601 exam 2 merged

2 examples of ASO therapy for RNA mis-splicing diseases

DMD and SMA. They work in kinda opposite ways - the ASO blocks an ESS relevant to DMD, but blocks an ISS relevant to SMA.

How does ASO therapy work for DMD?

The mutation that causes DMD does so by deleting a few exons, which causes a frameshift that results in a premature stop codon. The ASO blocks the ESE (exonic splicing enhancer) that causes exon 51 skipping. This prevents the frameshift and the premature stop codon, which increases the amount of functional dystrophin protein in the cell.

How does ASO therapy work for SMA?

SMA is caused by the lack of SMN1 protein. ASO binds to the ISS of SMN2 mRNA (a similar gene), and increases splicing of exon 7. This increases the amount of SMN protein overall, making up for the loss of SMN1.

What are the three steps of protein translation? Which one is rate limiting?

Initiation, elongation, and termination. Initiation is the rate limiting one.

What complex regulates translation initiation? How does it do so? What factors are important for the formation of that complex?

the eIF4F complex. It regulates initiation by recruiting the 40S (small subunit) of the ribosome. That makes sense because the ribosome is needed for protein translation. The 5’ cap and the 3’ polyA tail on the mRNA are important for the eIF4F complex to form: eIF4E (complex, cap) binds to the 5’ cap, and PABP (poly a binding protein, duh) binds to the 3’ tail.

What does the mTOR pathway promote? What activates it?

global protein synthesis and growth signaling; nutrients, IGF

How exactly does mTOR promote protein translation molecularly? Name 6 complexes, starting with mTOR twice.

mTOR phosphorylates 4E-BP1 (binding protein of the next step yay). This causes the dissociation of 4E-BP1 from eIF4E. Free eIF4E can now bind the 5’ cap of mRNA and help start protein synthesis.

mTOR also phosphorylates S6K, which phosphorylates S6 in the 40S ribosome. When the S6 of 40S is phosphorylated, this promotes interactions between the ribosome and the eIF4F complex.

miRNA: length in nucleotides, # encoded in the human genome, % of human protein coding genes regulated by them, potential target sites of genes they regulate

22 nucleotides

2500 encoded

>60% of human genome regulated

3’UTR, coding regions, or 5’UTR

steps in miRNA biogenesis (4)

Pri-miRNAs are formed from longer RNAs transcribed from the genome

The Drosha/DGCR8 complex cleaves the pri-miRNA to a pre-miRNA

dicer cleaves the hairpin loop off of a pre-miRNA, forming a mature miRNA duplex

one strand of the duplex is loaded into the RISC (RNA-induced silencing complex) and guides the complex to the target mRNA, silencing it.

mechanisms of miRNA in negative regulation of gene expression (2) and details about when each happens and what’s involved

degradation of target mRNA: complete match between miRNA and target mRNA will cause degradation

repression of translation initiation: miRNA can interfere with eIF4E binding to 5’ cap and/or block the 60S subunit from joining the initiation complex

lncRNA: length, how it regulates gene expression (5)

>200 nucleotides long

1. recruits chromatin modifiers

2. regulates chromatin looping

3. recruits TFs

4. regulates RNA splicing

5. sponges up miRNAs

siRNA vs miRNA (acronym, how made, strandedness)

small interfering vs micro

lab made vs encoded in the genome

double stranded vs single stranded (at least originally, miRNAs are ds when they join w/ RISC)

how do siRNAs compare to the miRNA biogenesis pathway

siRNAs are injected as the mature, processed miRNA duplex (ds). Then one strand of the duplex (!, just like miRNA) can join with RISC to recognize target mRNAs and cleave. Question to self: do siRNAs regulate translation initiation like miRNAs, or are they just for mRNA degradation?

Considerations when designing an siRNA

Make sure the sequence you design is very specific to the target (ie matches it 100%). Conversely, see if that target sequence is present in other genes. Both of these help limit off target effects.

Patisiran siRNA-based drug: 1st/2nd? what does it treat? what mRNA does it target?

1st

polyneuropathy (p) incited by hereditary transthyretin-mediated amyloidosis

the mRNA making the mutated transthyretin protein (interesting - it can be better to silence the mutant protein and live without it than experience the toxic buildup)

givosiran siRNA-based drug: 1st/2nd? what does it treat? what mRNA does it target?

2nd

acute hepatic porphyria (AHP) - results from buildup of toxic porphyrin molecules formed during heme production

ALAS1, an enzyme critical in heme production

main mechanistic difference between CRISPR and siRNA (both use lab generated targeting sequences)

CRISPR targets DNA, siRNA targets mRNA

Components of the CRISPR system (3)

sgRNA (3 components): a 20nt base pairing sequence, a 42 nt Cas9-binding hairpin, and a 40nt terminator

Cas9: does the cutting

PAM: a sequence in the DNA target sequence required for complex recognition

How does CRISPR cas9 work for gene knockout vs knockin

In both cases, the system creates a DSB in the target sequence it binds to

If the DSB is repaired by NHEJ, indels are generated, and gene knockout happens

If a homologous template is available (based on cell cycle or scientist presentation), the cell will use that template to repair the DSB, and gene knockin happens

How do CRISPRa and CRISPRi work?

Both systems use dCas9, which is enzymatically dead and cannot generate DSBs. They also both use guide RNAs to target genes of interest for changes to gene expression!

CRISPRa fuses dCas9 to a transcriptional activator to turn on gene expression

CRISPRi can be used by itself (dCas9) to block transcription initiation or elongation. dCas9 can also be fused to a transcriptional repressor (like KRAB) to turn of gene expression

How can we tell if RNA editing is happening post transcriptionally?

By comparing the DNA and RNA sequences (there will be an expected RNA sequence, and the actual one you see could be different)

2 major types of RNA editing

C to U mediated by deaminases APOBECs

A to I mediated by deaminase ADARs (adenosine deaminase acting on RNAs)

RNA editing definition

The changes that take place in the RNA sequence after transcription is completed

How would RNA editing for potential disease therapeutics work?

The MS2 system with ADARs (the enzyme that converts A to I) and a guideRNA complementary to the target sequence would find and edit.

pick a common genetic disease and explain how to fix it with a technology that makes sense

yas

What’s the difference between the two types of posttranslational modifications: physical and chemical

physical is like folding and aggregation. It changes the molecular structure of proteins without a chemical reaction. Kinda based on attraction chemistry.

chemical is changes in the molecular property via chemical reactions. like cleavage and adding/removing chemical groups

secondary vs tertiary vs quaternary protein folding

secondary: backbone interactions drive formation of alpha helices or beta pleated sheets

tertiary: R groups drive 3-D structure of the entire polypeptide chain

quaternary: multimerization, like hemoglobin. multiple proteins coming together to form a complex

What is the equation that describes the forces at work in secondary structure protein folding? What does each variable mean?

deltaG = deltaH - T*deltaS.

deltaG: gibbs free energy change. Negative values are thermodynamically favorable

deltaH: enthalpy change. this describes energy in the system, stored in H bonds that keep the protein together

T: temperature

deltaS: entropy change. this is the disorder/chaos in the system (movement)

(sequential) forces at work during protein folding

secondary structure formation: H bonds make helices and sheets

hydrophobic collapse: hydrophobic residues collapse towards the center of the protein due to entropy forces

nucleation (local, then 2ndary and 3ary structures): everything else falls into place “folding inducing folding or bonding”

general principles of secondary structure protein folding

diffusion and random collision help form secondary structures

structure formation is driven by enthalpy and bonds

protein folding is processive

Which parts of proteins are formed through tertiary folding?

The parts that require 3D structure - enzyme active centers, TF DNA binding grooves, etc. These parts also need to be stabilized with strong bonds, which is a part of tertiary folding

Chaperone proteins

Assist with tertiary structure formation. Can help proteins fold that would not otherwise fold properly. Loss of chaperones can cause aggregation.

What’s the difference between a protein domain and motif?

Domain: primary structure. Has a distinct property/function, like catalytic domains and DNA binding domains

Motif: secondary/3D structure with a distinct function, like DNA-binding motif, helix-turn-helix motif, zinc finger motif.

Multimer

protein complex with multiple subunits. ex heterodimer, homotetramer

regulatory region of a gene

umbrella term for sequences like the promoter, enhancer, silencers, TF binding sites, etc. other names in prokaryotes.

Anatomy of a gene: 5’ to 3’

5’ regulatory region (can differ), transcription start site (DNA), 5’ UTR (mRNA), AUG (mRNA), exons and introns (both), UAG (mRNA), 3’ UTR (mRNA), termination region (DNA)

mRNA processing steps

addition of the 5’ cap, removal of 3’ end (up til poly A signal), addition of poly A tail; intron splicing

purpose of the 5’ cap

exonuclease protection, ribosomal recognition

purpose of the poly A tail

enzymatic protection, nuclear transport, translation efficiency

coding region of a gene

from the start codon to the stop codon, exons only, doesn’t include the regulatory region

what’s more similar in terms of gene expression: a dog and a human brain or a human brain and a human liver

dog brain and human brain

two main categories of epigenetic modifications

histone modifications and cytosine methylation

molecular memory

histone and cytosine modifications can keep DNA in its current expressive state

the main driver of cell type variability

gene expression! they all have the same genome duh

different reasons gene expression should differ (4)

cell type, brain region for the same cell type (ie temporal vs frontal lobe neurons), species for the same cell type, developmental lineage (progenitor vs committed cell)

diseases with multiple seemingly unrelated affected systems could be explained by

a mutation in a progenitor cell common to all of those lineages

TATA box

a DNA sequence at the start of genes where the RNA polymerase assembly occurs. Easy to unwind (AT rich), and conserved in about 15% of human genes

TBP transcription machinery

TATA box binding protein: recognizes TATA box and recruits other transcriptional machinery (TFIIA thru TFIIH essentially)

TFIIB transcription machinery

recognizes BRE: TFIIB recognition element

TFIID transcription machinery

recognizes INR (initiator, contains the TSS) and DPE (downstream promoter element)

enhancer elements

can be upstream or downstream of a gene, and very far away. allow for additional transcription factors to bind and influence gene expression

components of the RNA polymerase assembled complex

pol II, TBP, TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

names for the complexes that bridge enhancers and promoters, especially when they’re very far away

activator (sequence-specific DNA binding protein) and mediator (bridge between activator and RNA polymerase)

transcription inititation

once the transcription initiation complex is assembled (RNAp in the right spot, enhancer binding proteins bridged, etc), RNAp begins to move, hydrolyzing ATP, bringing in bases and polymerizing them

components of the regulatory environment in eukaryotic cells

cis regulatory elements (enhancer, promoter); TF availability; histone modifications; nucleosome occupancy (histone presence/density); chromatin environment (remodeling, DNA accessibility)

molecular mechanism of DNA being locked into loops (transcription)

Condensin is a ring-shaped protein that slides along chromatin, pulling DNA out into a loop until it hits a bound CTCF protein. CTCF proteins are directionally specific and recognize specific DNA sequences. The loop is finalized based on when the directional CTCF protein locks it into place. The locked DNA loop can help the enhancer and promoter interact from far away and create local domains of gene expression or repression.

why might a cell want to lock DNA into loops (transcription)?

genes on loops often have common functionality, so they can be expressed or repressed together

smallest scale of DNA regulatory environment

compaction of DNA into chromatin, nucleosome wrapping

intermediate scale of DNA regulatory environment

chromatin loops and topologically associating domains (TADs) (can be made by CTCF); epigenomic compartments (active vs inactive)

large scale of DNA regulatory environment

chromosome territories, regulation of contact between them

in the location of CTCF binding, what would happen to DNA-DNA interactions

an absence! proteins are bound to the DNA and DNA gets looped out, both of which decrease DNA-DNA interactions in the area. It also helps with enhancer and promoter connection via activators and mediators.

How do DNA-DNA interactions look on a Hi-C map when plotted against CTCF and cohesin binding sites

In the areas with CTCF and cohesin binding, there are low DNA-DNA interactions. This looks like less colored areas that make up the bases of triangles. The peak of the colored triangles comes in the middle of large gaps without CTCF and cohesin binding sites.

How might the graph of CTCF and cohesin binding sites along with DNA-DNA interactions look different in different cell types?

Different cell types have different expression profiles and local expression domains, so their graphs of where CTCF and cohesin bind (decreasing DNA-DNA interactions) will differ! These maps can give you an idea of the different expression profiles of different cells.

most common modifications on histone tails

acetylation, methylation, phosphorylation

4 major classes of proteins that do work on histones

writers, erasers, readers, remodelers (remodel chromatin by moving histones)

examples of epigenetic writers

histone methyltransferase, histone acetyltransferase

examples of epigenetic erasers

histone demethylase, histone deacetylase

examples of epigenetic readers

bromodomains, chromodomains, tudor domains, PHD fingers, PWWP domains, malignant brain tumor domains (MBT). These are very often affected in cancer.

zinc finger, leucine zipper, helix-turn-helix (HTH/homeodomain), helix-loop-helix (HLH) are all what class of protein

TFs

domains possible for TFs to have

DNA bindgin domains, ligand binding domains (other proteins, histones, signals, etc), enzymatic activity domains

TF role in cell type identity

TFs help genes be expressed in a tissue specific manner. This establishes cell type identity and directs organism development

example of TF acting as intermediate between extracellular signaling and gene expression change

external ligand causes cAMP to activate a kinase that phosphorylates CREB (CRE = cAMP response element). Phosphorylated CREB is active and can bind DNA, activating transcription of target genes

sequence logo

visualization of the consensus sequence at a TF’s DNA binding site. height of the letters indicates how often that letter is seen at that position. Shows you how tightly the TF needs to match its target sequence in order to bind.

Body patterning is dictated by

spatial expression of specific transcription factors (classic ex is fruit fly, where the TFs are clustered on the chromosome in the order of the body segments)

Main difference between the body patterning TFs in simple and complex organisms

Expansion of families, like duplications that gained new related functions. Many of the ancestral genes are paralogs of the current human genes.

what drives the differentiation of pluripotent stem cells into their eventual cell types (and example)

transcription factors duhhhh (the Ets family of TFs play a large role in hematopoiesis)

yamanaka factors

TFs expressed very early in dvelopment can also reprogram mature cells back into stem cells (iPSCs, which can be redifferentiated for a variety of purposes)

Pioneer TFs

one of the first TFs to bind in a closed site, can open chromatin up by signaling readers and writers to reorganize epigenetic landscape. Then, other lineage-specific TFs can bind and do their jobs

Antagonistic TFs

A TF can come in and kick an already bound TF off the DNA. Gene expression stops if the kicked off TF was promoting it.

Synergy TFs

One TF is already bound and gene expression is on. The binding of another TF nearby increases gene expression.

Competition TFs

2 bound TFs are in competition for the help of a third TF? look this up, might not be this specific

Redistribution TFs

A TF comes in and causes a previously bound TF to bind at a different spot in the genome. The gene it was supporting is no longer transcribed, and the new TF promotes transcription nearby.

Modulation TFs

The opposite of synergy. A second TF coming in to bind decreases the activity of an already bound TF. Could be down to 0. Could support lineage branching (ex)

4 factors that affect the ability of TFs to do their jobs

Biochemical compatibility, spatial architecture, insulation, and chromatin environment

Biochemical compatibility of TFs

Can TFs bound at different spots on the DNA interact with each other based on their 3D protein shape?

Spatial architecture of TFs

Are TFs bound on different parts of the DNA able to interact with each other based on how the DNA is wrapped? Are they close enough to one another?

Insulation of TFs

Are bound TFs that need to interact with each other separated from each other by an insulating factor? Ie a protein that binds DNA or creates a DNA loop out?

Chromatin environment of TFs

If the TFs are bound, are they in an open chromatin environment that allows them to interact with other bound TFs to promote gene expression?

Enhancers (definition)

positive transcriptional control elements greater than 200 bp from TSS

Silencers

negative transcriptional control elements, similar to enhancers except they contain binding sites for repressors

Repressors

TFs that repress transcription

Insulators

DNA sequence bound by CTCF (CCCTC-binding factor) to serve an insulation function. Can block an enhancer from acting on its target gene

eRNA

enhancer RNA. Can be encoded by enhancer. eRNA can function as a scaffold to facilitate the assembly of transcriptional machinery on the promoter. Can also serve as a marker for the enhancer?

ncRNA in terms of enhancer and promoter sequences

ncRNA can facilitate interactions between enhancer- and promoter-bound proteins, helping the enhancer loop around to find the promoter

Regulation of transcriptional elongation: general

Pol II transcribes the first 20-60 nt, then pauses. Escape from pause is highly regulated, relevant for about half of mammalian genes.

Regulation of transcriptional elongation: players and their roles (3)

DSIF: DRB sensitivity-inducing factor; facilitates pausing

NELF: negative elongation factor; facilitates pausing

P-TEFb: positive elongation factor b; drives escape from pausing

Examples of diseases caused by mutations in TF binding sites, affecting gene expression

Alpha and beta thalassemia, hemophilia B, osteoporosis, hirschprung, preaxial polydactyly

Gene expression and Kabuki syndrome

mutations in KMT2D and KDM6A result in too much closed chromatin, leading to symptoms throughout the body

5’ capping: what happens and why

7 methylguanosine is added to 1st nucleotide of mRNA. promotes stability, transport to cytoplasm, and translation

3’ polyadenylation: what happens and why

poly A tail is added to the polyadenylation signal (PAS, AAUAAA). Promotes mRNA stability, transport to cytoplasm, and translation