Molecular Neuro Exam 1

Untranslatable Regions

5’ UTR: hairpin, uORF, IRES

3’ UTR: zip code, CPE

upstream open reading frame (uORF)

associates with ribosomes and regulates timing of translation

internal ribosome entry site (IRES)

translational control

ZBP-1

Zip code binding protein (3’ UTR)

Transports actin to axon growth cone during development

Dscam gene

Found in Drosophila

Keep dendrites of the same neuron from interacting with one another

Can produce 38,016 proteins via alternative RNA splicing

Examples of alternative RNA splicing

1 Cassette exon

2 Mutually exclusive exons

3 5’ splice

4 3’ splice

Kinds of RNA

Messenger RNA (mRNA)

Transfer RNA (tRNA)
Ribosomal RNA (rRNA)

Micro RNA (miRNA)

Long Non-coding RNA (lncRNA)

Piwi RNA

miRNA

Post-translational gene regulation

Degrades target mRNA

Origins of mRNA

Intergenic

Intragenic

• Transcribed from host gene promoter

• Transcribed from intronic promoter

pri-miRNA vs pre-miRNA

pri-miRNA = “primary miRNA” - processed by Drosha/DGCR8 into pre-miRNA

pre-miRNA = may be processed into multiple miRNAs

Role of miRNA in early neurogenesis

Positive AND negative roles in regulation

BAF53a repression

Mediated by sequences within its 3’ UTR

Target of mi-R9* and mi-R124

lncRNAs

Longer than 200 bp and don’t typically encode protein

Possible functions:

• Decoy

• Scaffold

• Guide

• Enhancer

Types of lncRNAs

Long intergenic NCRNA: associate with chromatin modifying enzymes

Natural Antisense Transcripts (NAT) - recruit epigenetic modifiers

Overlapping - open chromatin structure

Bidirectional - open chromatin structure, facilitate chromatin modification of target genes

Sense intronic - open chromatin structure, recruit epigenetic modifiers

RMST

lncRNA

Involved in neurogenesis

Inhibits SOX2, prevents it from maintaining neural stem cell identity

EVF2

lncRNA

Required for interneuron development

Regulates Dlx5/6 and Gad1

Transcription factors

Stabilize RNA polymerase on the promoter site of DNA

Histone Methylation

Can promote OR repress transcription

Histone Acetylation

Switches from repressive to permissive chromatin

Condensed vs Uncondensed nucleosomes

Condensed: histone tails largely methylated

Uncondensed: histone tails LARGELY unmethylated, and acetylated

“canonical gene”

one gene-one protein

continuous area on a chromosome that codes for one protein

Establishment of cell identity

Happens early on

Caused by exposure to morphogens, expression of selector genes, and cell:cell interactions

Contact signals in mammals and drosophila

wnt family (wingless), TGFbeta(dpp) and hh(hedgehog)

Hedgehog (hh) signaling from floor plate

specifies motor neuron identity

Does any one marker/selector specify a cell type

NO!!!

Site Specific Recombinase

A protein that triggers a recombination when it binds to two specific DNA sequences

Example: flip recombination targets

RNAseq

the ability to quantify the expression of each gene in a given tissue

tRNA processing steps

removal of leader and trailer sequences,

post-transcriptional modification of bases,

addition of 3-CCA ends

aminoacyl tRNA synthetase (aaRS)

charges tRNA with cognate amino acid,

AA-tRNA synthetase use various chemical features, such as charge, hydrophobicity, size, and shape, to distinguish between different amino acids.

steps of tRNA charging with cognate amino acid

1) Activation of amino acids by attachment of AMP to form an AA-adenylate

2) Transfer of aminoacyl group to 3’ hydroxyl group of ribose of terminal adenosine of the CCA tail of tRNA

Double-sieve model

Allows each AA-tRNA synthetase to charge the correct amino acid (?)

1st sieve: size

2nd sieve: hydrophilicity

Editing site of ribosome

Secondary site which serves as an alternative to the aminoacylation site, important for amino acids that tend to be mis-activated in the aminoacylation site

Increases fidelity of aminoacetylation reaction

Two primary functions of tRNA

1) they decode the mRNA through direct base-pairing interactions 

2) they participate in the synthesis of polypeptide, as they carry a chemically reactive amino acid at their 3’ end.

Wobble pairing

occurs at third position of anticodon

allows certain non-Watson-Crick pairing interactions to be accepted by the translational machinery

monosome vs polysome

monosome: single, complete 80s ribosome. preferred in neurites

polysome: multiple ribosomes. preferred in cell soma

40s

small ribosomal subunit

responsible for deciphering mRNA

60s

large ribosomal subunit

mediates formation of peptide bond between amino acids

A site (Aminoacyl)

Ribosome primary binding site that is closest to 3’ end of mRNA

Receives and binds the incoming "charged" aminoacyl-tRNA carrying the next amino acid to be added to the chain.

P site (Peptidyl)

Middle ribosome primary binding site

Holds the tRNA that is currently attached to the growing polypeptide chain.

E site (Exit)

Ribosome primary binding site that is closest to 5’ end of mRNA

Holds the empty (deacylated) tRNA just before it is released from the ribosome into the cytoplasm

Classical vs revised model of ribosome biogenesis and heterogeneity

Classical model: Eukaryotic ribosomal proteins are shipped into the nucleus for assembly into ribosomal subunits within the nucleolus

Revised model: Several individual ribosomal proteins in the ribosome can be replaced by free cytoplasmic ribosomal proteins. Ribosome heterogeneity exists across cells or across cellular compartments

Most tightly regulated step in protein synthesis

Translation initiation

m7G modification

Adding the 5’ cap to mRNA

Recognized by eIF4E, binds to cap structure which is vital for cap-dependent translation

eIF2

Initiation factor

GTP dependent G-protein

Associates with 40s subunit and other initiation factors after binding tRNAmet - this forms 43s initiation complex

eIF4F

Subunits:

eIF4E: binds m7-GTP cap

eIF4A: DEAD-box RNA helicase, unwinds secondary structures on 5’ end of mRNA

eIF4G: scaffolding protein that binds to poly-A binding protein and eIF3 (part of 43S)

Integrated Stress Response Regulators

GCN2 - amino acid deprivation

PKR - viral infection

PERK - endoplasmic reticulum stress

HRI - heme depletion

Effects of phosphorylating eIF2alpha (eIF2 subunit) in the Integrated Stress Response

Inhibits eIF2B → prevents eIF2B from converting eIF2-GDP into eIF2-GTP. This conversion is essential for the initiation of protein translation.

Upregulates ATF4 by limiting availability of the eIF2 ternary complex, which prevents ribosome from stopping at the uORF that precedes ATF4

ATF4 is both a transcription factor and transcription repressor. it both represses CREB genes and upregulates stress response genes

Anabolic Signaling

Increases translation initiation

ERK/MAPK and mTORC1

mTORC1 phosphorylates/inhibits 4E-BP(4E binding protein). This inhibition leaves eF4E free to bind to the 5’ cap and allows for protein translation to initiate

Additionally, both mTORC1 and ERL/MAPK phosphorylate S6K1, which increases 43s formation

Translation Elongation

Occurs in three steps and requires 3 elongation factors:

GTP-bound eEF1A binds to the aa-tRNA and delivers it to the ribosome

Ribosomal peptidyl transferase (composed entirely of rRNA) catalyzes peptide bond formation.

eEF2 catalyzes coordinated translocation of both the ribosome and the mRNA/tRNA complex during protein synthesis

eEF2 regulation

eEF2 is highly regulated

eEF2k - inhibits eEF2 by phosphorylating it

Increased calcium or AMPK activity increases eEF2k activity

ERK phosphorylates and inhibits eEF2

mTORC1 phosphorylates S6K1, which phosphorylates and inhibits eEF2

Causes of ribosome stalling

mRNA defects (ex: truncations within coding sequences and specific mRNA sequences such as poly(A) tracts)

Ribosome rescue factors

Detect molecular signatures of stalling, such as empty A site or ribosome collisions

3 stop codons

UAA, UAG, UGA

Release factors

Required for termination of protein synthesis

Bind to A-site of ribosome

eRF1

release factor

structural mimic of tRNA, identifies stop codons and binds eRF3 and GTP

eRF3

release factor

GTPase activity

triggers hydroysis of bond between completed protein and final tRNA at P-site

ABCE1

Splits ribosome into free 60s subunit and mRNA and tRNA bound 40s subunit

Steady-state proteosome

all proteins present in cell + their interactions

Nascent proteosome

newly synthesized proteins

Local nascent proteosome

specific cell regions, axons/dendrites

Cell-type proteosome

Cell-specific nascent proteins

BCA (bicinchoninic acid) Assay

measure total protein content in sample

candidate POI (protein of interest) approach

Quantitative Mass Spectrometry

measures steady state proteosome

mass/charge ratio

bottom up: more established, can identify a greater number of proteins, and is more sensitive, but can lose information on post-translational modifications (PTMs)

vs

bottom down: provides a more complete picture of the functional, intact protein but is more challenging to perform and has lower throughput.  

Radioactive metabolic labeling

Nascent proteome

measures bulk translation

SILAC (stable (non-radioactive) isotope labeling in cell culture)

unbiased nascent proteomics

more modern than radioactive metabolic labeling and does not rely on radioactive materials

relies on incorporation of “light” or “heavy” form of amino acid into new proteins

weakness: peptides cannot be purified/enriched and method cannot capture rapid fluctuations

SUnSET (surface sensing of translation)

measures bulk AND protein of interest translation

puromycin mimics tyrosyl tRNA and occupies the A site when the codon codes for tyrosine.

puromycylated peptides can be identified using Western blot with antibody for puromycin

weakness: proteins generally truncated and nonfunctional

PunchP

Similar to SUnSET

Based on biotinylated puromycin

Involves the isolation of ribosomes followed by labeling, capture and mass-spectrometric analysis of nascent polypeptide chains

NCAT (non-canonical amino acid tagging)

nascent proteome

Azidohomolanine (AHA) fits into the binding pocket of Methionine aaRS then methionyl tRNA gets charged with AHA instead of methionine (Met)

Click chemistry

interaction between azide and alkyne

needs copper as a catalyst

results in formation of a covalent bond (triazole).

FUNCAT

varient of NCAT

utilizes click chemistry

uses a fluorophore-conjugated alkyne that bonds with AHA

especially useful for measuring protein synthesis load at the level of single cells using microscopy.

single-cell but not single-peptide resolution

measuring cell-type specific proteome with NCAT

ANL instead of AHA

ANL is only recognized by endogenous MetRS and is only recognized by mutant tRNA synthetase that is only expressed in target cell-type

Sucrose density gradient

Old technique used for polysomal profiling

continuous sucrose gradient of 15% to 45%.

using a UV detector to measure absorbance across the gradient at 254 nm (the wavelength that is absorbed by hydrocarbons), the gradient can be separated into fractions

heaviest fractions indicate polysomes

Ribosome tagging

cell-type specific translational profile

Express fluorescent/peptide tag for ribosomal subunit in specific cell-type

Perform translating ribosome affinity purification by using an anti-GFP (or HA) antibody to pull down mRNA-polysome complexes followed by mRNA purification and RNA-seq

x-rays/radiation

high mutation frequency

large chromosomal changes (insertion,deletion,inversion)

chemical mutagens

high mutation frequency

unbiased in which genes they affect

missense mutations, nonsense mutations, and frameshift mutations

insertional mutagenesis

ease in cloning genes

biased in which genes the affect

insertion frequency is low for transposons and retroviruses

forward vs reverse genetics

forwards: starts with phenotype and identifies genotype

reverse: starts with genotype and identifies phenotype

diploid screen for recessive mutations

screening approaches

morphology

mRNA/protein expression

fluorescent labeling of live cells

behavioral assays (ex:optokinetic response)

complementary testing to sort alleles

can tell you if mutations are on the same gene

DNA microarray for singlue nucleotide polymorphism detection (SNP)

Loss of heterozygosity

1 copy of a gene is lost