ribosome

• Ribosomes: polypeptide (translated in the cytosol) and rRNAs. 

• Ribosomal RNA genes transcribed at the interface between fibrillar center & dense fibrillar component of nucleolus → pre-rRNAs that undergo initial processing steps within dense fibrillar component. 

• Ribosomal proteins imported into nucleolus from cytoplasm → begin to assemble on pre-rRNA during their processing. 

• As the pre-rRNA is further processed = more ribosomal proteins & 5S rRNA assemble ⇒ pre-ribosomal particles. 

• Maturation: export of pre-ribosomal particles into cytoplasm ⇒ 40S and 60S ribosomal subunits. 

Polycomb bodies

Biomolecular condensates of repressed gene

• PCR2: catalyzes H3K27 trimethylation is also localized to Polycomb bodies. 

• Polycomb bodies formed by liquid-liquid phase separation mediated by inherently disordered domains (IDR) of PCR1 bound to H3K27me3. ⇒ PCR1 binding to the mark compacts chromatin = block transcription initiation. 

• So together = maintain stable, heritable gene silencing within Polycomb bodies. 

Cajal bodies

In the nucleus: small, round nuclear structures found in nuclei of eukaryotic cell

• RNA processing, later stages of snRNP (splicing) and snoRNP (rRNAs processing) maturation 

• Histone gene transcription, especially during DNA replication.

• Telomerase assembly

• Pseudouridylation (post-transcriptional modification of RNA) 

• Nuclear organization: organization and compartmentalization of cell nucleus.

Nuclear speckles

subnuclear structures found within the interchromatin regions of the nucleoplasm of eukaryotic cell. 

• Storage or reservoir site for splicing factors for pre-mRNA

• Active genes can be found located near or at the periphery of speckles, suggesting a functional relationship between speckles and transcriptional activity.

• RNA Processing and Modification

• Composition and morphology of nuclear speckles can change in response to cellular activity. Transcriptional inhibition=the size of speckles increases

• Contribute to the organization of the nuclear space