Exam Two

Pre-mRNA Splicing

removal of interrupting non-coding segments (introns) from messenger RNA precursors and the splicing together of the protein-coding segments (exons) to form mature RNAs

Introns

interrupting non-coding regions found in eukaryotic genes

prokaryotic genes do not contain these

Exons

the regions of the gene that will be expressed and used to make proteins

Step #1 of Intron Splicing (Branching)

2’ hydroxyl of the branch point adenosine attacks the 5’ splice site phosphodiester bond

Step #2 of Intron Splicing (Exon Litigation)

3’ hydroxyl of the 5’ exon attacks the 3’ phosphodiester bond

Spliceosome

does the pre-mRNA splicing

contains five small nuclear RNAs, Sm proteins, and some other proteins like helicases

assembly is highly ordered

Alternative RNA Splicing

the cell can pick and choose the exons it wants to splice together

one pre-RNA can be spliced into different mRNAs that encode different proteins

Splicing Enhancers

communicate with U1 and U2 to promote exon inclusion

bind to serine/arginine-rich proteins

Splicing Silencers

compete with SR proteins to repress splice site selection

bind to heterogeneous nuclear ribonucleoproteins

Mutually Exclusive Exons

if one exon is spliced out, the other must be retained and vise versa

Down Syndrome Cell Adhesion Molecule (Dscam)

controls nervous system development with axon guidance

undergoes mutually exclusive splicing

required for proper axon targetting

Steps of Axonal Guidance

1. axons travel a route that leads them to a region in the embryo

2. axons recognize and bind to a set of possible target cells

3. the initial pattern is refined such that the axon binds to a small subset of possible targets

Pioneer Nerve Fibers

axons that go ahead of other axons and serve as guides for them

Growth Cone

motile tip of a neuronal axon that leads to nerve outgrowth

attach to the substrate and pulls the axon forward

can sense external guidance cues

Fasciculation

one axon binding to another and using it for growth

Is the splicing of Exon 4 in the Dscam assignment a regulated or random process? Why?

It is a random process. The animals express the variants in different areas from one another.

Autism Spectrum Disorders (ASDs)

a group of complex neurodevelopmental disorders characterized by repetitive and characteristic patterns of behavior and difficulties with social communication and interaction

exhibits high heritability

Multifactorial Condition

a complex disorder/disease without a single genetic cause but is associated with disturbances in multiple genes in combination with lifestyle and environmental factors

Concordance

genetic term used to describe presence of the same trait in both twins if one twin exhibits the trait

Polymorphisms

two or more genetic differences in or between populations

many forms

Single Nucleotide Polymorphisms (SNPs)

one nucleotide in the DNA sequence differs between individuals

Copy Number Variants (CNVs)

the number of copies of parts of genes, entire genes, or sets of genes that differ in humans

DNA segments larger than 50 base pairs that are present at a variable copy number

estimated 4.8-9.5% of the human genome

some are linked to human ASD

De Novo Mutations

present in the offspring but was not present in the genomes of the parent

Genome-Wide Association Studies (GWAS)

a research technique identify associations between genotypes and phenotypes by comparing differences in genetic variants between individuals who differ phenotypically

Polygenic Risk Score (PRS)

a numeric value that estimates an individual’s genetic predisposition to a complex trait or disease

What are the core symptoms of ASD in humans?

social deficits

repetitive behaviors

language deficits

Links Between ASD and GI Issues

children with ASD are 3.5 times more likely to have GI issues

these issues are associated with changes to the microbes living in the gut

What technique did the researchers use to look at gut bacteria in ASD mice?

quantitative PCR

What effect does a maternal high-fat diet have on mouse social behavior and levels of L. reuteri?

induces social deficits

reduces levels of L. reuteri

L. reuteri Bacteria

gut bacteria lacking in mice with ASD

modulates social behavior and related changes in synaptic function within social reward circuits via the oxytocinergic system

does this independent of other gut microbes and in a vagus nerve-dependent manner

treatment with this can reverse social deficits in ASD mice with many causes of ASD

How would you determine if the effects of L. reuteri on social behavior are vagus nerve-dependent?

remove it

Epigenetics

control of heritable or non-heritable phenotypic changes due to internal or external (environmental)- induced modifications of gene expression and in the absence of changes with the DNA sequence

Euchromatin

loosely packed chromatin

transcribed since transcriptional machinery has access to the promoter

Heterochromatin

highly packed, condensed chromatin

not transcribed since transcriptional machinery does not have access to the promoter

Nucleosome

fundamental unit of chromosome packaging

80% of human DNA packaged like this

Histone

disc proteins DNA is wrapped around

eight of these make up a nucleosome core particle

rich in positively charged amino acids

Histone Modifications

acetylation, methylation, phosphorylation, ubiquitination, sumoylation

reduces positive charges on histones to decrease histone/DNA binding

recruits transcriptional activators or repressors

Ubiquitination

tags proteins for degradation

Sumoylation

affects functions of proteins

Writers

put on modifications

Erasers

take off modifications

Readers

recognize modification and recruit appropriate proteins

Heterochromatin-Specific Proteins

cause heterochromatin spreading

recruited to modify neighboring nucleosomes

Chromatin-Remodeling Complexes

hydrolyze ATP to slide nucleosomes

DNA Methylation

methyl groups added to cytosines (CpG islands)

Methylation of Promoters and Transcriptional Starts Sites

repressed transcription by recruiting transcriptional modifiers

Methylation of Gene Bodies

activates transcription

Long Non-Coding RNAs (lcnRNAs)

can act as epigenetic regulators by acting as scaffolds at recruit chromatin modifying enzymes

larger than 200 nucleotides in length

roughly 5,000 in human genome

used to inactivate X chromosomes

What specific type of DNA methylation do we see in our genomes?

5-methylcytosine (5mC)

usually at CpG dinucleotides

How does promoter methylation promote embryonic development?

represses self-renewal genes allowing for differentiation

What specific type of RNA methylation do we see in our transcriptome?

N⁶-methyladenosine (m⁶A)

How does mRNA methylation promote embryonic development?

downregulates pluripotency regulators allowing for differentiation to occur

What kind of model allows for m⁶A in a mRNA to contribute to chromatin structure and gene expression?

the METTL3-METTL14 complex pulls the methyl group off of the histone at the same time transcription is occurring

Through which mechanism does the METLL3-METTL14 complex lead to epigenetic modifications of a gene?

it recruits the 5mC writer DNMT1 that then marks the gene body

What do the authors of the RNA methylation paper conclude about the co-occurrence of m⁶A and 5mC?

they are a distinct method of gene expression regulation involving both transcriptional and post-transcriptional effects

What model do the authors of the RNA methylation paper put forth to explain how combinations of m⁶A and 5mC levels promote the differentiation of embryonic stems cells to embryoid bodies?

a shift in the balance influences expression of differentiation genes

What method can be used to see what in methylated in the genome?

bisulfide treatment converts cytosine to uracil, but methylated cytosines do not get converted